The level of protein in the maternal murine diet modulates the facial appearance of the offspring via mTORC1 signaling

The level of protein in the maternal murine diet modulates the facial appearance of the offspring via mTORC1 signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The level of protein in the maternal murine diet modulates the facial appearance of the offspring via mTORC1 signaling Andrei Chagin, Meng Xie, Marketa Tesarova, Yaakov Gershtein, Daniela Schnyder, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2542333/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Mar, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The development of craniofacial skeletal structures is fascinatingly complex and elucidation of the underlying mechanisms will not only provide novel scientific insights, but also help develop more effective clinical approaches to the treatment and/or prevention of the numerous congenital craniofacial malformations. To this end, we performed CAGE-sequencing of the facial mesenchyme of human embryos and cross-checked the active enhancers thus identified against genes, identified by GWAS for the normal range human facial appearance. Among the identified active cis-enhancers, several belonged to the components of the mTORC1 (Mechanistic Target of Rapamycin Complex 1) pathway. To assess the functional role of this pathway, we manipulated it both genetically and pharmacologically in mice and zebrafish. These experiments revealed that mTORC1 signaling modulates craniofacial shaping at the stage of skeletal mesenchymal condensations, with subsequent fine-tuning during clonal intercalation. This ability of mTORC1 pathway to modulate facial shaping, along with its evolutionary conservation and ability to sense external stimuli, in particular dietary amino acids, indicate that the mTORC1 pathway may play a role in facial phenotypic plasticity. Indeed, the level of protein in the diet of pregnant female mice influenced the activity of mTORC1 in fetal craniofacial structures and altered the size of skeletogenic clones, thus exerting an impact on the local geometry and craniofacial shaping. Overall, our findings indicate that the mTORC1 signaling pathway is involved in the effect of environmental conditions on the shaping of craniofacial structures. Biological sciences/Genetics/Gene regulation Biological sciences/Developmental biology/Morphogenesis/Cell lineage Craniofacial neural crest skeleton mTORC1 protein shape mesenchymal condensation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction A key aspect of most social communication between humans is facial recognition 1 . Accordingly, congenital craniofacial malformations, including cleft palate, craniosynostosis, and craniofacial skeletal hypoplasia, which together account for more than one-third of all congenital birth defects, can have a profound influence on social interactions 2 , 3 . From an evolutionary perspective, the viscerocranium harbors vital structures such as the feeding apparatus and supports sensory organs. Its precise sculpting and inheritable reproducibility are of unequivocal importance for survival. At the same time, adaptability of viscerocranium and particularly of the feeding apparatus to the environmental cues allows adjusting to environmental changes, e.g., feeding sources, or even concurring novel ecological niches. The latter type of adaptation is less common in mammals than in several classes of Gnathostomata, e.g., Actinopterygii 4 . The craniofacial skeleton, one of the most complex and sophisticated part of the skeletal system, is composed of many different parts and formed via the interplay of a variety of genetic, epigenetic, and environmental factors 5 – 7 . Studies involving families with monozygotic and dizygotic twins indicate that the genetic inheritability score for craniofacial morphology in humans varies widely among different facial features, e.g., from 0.8 for the distance between the inner corners of the eyes to approximately 0.5 for the position of the point midway between the nose and upper lip, as well as for nasal protrusion 8 – 11 . One well-known environmental influence on facial morphogenesis in humans is alcohol consumption during pregnancy 12 . In all Gnathostomata , including zebrafish, mice, and humans, the viscerocranium develops from descendants of the neural crest cells (NCCs), a transient and multipotent population of embryonic progenitors 13 . Multiple subpopulations of the mesenchyme derived from NCCs condense and then differentiate further into chondrocytes and osteoblasts, the major types of skeletal cells in the craniofacial region 14 – 16 . Other developmental subpopulations derived from NCCs, such as Schwann cell precursors, also contribute to the formation of chondrocytes and osteoblasts, but to a relatively limited extent 17 . The shapes of the craniofacial skeletal elements are determined primarily by the shape of these mesenchymal chondrogenic condensations during development, with subsequent fine-tuning by localized intercalation of new chondrogenic clones arising from the surrounding mesenchyme 18 , 19 . A complex interplay between NC cells, the facial ectoderm, placodes, endoderm and neuroepithelium orchestrates accurate sculpturing of the viscerocranium. Not surprisingly, this process involves continuous changes in the expression of thousands of different genes 20 . Genome-wide association studies (GWAS) have implicated more than 100 loci in the formation of facial morphology within the normal range and more than two hundred single nucleotide polymorphisms (SNPs) that exert a significant impact on this formation 21 – 23 . In association with abnormal morphology of the human facial skeleton, the HPO database ( http://hpo.jax.org ) lists 1165 genes 24 . Although many of these are associated primarily with other systems, such as hematopoiesis and neurogenesis, their large number reflects the complexity of facial morphogenesis. At the same time, the proper migration and differentiation of NCCs, as well as their interaction with surrounding tissues during facial development involves limited number of developmentally and evolutionarily conserved signaling pathways. Among those are hedgehog (HH), fibroblast growth factors (FGF), bone morphogenic proteins (BMPs), WNT, retinoic acid (RA) and platelet-derived growth factor (PDGF) pathways 25 . These pathways and associated morphogens form a hardware system, genetically responsible for sculpting of viscerocranium. However, the signaling pathways, capable to sense environmental clues and integrate these signals into genetical hardware of facial morphogenesis are rather unknown. One proposed system is HH signaling pathway, which may sense mechanical forces via cilium 26 and modify craniofacial formation as it has been shown in bony fish 27 . Clearly, a major factors underlying natural selection has been the availability of nutrition 28 and the feeding apparatus, part of the viscerocranium, is of particular importance in this context. Nutritional sensing by the Mechanistic Target of Rapamycin Complex 1 (mTORC1) signaling pathway has been highly conserved evolutionarily 29 . Budding yeasts sense the availability of amino acids via mTORC1 and, in response to this information, shift towards the synthesis of proteins or autophagy 29 . Although this pathway plays a similar role in multicellular organisms 30 , 31 , in this case levels of oxygen, energy and growth factors (primarily those transducing via P13 kinase and Akt 30 ) also exert an influence. On the systemic level, mTORC1 can be regarded as belonging to an endocrine network of both up- and downstream of insulin-like growth factors (IGFs) that regulates a variety of processes in response to the availability of nutrition 30,32−34 . Changes in the activity of mTORC1 can alter the shape of craniofacial structures 35 , 36 and, in addition, the mTORC1 pathway interacts with the HH, BMPs, and Wnts 37 – 39 pathways, which are strongly involved in sculpting of the viscerocranium. Accordingly, we hypothesize that the mTORC1 signaling pathway may play a role in mediating interactions between certain environmental factors and the inherited program of craniofacial morphogenesis. Results The PI3K/mTORC1 pathway is associated with facial appearance in humans To identify enhancers actively involved in human facial development, embryonal facial material was CAGE-sequenced (Fig. 1 A) and all enhancers actively transcribed during human facial development between week 3 and 12 of gestation were identified (GEO #xxx, supplementary metadata file). All thus identified enhancers were further cross-checked and enriched against enhancers previously identified in ENCODE project 40 (see the Methods). The resulting pool of enhancer coordinates was overlapped with published GWAS hits 22 (see the Methods) identified to be associated with normal-range facial morphology (Fig. 1 A). Among enhancers thus identified there was a clear enrichment in components of the PI3K/mTORC1/autophagy pathway (Fig. 1 B, C, Extended data Fig. 1 ). Predictions based on the STRING database (Fig. 1 D) and the specific facial phenotype related to each individual polymorphism (Fig. 1 E) indicated that among the major enhancers active during sculpting of the human face, the mTORC1 pathway was clearly enriched. Thus, this approach identified PI3K/mTORC1 pathway as a potentially important player in human facial morphogenesis. To explore the mechanism(s) underlying involvement of this pathway in craniofacial shaping, we manipulated the mTORC1 pathway during facial development in experimental animals. mTORC1 modulates the shaping of chondrogenic condensations in the mouse First, we activated the mTORC1 pathway in neural crest cells (NCCs) by crossing Tsc1 floxed mice with the Sox10 CreERT 2 strain 41 , in which a pulse of tamoxifen on embryonic day 8.5 (E8.5) causes recombination in NCCs 15 . Reconstruction of the developing craniofacial structures in the offspring utilizing 3D µ-CT images with enhanced contrasting of soft tissues revealed alterations in the thickness of skeletal elements, as well as minor developmental abnormalities already on E17.5 (Fig. 2 A-D). Overlay of the reconstructed cartilage of Tsc1 cKO and control ( Tsc1 heterozygous) embryos revealed enlargement of a variety of elements of the craniofacial skeleton, as well as enhanced thickness of all components of the nasal cartilage (Fig. 2 E, F). These observations confirmed the involvement of the mTORC1 pathway in craniofacial shaping 35 , 36 and, in addition, showed that this pathway is involved during early development. Previously, we demonstrated that in mice craniofacial shape is established at the time of mesenchymal condensation (E12.5-E13.5), with subsequent fine-tuning via intercalation of new clones 18 , 19 . To reveal the shape of these mesenchymal condensations, KO embryos were stained for Sox9 on E12.5 and, although the overall shape was preserved, their nasal prominence and nasal capsule compartments were thicker (Fig. 2 G-I). We then bred in the R26R Confetti reporter transgene, which allows clonal behavior to be assessed. Analysis of Sox10 CreERT 2 ; Tsc1 flfl ; R26R Confetti embryos on E17.5 (with previous pulsing on E8.5) revealed that in the absence of the Tsc1 gene, the clones of nasal chondrocytes appeared as bulky large clusters (Fig. 2 J-O), with more extensive dispersion and misalignment (Fig. 2 P-S ), compared to the individual columns observed in the Sox10 CreERT2 ; Tsc1 fl+ ; R26R Confetti heterozygotes (Fig. 2 J-S). Thus, activation of the mTORC1 pathway in murine NCCs modulated both chondrogenic condensation and clonal arrangement. Modulation of the activity of the mTORC1 pathway at the stage of intercalation influences shaping of the craniofacial skeleton to a relatively minor extent To explore the influence of the mTORC1 pathway during intercalation of new clones into existing mesenchymal condensations 19 , we injected tamoxifen on E12.5, the stage at which Sox10 CreERT 2 targets perichondrial cells surrounding cartilage elements and Schwann cell precursors 42 . Surprisingly, ablation of Tsc1 at this developmental stage did not change the shape of the craniofacial skeleton (not shown) and increased clonal size only slightly (Fig. 3 A-J). To further verify this observation, we targeted chondro-progenitors involved in early mesenchymal condensation employing Col2 CreERT mice coupled with both the Tsc1 floxed and R26R Confetti strains and pulsed with tamoxifen on E12.5. In line with the previous observation, activation of mTORC1 signaling by Col2 CreERT at this developmental stage did not alter the structure of the craniofacial skeleton and increased clonal size slightly (Fig. 3 K-O). At the same time, ablation of mTORC1 signaling in chondro-progenitors by crossing Raptor floxed mice with the Col2 CreERT and R26R Confetti strains and pulsing with tamoxifen on E12.5 augmented facial length on E17.5 (as detected by µCT, see Fig. 3 P-R) without affecting any other skeletal parameters (Extended data Fig. 2 A-C). Ablation of Raptor under these same conditions lowered the number of large clones somewhat and enhanced the number of cells that were single-labeled (Fig. 3 S-Y). Successful manipulation of mTORC1 activity in these various strains was confirmed by assessment of S6 phosphorylation (Extended data Fig. 2 D-E and data not shown). These observations indicate that in mice the mTORC1 pathway is involved in craniofacial shaping predominantly prior to and/or during the stage at which chondrogenic condensations occur. Inhibition of mTORC1 immediately prior to chondrogenic condensations alters the formation of cartilaginous facial structures in both mice and zebrafish To establish the stage of craniofacial skeletogenesis during which the role of mTORC1 signaling is most important, we inhibited mTORC1 with a single injection of rapamycin into pregnant animals on E10.5, when migration of cranial NCCs has been completed, but chondrogenic condensation has not yet begun 15 , 17 . This resulted in a slightly elongated snout in the embryos on E17.5 in comparison to the controls injected with DMSO (Fig. 4 A-C). Moreover, the thickness of chondrogenic mesenchymal condensations on E12.5 was reduced (Fig. 4 D-F). Clonal lineage tracing of chondro-progenitors in these same embryos beginning on E12.5 revealed disorganized clones, with relatively fewer elongated clones containing more than three chondrocytes and a relatively higher number of labeled cells that had not divided (i.e., in which recombination had occurred, but which did not proliferate during the period of tracing) (Fig. 4 G-M). To determine whether this influence of mTORC1 signaling on mesenchymal condensation is conserved among species, we also studied zebrafish, in which shaping of the craniofacial skeleton also occurs via chondrogenic condensation and intercalation of chondro-progenitors into the primary cartilage anlagen 43 . In these animals the craniofacial skeleton begins to develop between 48 and 72 hours post-fertilization (hpf) 44 and the first Sox9- and Col2-positive cells appear at 48 and 53 hpf, respectively 17 . Col2a1aBAC:mcherry zebrafish larvae were exposed to rapamycin at various time-points in their development, washed free of this compound, and then allowed to develop until 120 hpf. Exposure prior to (14–22 hpf, 24–32 hpf) or during (32–56 hpf) chondrogenic condensation did not affect the overall size of the facial skeleton, but led to narrowing of cartilaginous structures (Fig. 4 N-S). Interestingly, significant elongation of the face occurred when the larvae were exposed to rapamycin at 14–22 hpf or 32–48 hpf (Fig. 4 R). Furthermore, exposure prior to chondrogenic condensation resulted in slight curvature of the ethmoid plate (ETH) and re-positioning of several other elements of the cartilage (MC, PQ and CH) (Fig. 4 P,Q). Altogether, these findings indicate that in both mice and zebrafish the mTORC1 pathway modulates the shape of craniofacial structures by regulating the recruitment and clonal expansion of mesenchymal derivatives of neural crest cells. Interestingly, even transient inhibition of mTORC1 activity early during development altered the clonal behavior of NCC progeny, thereby leading to subsequent modulation of the shape of the craniofacial skeleton. Dietary interventions that modulate mTORC1 activity also alter the skeletal structure by impairing clonal dynamics The evolutionary conservation and mild variability described above indicate that mTORC1-dependent modulation of craniofacial structures, and particularly those of the feeding apparatus, may be an important adaptive mechanism. As also mentioned above, the activity of the mTORC1 pathway is regulated by nutritional status and, in particular, by dietary levels of amino acids, which act both directly at the cellular level through receptors for arginine and leucine and systemically via pathways involving growth hormone and insulin growth factors (IGFs), which are themselves also controlled by amino acids levels 30 , 32 . Accordingly, we examined whether alteration of mTORC1 activity through feeding diets containing different levels of protein to pregnant dams might modulate craniofacial shaping in the offspring. For this purpose, starting on E6.5 pregnant C57BL/6J mice consumed isocaloric diets containing either 20% protein (a level similar to that in standard mouse chow = the control), 4% (low) protein or 40% (high) protein, with subsequent analysis of at least 4 different litters of embryos from each group. As expected, mTORC1 activity (as reflected in the level of pS6) was lowest in the control embryos and most pronounced in the high group (Extended data Fig. 3 A,B), with no differences in body weight (Extended data Fig. 3 C). µCT scans of embryos on E17.5 utilizing phosphotungstic acid (PTA) to augment contrast (Fig. 5 A) revealed that both the length and width of the nasal capsule (Fig. 5 B,C), as well as the length of the Meckel’s cartilage (Fig. 5 D) were all influenced by the level of protein in the maternal diet. Thus, comparison of 3D segments of the chondrocranium cartilage showed that both the nasal capsule and mandible were slightly smaller in the embryos whose dams received 4% dietary protein (Fig. 5 E). In addition, the thickness of the cartilage of the nasal capsule was elevated by the higher level of dietary protein (Fig. 5 F). To confirm these observations, the same experiment was performed, but utilizing Hexabrix 320 for contrast in connection with the µCT, and similar changes in craniofacial structures were observed (Extended data Fig. 3 D-M). Lowered proliferation of cells within skeletal elements was observed only in embryos whose dams received the lowest level of dietary protein (Extended data Fig. 3 N-T) and there were no differences between the groups with respect to the extent of cell death within cartilaginous elements (Extended data Fig. 3 Q-U). When the level of protein in the diets was manipulated in this same manner in pregnant Sox10 CreERT 2 ; Tsc1 flfl dams (pulsed with tamoxifen on E8.5 and, accordingly, having embryos with constitutively active mTORC1 in all their NCCs-derived cells) and the craniofacial structures of these embryos analyzed on E17.5, again by µCT scans utilizing PTA for contrasting, no differences in any of craniofacial parameters were detected (Fig. 5 G-J). Power analysis showed that at least 320 pups need to be analyzed to detect significant difference in the length of nasal capsule with a power of 0.95 and 55 pups for its width, which is beyond the feasibility in 3D reconstructions of PTA-enhanced embryos. Thus, the lack of any alteration in the craniofacial parameters examined (Fig. 5 H-J), together with the changes in pS6 activity observed above (Extended data Fig. 3 A,B), indicate that the alterations in craniofacial structures in response to the different levels of dietary protein were mediated by mTORC1 signaling. Next, when the level of protein was manipulated in this same manner in the diets of Col 2 CreERT 2 ; R26R Confetti mice and these animals injected with tamoxifen on E12.5 and E13.5, both the low and high levels of protein caused remarkable disorganization of the clones within developing cartilaginous elements (Fig. 5 K-L). This finding is in agreement with the conclusion above that both elevation and attenuation of mTORC1 activity disturbs clonal organization within developing cartilage. Finally, incorporation of the average values obtained in mice with the low and high protein diets to a mathematical model of human skulls for visualization purpose indicated slight, but clear alterations in multiple elements of the craniofacial skeletons (Extended data Fig. 4 , see the Methods for further details). Altogether, these findings indicate that the level of protein in the maternal murine diet during pregnancy influences embryonic shaping of craniofacial cartilage by altering the activity of mTORC1, which in turn changes the clonal dynamics of neural crest progeny. Discussion Here, we have revealed cellular mechanisms underlying mTORC1-dependent shaping of elements of the craniofacial skeleton and demonstrated in both zebrafish and mice that this shaping occurs predominantly in association with mesenchymal chondrogenic condensations, with subsequent fine-tuning to a lesser degree via intercalation. In addition, we have demonstrated that mTORC1 activity in embryos of these species is modulated by the level of protein in the maternal diet, with associated effects on the chondro-cranium and fine-tuning of the shape of the craniofacial skeleton. In greater detail, we show here that alterations in the behavior of progeny of NCCs influence skeletal shaping, both at the stage when chondrogenic mesenchymal condensations occur and when the 3D morphology of the cranial skeleton is fine-tuned via clonal intercalation. The finding that the shape of mesenchymal condensations largely determines the subsequent shape of cartilaginous and, later, bony structures 19 provides a link between the expansion of ectomesenchyme derived from murine NCCs lacking Tsc1 and resulting changes in craniofacial shape 35 . It is noteworthy that mTORC1 activity influences the shaping of different chondrogenic mesenchymal condensations to different extents, with imperfect preservation of the rough 3D geometry of the entire chondrocranium. For example, constitutively active mTORC1 increases the thickness of the condensations underlying the nasal prominence and nasal capsule, while changing the patterning of the nasal septum to a much more limited degree. Therefore, specific mechanisms or processes appear to be localized within distinct regions of the chondrocranium. The potential underlying mechanism(s) may involve the known interactions between the mTORC1 pathway and the major morphogens involved in the shaping of craniofacial structures, including HHs (hedgehogs), FGFs (fibroblast growth factors), BMPs (bone morphogenetic proteins), WNTs (Wingless/Integrated family of morphogens), RA (retinoic acid) and PDGFs (platelet-derived growth factors) 25,39,45−49 . For instance, ablation of mTOR specifically in NCCs reduces the activities of the canonical Wnt and BMP pathways 36 . SHH, which is secreted in localized regions by the neuroepithelium and the brain, participates in shaping the anterior chondrocranium in a highly specific manner, e.g., by inducing or permitting formation of the nasal septum 18 . At the same time, S6K1, a kinase downstream of mTORC, augments HH signaling by phosphorylating GLl1 39 . Thus, the differential chondrogenic activity of SHH, in combination with its functional interactions with the mTORC1 pathway, may contribute to the difference in the consequences of chondrogenic condensations at different locations in the developing face. Furthermore, our present findings indicate that the mTORC1 pathway influences facial skeletal shaping by modulating the clonal expansion of committed chondro-progenitors. Previously, we reported that the growth of facial skeletal elements depends on intercalation of chondrocyte clones originating from committed chondro-progenitors within the perichondrium surrounding these elements and oriented transversally into pre-formed cartilage 19 . This intercalation and subsequent expansion of chondrogenic clones plays a key role in controlling the final thickness and geometry of cartilaginous elements. Here, we show that manipulation of mTORC1 activity prior to formation of the perichondrium and committed chondro-progenitors alters the formation of these oriented clones later in development. With attenuated mTORC1 activity, the intercalated clones in the nasal cartilage of embryos were smaller, whereas elevation of mTORC1 activity in chondro-progenitors via deletion of the Tsc1 gene resulted in intercalation of bulky clonal clusters rather than individual clonal columns. Intercalation of these aberrant clones likely underline the altered length and thickness of the nasal cartilage, which eventually influenced the overall craniofacial shape. Thus, during mesenchymal condensation mTORC1 activity regulates the overall geometry of facial cartilage, whereas with respect to committed chondro-progenitors this activity influences individual cartilaginous elements. Therefore, modulation of mTORC1 activity at different time-points may result in a spectrum of somewhat different craniofacial shapes, perhaps thereby also contributing to the variety of defects in patterning observed. It is worth pointing out that the mTORC1 pathway is also involved in chondrogenesis in the limbs, with ablation of Raptor in the limb bud mesenchyme resulting in growth impairment 50 . However, modulation of mTORC1 activity in mature chondrocytes does not influence limb growth 51 , 52 . These observations indicate that the appropriate level of mTORC1 activity in chondro-progenitors, rather than in mature chondrocytes, is important for skeletogenesis, in line with our present results. Since mTORC1 is primarily involved in adjusting cellular responses to the nutrition available 53 , either being enhanced directly by amino acids 54 or via insulin and insulin-like growth factors (all of which are tightly regulated by nutritional levels 32 , 33 , 55 ), it is not surprising that we found that modulation of the level of protein in the maternal diet regulates mTORC1 activity resulting in subtle, but distinct changes in the craniofacial shape of the embryos. Availability of nutrition is a major factor in connection with natural selection and such a spectrum of closely related craniofacial shapes may reflect adaptive phenotypic plasticity, and, accordingly, allow various feeding strategies. Phenotypic plasticity in the feeding apparatus of teleost fish has been observed, both in the wild 56 and under experimental conditions 4 . Recently, it has been reported that the HH pathway mediates plasticity of the feeding apparatus in response to the mechanical properties of the foraging species 27 , with mechanical sensing being, at least in theory, mediated by cilia, a mechanical sensor that is a key component of the HH pathway 26 , 57 , 58 . Thus, interactions between nutritional sensing by the mTORC1 pathway and mechanical sensing by the HH pathway may mediate phenotypic plasticity of the feeding apparatus in response to external conditions. Interestingly, mTORC1 is also involved in regulating the phenotypic plasticity of skeletal muscles 59 , as well as in long-term synaptic plasticity 60 . In humans, craniofacial plasticity has been described in response to the consistency of the diet and alcohol consumption by the mother during pregnancy, as well as to the climate 12 , 61 , 62 . Thus, plasticity of the feeding apparatus, as well as of the entire facial skeleton, may be an evolutionarily conserved characteristic of all gnathostomes, including humans. On the basis of the findings of others and the data documented here, we propose that the mTORC1 pathway is a key part of the molecular machinery that adapts craniofacial structures to nutritional conditions. In summary, we have demonstrated here that the mTORC1 pathway modulates the embryonic shaping of craniofacial skeletal elements at the stage of chondrogenic condensations, with subsequent fine-tuning during intercalation of chondro-progenitors. Furthermore, we provide evidence for an impact of maternal protein intake during pregnancy on the shaping of fetal craniofacial cartilage. These findings provide novel and important insights into the mechanisms underlying craniofacial shaping and, potentially, the phenotypic plasticity of this process as well and, in addition, help elucidate the role of material dietary protein during pregnancy in this context. Experimental Procedures Human embryos Human fetal tissue collection was reviewed and approved by the local ethics committee of Institute of Fundamental Medicine and Biology of Kazan Federal University (No. 8, May 2018). Written informed consent was obtained from the patients subjected to medical abortion. To identify enhancers actually transcribed in human embryonic faces, human facial material was collected between weeks 3 and 12 of development, time-window that potentially influence human facial individuality. Next, we performed CAGE-sequencing on embryonic human facial material and compared the transcriptional start sites, proximal promoters and distal transcribed enhancers 63 thus identified to loci indicated as being involved in human facial variability by genome-wide sequencing 22 ( http://portaldev.sph.umich.edu/docs/api/v1/#introduction ). For our CAGE-sequencing, total RNA (2–3 mg) was extracted from the facial portion of human embryos, preserved in RNAlate and stored at -80 o C using the RNeasy Fibrous Tissue Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol. The concentration and purity of extracted RNA were determined on the basis of absorption employing the NanoDrop™ 8000 Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) and quality verified with the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Libraries were then prepared utilizing the standard nAnT-iCAGE (non-Amplified non-Tagging Illumina Cap Analysis of Gene Expression) protocol 64 , employing 2.5–3 µg total RNA as a template for synthesis of the first cDNA strand (nAnT-iCAGE Library Preparation kit DNA form, Yokohama, Japan and SuperScript III Reverse Transcriptase, Invitrogen, Waltham, MA, USA). This cDNA was subsequently biotinylated at its 5´- end (nAnT-iCAGE Library Preparation kit, DNA form, Yokohama, Japan), which allowed selection of the 5´-cap containing molecules with streptavidin beads (Dynabeads M-270 Streptavidin, ThermoFisher Scientific, USA). In this manner, rRNA, as well as truncated or not fully transcribed RNA was eliminated. For more complete removal of RNA, the cDNA was treated with RNase I and H (nAnT-iCAGE Library Preparation kit, DNA form, Japan) and then purified using RNACleanUP (Beckman Coulter, Brea, CA, USA). Next, linkers were ligated to the 5′and 3′ ends (nAnT-iCAGE Library Preparation kit, DNAform, Japan) of the cap-trapped cDNA. The 5′- linker employed contained recognition sites for the XmaJI restriction endonuclease (nAnT-iCAGE Library Preparation kit, DNA form, Japan) and the MmeI class II restriction enzyme (nAnT-iCAGE Library Preparation kit, DNA form, Japan), as well as a barcode for multiplexing. The 3′- linker contained a recognition site an XbaI restriction enzyme (nAnT-iCAGE Library Preparation kit, DNAform, Japan). Treatment with these restriction enzymes yielded short CAGE tags to which a sequencing primer was ligated. In the final stage, a second cDNA strand was synthesized from these short CAGE tags??? (nAnT-iCAGE Library Preparation kit, DNAform, Japan). The concentration of the resulting libraries was determined by the PicoGreen Assay in a GloMax® Multi Detection System (Promega, Madison, WI, USA) and their quality assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Finally, the libraries were validated using real-time PCR (KAPA Library Quantification Kits Illumina, KAPA Biosystems, Wilmington, MA, South Africa) and sequencing on a HiSeq 2500 platform (Illumina, San Diego, CA, USA) using the HiSeq v4 reagent kit (HiSeq SR Cluster Kit v4 cBot and HiSeq SBS Kit v4 50 cycles, Illumina, San Diego, CA, USA) in the 50-bp single-end mode. Single-read sequences were analyzed for quality and over-represented adapter sequences identified with the FastQC tool. Quality filtering trimming was performed with the fastx_trimmer (FASTX Toolkit 0.0.13.2) and Trimmomatic-0.39 and RNAdust 1.06 utilized as adapters and for removal of rRNA removal. Read mapping on human genome hg38 and mouse genome mm10 was performed with BWA-0.7.10, with unmapped reads being realigned using Hisat2-2.2.1. Aggregation of CAGE tag start sites (CTSS) for each sample, with subsequent peak clustering, were carried out employing the PromoterPipeline script from the C1 CAGE protocol 63 . Bidirectional enhancers were identified using the pipeline described by Andersson and colleagues (2014) 65 . The statistical significance of the differential expression of CAGE peaks was calculated using the edgeR package for R. Triple overlap of GWAS-derived data of coordinates of face shape-affecting loci with already pre-identified and annotated enhancers (from genome annotation 40 ; https://genome.ucsc.edu/cgi-bin/hgTrackUi?db=mm10&g=encodeCcreCombined ; ENCODE Project Consortium) and our human CAGE-seq-derived coordinates of active facial human embryonic enhancers was done involving a specific prior filtration step such as: we selected polymorphisms falling within 5 kilo-base pairs distance from the CDS in any direction for GWAS-identified genes. Mice All animal experiments were pre-approved by the Stockholm North Ethical committee and performed in accordance with the guidelines of the Swedish Animal Agency. The Sox10-CreERT2, Col2-CreERT2, R26Confetti, Tsc1 flox, and Raptor flox strains of mice employed have been described in detail previously 41,66−69 . Embryonic Cre recombination was induced by intraperitoneal (i.p.) injection of 1–3 mg tamoxifen (Sigma) into each pregnant dam. The day on which the plug was detected was defined as embryonic day 0.5 (E0.5). Rapamycin (0.02 mg, LC Laboratories) was injected i.p. into each pregnant dam. Zebrafish The Col2a1aBAC:mcherry strain of zebrafish was kindly provided by Prof. Chrissy Hammond (University of Bristol, UK) and has been utilized as described in detail elsewhere 70 – 72 . Zebrafish larvae were exposed to 400 nM rapamycin at the time-points indicated. Manipulation of the maternal murine diet Pregnant dams received standard mouse chow containing 4% protein until E6.5 and thereafter an isocaloric diet containing 4%, 20% or 40% protein (TD. 93032, TD. 91352 and TD. 90018 from Envigo) until the day of sacrifice. X-ray computed microtomography (µCT) with enhanced contrast achieved with phosphotungstic acid (PTA) On E17.5, the heads of mouse embryos were placed in a 1% PTA/methanol solution to enhance the contrast of soft structures, as described previously 73 . µCT scans were performed with the GE phoenix v|tome|x L 240 system equipped with a nanofocus X-ray tube (180 kV/15 W maximal power) and high flat panel (dynamic 41|100 with 4000 × 4000 pixels, each 100 × 100 µm in size). Acquisition involved the use of a 0.2-mm aluminum filter to soften the beam; 60 kV and 200 µA; exposure for 600 ms; and averaging of 3 projections to reduce noise. 1800 images were acquired over 360°, requiring a scanning time of one hour per sample. The isotropic voxel size was 6.2 µm in all cases. The tomographic reconstructions were performed in the GE phoenix datos|x 2.0 3D computed tomography software. Segmentation of craniofacial structures was performed manually using a combination of the Avizo (Thermo Fisher Scientific, USA) and VG Studio MAX 3.2 software (Volume Graphics GmbH, Germany), as described elsewhere 73 . High-resolution microfocus computed tomography using enhanced contrast with Hexabrix (CE-HRµCT) and subsequent image processing and 3D analysis Following fixation, samples were stored in PBS at 4° C. Prior to scanning, these samples were transferred to Eppendorf tubes containing 1.5 ml 30% Hexabrix 320 in PBS (Guerbet Nederland B.V.); incubated for two weeks with continuous gentle shaking at 4° C; and then scanned while still inside the same tubes. Hexabrix 320, a negatively charged ioxaglate, is repelled by the anionic sulfated-glycosaminoglycan (sGAG), resulting in negative staining of cartilage, while still providing good contrast between mineralized tissues and the background. For acquisition of all images, the NanoTom M (GE Measurement and Control Solutions, Germany) system in combination with a diamond-coated tungsten target was employed with the following conditions: a 0.2-mm aluminum filter to soften the beam; 60 kV and 300 µA; exposure for 500 ms; and averaging of each sample individually and a skip of 0 (‘fast scan mode’). 2400 images were acquired over 360°, requiring a scanning time of 20 minutes per sample. In all cases the isotropic voxel size was 5 µm. Reconstruction was performed using the Phoenix datos|x CT software, applying a correction of 5 for beam hardening and a Gaussian filter (radius 3) to reduce noise. The transaxial, coronal and sagittal cross-sections of each sample were visualized with the DataViewer (Bruker MicroCT, Belgium); while 3D visualization of the cartilage and mineralized tissue and quantification of their volumes were performed with the Mimics Innovation suite (Materialise NV, Belgium). Briefly, two threshold values were selected manually to distinguish between non-mineralized cartilage and mineralized tissues (i.e., mineralized cartilage and subchondral bone) and these thresholds then fine-tuned with dynamic region-growing and multi-slice edit. Using these adjusted threshold values, 3D models based on marching-cubes were generated and the volumes of mineralized tissue versus non-mineralized cartilage and the ratio between these volumes calculated. In addition, the length and width of the nasal capsule and the Meckel cartilage were measured. Immunohistochemical analyses Embryos were fixed in 4% paraformaldehyde (PFA) for 6 hours at 4° C and tissues then embedded in OCT (Tissue-Tek) on dry ice for sectioning. Thereafter, the 30-µm frozen sections were blocked in PBST (PBS + 0.01% Tween20) + 3% normal horse serum (Vector laboratories) for one hour prior to incubation with the primary antibody (anti-pS6 (Cell Signaling), anti-PAR3 (Millipore), anti-acetylated-tubulin (Santa Cruz), anti-PKCζ (Santa Cruz), or anti-β-catenin (Santa Cruz)) overnight. TUNEL staining 30-µm tissue sections were treated with 10 µg/ml proteinase K (Ambion) for 40 minutes at 37° C before applying the TUNEL reaction mix (Roche Inc.) for 90 minutes. The cell nuclei were then counterstained with DAPI. Staining with haematoxylin and eosin 15-µm frozen sections were stained with haematoxylin for 30 seconds and 0.02% eosin for 2 minutes. Microscopy and image analysis Images were acquired with a LSM710 confocal microscope. 3D visualization and all quantification were performed utilizing the IMARIS (Bitplane) and ImageJ software. Mathematical modelling of human craniofacial anatomy To examine how the morphological changes observed in mice might be manifested in humans, we transformed polygon data on human craniofacial anatomy extracted from full-body MRI (BodyParts3D, Database Center for Life Science, Japan, Tokyo). The skull was divided into 53 high-resolution images of teeth, bones and ligaments and transformation carried out in Mathematica 11.0 (Wolfram Research, USA, Illinois) using custom-written code. The non-linear 3D transform was designed as a three-dimensional 'magnifying glass' (adopted from 74 ) with a radius of 3 cm and centered on the nasal cavity. The magnification was adjusted so that the width of the nasal cavity increased in the same manner as observed in mice. The algorithm allowed us to selectively magnify defined anatomical regions of the skull, while maintaining the rest of the craniofacial anatomy unchanged. Declarations ACKNOWLEDGEMENT We wish to thank Olga Kharchenko for the artwork included and Ostap Dregval for technical assistance. This study was supported by the Swedish Research Council (Projects 2020-02298 to A.S.C., 2018-02713 to I.A., 2022-00611 to K.F. and 2021-01805 to M.X.), an ALF-agreement (ALFGBG-966178 to A.S.C.) and the NovoNordisk Foundation (NNF21OC0070314 to A.S.C.). M.X. was supported by a long-term postdoctoral fellowship from the European Molecular Biology Organization and by Stiftelsen Frimurare Barnhuset i Stockholm. E.I. was supported by a grant from the Russian Basic Science Foundation (#19-29-04115 to A.S.C). M.T., T.Z. and J.K. acknowledge financial support in the form of project CEITEC 2020 (LQ1601) from the Ministry of Education, Youth and Sports of the Czech Republic under the National Sustainability Program II and help from the CzechNanoLab Research Infrastructure supported by MEYS CR (LM2018110). 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Gene maps showing SNPs associated with facial appearance within the normal range and CAGE-identified active enhancers for genes associated with PI3K/mTORC1/autophagy. Gene maps from the UCSC Genome browser showing the SNPs and CAGE-identified cis-enhancers for AKT1 (A), ACTB (B), RRAGA (C), PIK3C2A (D), CAMKK1 (E), UGT2A3 (F). FigureS2.pdf Extended data Figure 2 Craniofacial parameters in mice lacking Raptor gene in their chondro-progenitors and mTORC1 activity in cartilage elements The size of the craniofacial skeleton of Col2 CreERT ;Raptor fl/fl mice pulsed with tamoxifen on E12.5 was determined on E17.5 (A-C). The activity of the mTORC1 pathway in elements of the nasal cartilage of either Col2 CreERT ;Raptor fl/fl mice (D) pulsed with tamoxifen on E12.5 or Sox10 CreERT2 ;Tsc1 fl/fl ;R26R Confetti mice (E) pulsed with tamoxifen on E8.5 was assessed on the basis of the level of S6 phosphorylation. All embryos were analyzed on E17.5. The controls are designated Cre- and the knock-outs Cre+. In D and E phosphorylated S6 is colored pink and the nucleus counterstained with DAPI (blue). The white dashed lines outline the cartilage. Means ± SD are presented in A-C, with individual values also indicated. Statistical analysis was performed utilizing the unpaired t-test. FigureS3.pdf Extended data Figure 3 The effect of manipulation of protein levels in maternal diet on craniofacial parameters, mTORC1 activity, cell proliferation and apoptosis in the embryos Pregnant C57BL/6J dams were placed on isocaloric diets containing either 4%, 20% (control, standard chow=22%) or 40% protein from E6.5 of pregnancy. The level of mTORC1 activity was assessed on the basis of S6 phosphorylation (A) in craniofacial structures of the embryos on E17.5 and quantified (B). The embryos were also weighed on E17.5 (C). Reconstruction of the craniofacial structures of embryos on E17.5 employing µCT with contrast enhancement by Hexabrix is shown, with cartilage and mineralized bone in blue and red, respectively (D-I). The green and yellow lines in (D-F) depict the lengths of the nasal capsule and Meckel cartilage, respectively, and these lengths were quantified (J,L). The green and yellow lines in (G-I) depict the width of the nasal capsule, quantified (average of two determinations) in (K). The ratio between bone and cartilage was also quantified (M). The number of proliferating KI67-positive cells (N-P) and apoptotic TUNEL-positive cells (Q-S) was quantified in the cartilage of E17.5 embryos whose mothers consumed diets containing the different levels of protein (T, U). Means ± SD are presented, with individual values also indicated. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparisons test. FigureS4.pdf Extended data Figure 4 Mathematical prediction of human craniofacial changes upon protein levels in maternal diet, projected from mouse observations (A-H) Application of the average craniofacial values for embryos whose dams consumed diets containing low or high levels of protein to a model of the human skull resulted in several structural changes in the craniofacial skeleton. (E-H) depict schematic outlines of the different skeletal elements shown in (A-D), respectively. The algorithm utilized for this conversion is described in the Methods. Cite Share Download PDF Status: Published Journal Publication published 26 Mar, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2542333","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":175272645,"identity":"01e837ba-d2c3-47f4-ac13-241a155c4ea5","order_by":0,"name":"Andrei Chagin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYHACAxhiOMBQQbqWM0RrgZGMbUSo55/dvO3DhwIGeXP24xcPF86rTWzgP3wArxaJO8eKZ84wYDDc2ZNTcHjmtuOJDRJpCfituZFjzMxjwJBgcCAn4TDvtmNALTwGeHXIg7T8AWk5/waoZQ5QC//5D3i1GIC0MIC03Eg/cJi3oSaxgSEHv7sMb6QVM/YYSBhuuPGG4TDPsQPGbRJp+B0mdyN5M8OPPzbyBufTH3/mqamT7ec//AC/NRAgAcRgXx9mYCNGPRSwgwyvI0HDKBgFo2AUjBQAAPxYSek3aQAcAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2696-5850","institution":"Gothenburg University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Andrei","middleName":"","lastName":"Chagin","suffix":""},{"id":175272646,"identity":"d50b60b2-0fb3-4f6c-a476-1efc30283979","order_by":1,"name":"Meng Xie","email":"","orcid":"","institution":"Peking University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Xie","suffix":""},{"id":175272647,"identity":"65740b83-a1e0-4360-a7b6-51f70b7f9cc1","order_by":2,"name":"Marketa Tesarova","email":"","orcid":"https://orcid.org/0000-0002-5200-7365","institution":"Central European Institute of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marketa","middleName":"","lastName":"Tesarova","suffix":""},{"id":175272648,"identity":"229728b9-cfa9-4a52-9dbd-0190621ca863","order_by":3,"name":"Yaakov Gershtein","email":"","orcid":"","institution":"Medical University of Vienna","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yaakov","middleName":"","lastName":"Gershtein","suffix":""},{"id":175272649,"identity":"50adffad-32ee-42e5-97ac-e475df42c183","order_by":4,"name":"Daniela Schnyder","email":"","orcid":"","institution":"Gothenburg University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Schnyder","suffix":""},{"id":175272650,"identity":"36ccb27a-76f3-432f-8768-7ae490897e16","order_by":5,"name":"Ruslan Devyatirov","email":"","orcid":"","institution":"Kazan Federal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruslan","middleName":"","lastName":"Devyatirov","suffix":""},{"id":175272651,"identity":"a6132282-756f-47e7-a740-bcabb95a06b2","order_by":6,"name":"Guzel Gazizova","email":"","orcid":"","institution":"Kazan Federal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guzel","middleName":"","lastName":"Gazizova","suffix":""},{"id":175272653,"identity":"e6543dca-d4b8-448f-9398-d458bd654ae7","order_by":7,"name":"Elena Shagimardanova","email":"","orcid":"","institution":"Kazan (Volga Region) Federal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Shagimardanova","suffix":""},{"id":175272654,"identity":"c9c1fdf6-141f-40e1-b38a-2f064b415eea","order_by":8,"name":"Tomas Zikmund","email":"","orcid":"","institution":"Brno University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tomas","middleName":"","lastName":"Zikmund","suffix":""},{"id":175272656,"identity":"297ee24e-b2b4-4b2a-b5fd-d25c82a1b6eb","order_by":9,"name":"Greet Kerckhofs","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Greet","middleName":"","lastName":"Kerckhofs","suffix":""},{"id":175272657,"identity":"2ec2636f-575e-4fc0-b33c-ac987ca11915","order_by":10,"name":"Evgeny Ivashkin","email":"","orcid":"","institution":"I.M. Sechenov First Moscow State Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Evgeny","middleName":"","lastName":"Ivashkin","suffix":""},{"id":175272659,"identity":"6e4365a6-e5be-4470-be47-87a99624e16d","order_by":11,"name":"Dominyka Batkovskyte","email":"","orcid":"https://orcid.org/0000-0002-0492-1259","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dominyka","middleName":"","lastName":"Batkovskyte","suffix":""},{"id":175272660,"identity":"9c08d1a8-c073-434d-963f-f9d83db90326","order_by":12,"name":"Phillip Newton","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Phillip","middleName":"","lastName":"Newton","suffix":""},{"id":175272662,"identity":"15840b6b-588c-46a3-910e-7228eac0a175","order_by":13,"name":"Olov Andersson","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Olov","middleName":"","lastName":"Andersson","suffix":""},{"id":175272663,"identity":"100eae82-bc9b-47b0-bdf3-453385df0b37","order_by":14,"name":"Kaj Fried","email":"","orcid":"","institution":"Karolinska Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kaj","middleName":"","lastName":"Fried","suffix":""},{"id":175272664,"identity":"04d1b1cf-df55-4331-8b4b-a533fabc859c","order_by":15,"name":"Oleg Gusev","email":"","orcid":"","institution":"Juntendo University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Oleg","middleName":"","lastName":"Gusev","suffix":""},{"id":175272666,"identity":"9a381f2b-3c2b-4658-b3f9-136184b927d2","order_by":16,"name":"Hugo Zeberg","email":"","orcid":"https://orcid.org/0000-0001-7118-1249","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hugo","middleName":"","lastName":"Zeberg","suffix":""},{"id":175272668,"identity":"126eaf7c-63cb-49e4-8dc6-84b86ff64cb1","order_by":17,"name":"Jozef Kaiser","email":"","orcid":"","institution":"Brno University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jozef","middleName":"","lastName":"Kaiser","suffix":""},{"id":175272672,"identity":"3c959cc2-3024-4ca8-811b-f4c26ad73e4e","order_by":18,"name":"Igor Adameyko","email":"","orcid":"","institution":"Medical University of Vienna","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Igor","middleName":"","lastName":"Adameyko","suffix":""}],"badges":[],"createdAt":"2023-02-02 10:43:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2542333/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2542333/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-46030-3","type":"published","date":"2024-03-26T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":34324322,"identity":"d5fc078c-9bcc-419b-a5de-2aa6261ceee3","added_by":"auto","created_at":"2023-03-15 23:32:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":896738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eActive enhancers in human embryonic tissue associated with facial individuality.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental layout: embryonic human facial mesenchyme was collected and CAGE-sequenced to identify transcribed enhancers, which were subsequently compared to SNPs associated with facial morphology within the normal range as indicated by genome-wide association sequencing (GWAS). \u0026nbsp;(B) Distribution of the genes located proximally to the enhancers identified \u0026nbsp;in relationship to the GWAS probability. Genes related to the PI3K/mTORC1/autophagy pathway are highlighted in red. (C) Illustration of the mTORC1 pathway (based on refs. 29 and 44 and the Autophagy database http://www.tanpaku.org/autophagy/) highlighting the genes identified. (D) Protein interaction network generated by the STRING database for the genes related to the development of individual facial features. The six genes indicated in B are highlighted in yellow. (E) Distribution of the individual facial phenotypes revealed by GWAS to be associated with each of the six genes encoding components of the PI3K/mTORC1/autophagy pathway.\u003c/p\u003e","description":"","filename":"Figure1v2copy.png","url":"https://assets-eu.researchsquare.com/files/rs-2542333/v1/56000870debca1cce5b8ddcf.png"},{"id":34323966,"identity":"5c7798d7-1a1c-4c88-9ad6-c53699345064","added_by":"auto","created_at":"2023-03-15 23:24:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4218841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eActivation of the mTORC1 pathway in the neural crest cells of mice results in thickening of the nasal cartilage, mesenchymal condensations, and the formation of bulky clonal clusters.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe mTORC1 pathway in \u003cem\u003eSox10\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;Tsc1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;R26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e mice was activated by ablation of Tsc1 through exposure to tamoxifen on E8.5. 3D reconstruction of the entire chondrocranium on the basis of µCT with enhancement by PTA allowed comparison of the thickness of various structures in control (A,C) and Tsc1cKO (B,D) mice. The surface of the skeletal structures as revealed by these reconstructions in control and Tsc1cKO mice are shown in (C and D). The arrow in D points toward the curved nasal septum. Overlay of 3D-reconstructions revealed differences in the size and shape of the chondrocranium in control and Tsc1cKO mice (E). Quantification of the thickness of the cartilage in the three major nasal compartments is quantified in (F). Representative images of chondrogenic condensations (revealed by SOX9 staining on E12.5) in control (G) and Tsc1 cKO (H) mice are presented in (G and H) and the condensation thickness of the three major nasal compartments quantified in (I). Clonal arrangements in facial cartilage on E17.5 in the presence of one (J-L, control) or no (M-O) copy of the \u003cem\u003eTsc1 gene\u003c/em\u003e (Tsc1 cKO) are depicted. The images shown are representative for the three major types of nasal cartilage - the septum (J,M), prominence (K,N) and capsule (L,O). Clones within cartilage were characterized with respect to the number of cells per clone (P), volume occupied by each clone (Q), average distance between cells within each clone (R), and dispersion of the clones as reflected in the standard deviation of distances between cells\u0026nbsp; (S). Means ± SD are presented, with individual values for every mouse also indicated in F and I and for every clone (from at least 3 different mice) in P-Q. Student’s unpaired t-test was applied to the values in F, I, Q, R, S.\u003c/p\u003e","description":"","filename":"Figure2final.png","url":"https://assets-eu.researchsquare.com/files/rs-2542333/v1/38758fecc9f56722a989ad9e.png"},{"id":34324323,"identity":"fe4aa95b-5ba3-47b7-9b48-5394569f0005","added_by":"auto","created_at":"2023-03-15 23:32:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3743057,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eModulating mTORC1 activity at the stage of intercalation alters craniofacial shape and the size of chondrocyte clones to a minor extent.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll mice were pulsed with tamoxifen on E12.5 and analyzed on E17.5. (A-J) The mTORC1 pathway was activated by ablation of Tsc1 in chondroprogenitors employing \u003cem\u003eSox10\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;Tsc1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;R26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e mice. The images shown are representative for the three major types of nasal cartilage - the septum (A,D), prominence (B,E) and capsule (C,F). Clones within cartilage were characterized with respect to the number of cells per clone (G), volume occupied by each clone (H), average distance between cells within each clone (I), and dispersion of the clones as reflected in the standard deviation of distances between the cells in each clone (J). The mTORC1 pathway was activated in chondroprogenitors by ablation of Tsc1 employing Collagen type 2-driven CreERT recombinase (\u003cem\u003eCol2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;Tsc1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;R26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e). Representative sagittal sections stained with HE (K,L), clonal appearance (M, N) and quantification of clonal size (O) revealed little difference between the heads of control (\u003cem\u003eCre+;Tsc1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;R26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e) (K,M) and \u003cem\u003eTsc1\u003c/em\u003e cKO (\u003cem\u003eCre+;Tsc1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;R26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e) (L,N) embryos. (P-Q) The mTORC1 pathway was inactivated in chondroprogenitors by ablation of the Raptor gene employing Collagen type 2-driven CreERT recombinase (\u003cem\u003eCol2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;Raptor\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e). Reconstruction of cartilaginous and bony structures in the heads of (P) control (Cre-negative, \u003cem\u003eRaptor\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e) and (Q) \u003cem\u003eRaptor\u003c/em\u003e cKO\u003cem\u003e \u003c/em\u003e(\u003cem\u003eCol2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;Raptor\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e) mice was examined by mCT enhanced with Hexabrix. The green line indicating nasal length was quantified (R). Clonal reporter R26R\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e was bred into \u003cem\u003eCol2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;Raptor\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003emice (\u003cem\u003eCol2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;Raptor\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;R26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e) and clonal appearance assessed in the nasal septum (S,V), nasal prominence (T,W) and nasal capsule (U,X). The number of cells per clone was quantified (Y).\u0026nbsp; Means ± SD are presented, with individual values also indicated and determination of statistical significance using the unpaired t-test.\u003c/p\u003e","description":"","filename":"Figure3final.png","url":"https://assets-eu.researchsquare.com/files/rs-2542333/v1/6c866e8ecc14f9e4b1aedc6d.png"},{"id":34323969,"identity":"1da32bee-291a-4e97-81fb-24dc6489b345","added_by":"auto","created_at":"2023-03-15 23:24:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2377540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eInhibition of mTORC1 at various stages of embryonic development modulates the shape of craniofacial structures in both mice and zebrafish.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePregnant C57BL/6J dams were exposed to a single dose of either DMSO or rapamycin on E10.5 and stained with hematoxylin-eosin on E17.5. Representative images of the general appearance (A) or sagittal sections of their heads (B-C) are shown. Embryos were treated as in A, but stained for SOX9 on E12.5 to reveal chondrogenic condensations (D,E), the thickness of which in the three major nasal compartments was quantified (F). The same treatment as in A was applied to\u003cem\u003e Col2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;R26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e mice pulsed with tamoxifen on E12.5 and E13.5 and clonal appearance analyzed on E17.5 (G-L) and clonal size quantified (M). (N-S) \u003cem\u003eCol2a1aBAC:mcherry\u003c/em\u003e zebrafish larvae were exposed to DMSO or rapamycin at various periods in development. Ventral (N-O) and lateral (P-Q) views of the larvae exposed to DMSO (N,P) or rapamycin (O,Q) 14-22 hours post-fertilization (hpf) and imaged at 120 hpf are shown. The facial length (R) and width (P) of 120-hpf-old \u003cem\u003eCol2a1aBAC:mcherry\u003c/em\u003e zebrafish larvae exposed to rapamycin during the intervals of time indicated were quantified. Means ± SD are presented, with individual values also indicated. The unpaired t-test was employed to compare the values in F and M, and one-way ANOVA followed by Dunnett's multiple comparisons test in R and S.\u003c/p\u003e","description":"","filename":"Figure4final.png","url":"https://assets-eu.researchsquare.com/files/rs-2542333/v1/f7b9c9b65492e19ec1036519.png"},{"id":34323973,"identity":"167dcaff-6e6a-4d80-a433-4cac526c4155","added_by":"auto","created_at":"2023-03-15 23:24:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3102913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eThe level of protein in the maternal diet alters the structure of the facial skeleton of their embryos.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A-I) Genetically identical wild-type pregnant C57BL/6J dams consumed isocaloric diets containing different levels of protein (4%, 20% (standard chow=22%), 40%) from E6.5 of pregnancy and on E17.5 the skeleton in the heads of their embryos was reconstructed employing µCT with PTA enhancement (A). The following parameters indicated in A were measured: the thickness (1) and length (2) of the nasal capsule, the thickness (3) and length (4) of the entire chondrocranium, and the length of the right (5) and left (6) portions of the Meckel cartilage. The thickness (B) and length (C) of the nasal capsule and the average length of the Meckel cartilage (D) are shown. Overlayed 3D reconstructions (E) and the thickness of craniofacial structures (F) from embryos whose mothers consumed diets containing different levels of protein are presented for direct comparison. (G-J) Pregnant Sox10\u003csup\u003e\u003cem\u003eCreERT2\u003c/em\u003e\u003c/sup\u003e;Tsc1fl/fl dams consumed isocaloric diets containing either 4% or 40% protein from E6.5 of pregnancy and Tsc1 was ablated in the neural crest cells of their embryos by pulsing with tamoxifen on E8.5. The skeleton in the heads of their embryos was reconstructed on day E17.5 employing µCT with PTA enhancement (G). Quantification of the width (H) and length (I) of the nasal capsule and the average length of the Meckel cartilage (J) are depicted. The same diets as in A-F were administered to pregnant \u003cem\u003eCol2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;R26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e dams pulsed with tamoxifen on E12.5 and E13.5. Representative images of clonal appearance (K) and quantification of clonal size (L) in the entire nasal cartilage are shown. Means ± SD are presented, with individual values also indicated. One-way ANOVA followed by Tukey's multiple comparisons test was employed to compare the values in B-D and L, and the unpaired t-test in H-J. The white dashed lines in K outline the cartilage.\u003c/p\u003e","description":"","filename":"Figure5final.png","url":"https://assets-eu.researchsquare.com/files/rs-2542333/v1/ea2e0a405b51ded3abe2cdec.png"},{"id":53533901,"identity":"46a693be-7fc0-4540-8200-27535b6dd340","added_by":"auto","created_at":"2024-03-27 07:05:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4400252,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2542333/v1/955cbab5-7264-4f5c-ab5d-0c07784e5932.pdf"},{"id":34324324,"identity":"cf457944-6710-4987-9dd7-8d71cf7a7251","added_by":"auto","created_at":"2023-03-15 23:32:13","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":260547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended data Figure 1. \u003c/strong\u003e\u003cem\u003eGene maps showing\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eSNPs associated with facial appearance within the normal range and CAGE-identified active enhancers for genes associated with PI3K/mTORC1/autophagy.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGene maps from the UCSC Genome browser showing the SNPs and CAGE-identified cis-enhancers for AKT1 (A), ACTB (B), RRAGA (C), PIK3C2A (D), CAMKK1 (E), UGT2A3 (F).\u003c/p\u003e","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2542333/v1/21c5ed5075437d723b98101c.pdf"},{"id":34323970,"identity":"e3bcfc38-10f0-4ecf-8b44-61e289fb3124","added_by":"auto","created_at":"2023-03-15 23:24:13","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":823780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended data Figure 2 \u003c/strong\u003e\u003cem\u003eCraniofacial parameters in mice lacking Raptor gene in their chondro-progenitors and mTORC1 activity in cartilage elements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe size of the craniofacial skeleton of \u003cem\u003eCol2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;Raptor\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice pulsed with tamoxifen on E12.5 was determined on E17.5 (A-C). The activity of the mTORC1 pathway in elements of the nasal cartilage of either \u003cem\u003eCol2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;Raptor\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (D) pulsed with tamoxifen on E12.5 or \u003cem\u003eSox10\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;Tsc1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;R26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e mice (E) pulsed with tamoxifen on E8.5 was assessed on the basis of the level of S6 phosphorylation. All embryos were analyzed on E17.5. The controls are designated \u003cem\u003eCre-\u003c/em\u003e and the knock-outs \u003cem\u003eCre+.\u003c/em\u003e In D and E phosphorylated S6 is colored pink and the nucleus counterstained with DAPI (blue). The white dashed lines outline the cartilage. Means ± SD are presented in A-C, with individual values also indicated. Statistical analysis was performed utilizing the unpaired t-test.\u003c/p\u003e","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2542333/v1/b0de609033e2d8d84031e598.pdf"},{"id":34324325,"identity":"cbaa6a05-221e-463a-b07b-048b72dd82c8","added_by":"auto","created_at":"2023-03-15 23:32:13","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3106764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended data Figure 3 \u003c/strong\u003e\u003cem\u003eThe effect of manipulation of protein levels in maternal diet on craniofacial parameters, mTORC1 activity, cell proliferation and apoptosis in the embryos\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePregnant C57BL/6J dams were placed on isocaloric diets containing either 4%, 20% (control, standard chow=22%) or 40% protein from E6.5 of pregnancy. The level of mTORC1 activity was assessed on the basis of S6 phosphorylation (A) in craniofacial structures of the embryos on E17.5 and quantified (B). The embryos were also weighed on E17.5 (C). Reconstruction of the craniofacial structures of embryos on E17.5 employing µCT with contrast enhancement by Hexabrix is shown, with cartilage and mineralized bone in blue and red, respectively (D-I). The green and yellow lines in (D-F) depict the lengths of the nasal capsule and Meckel cartilage, respectively, and these lengths were quantified (J,L). The green and yellow lines in (G-I) depict the width of the nasal capsule, quantified (average of two determinations) in (K). The ratio between bone and cartilage was also quantified (M). The number of proliferating KI67-positive cells (N-P) and apoptotic TUNEL-positive cells (Q-S) was quantified in the cartilage of E17.5 embryos whose mothers consumed diets containing the different levels of protein (T, U). Means ± SD are presented, with individual values also indicated. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparisons test.\u003c/p\u003e","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2542333/v1/191837b16019b4ae5bcd95fa.pdf"},{"id":34323974,"identity":"f042526c-74f9-4de4-8984-bc8c39b6794a","added_by":"auto","created_at":"2023-03-15 23:24:13","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":345389,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended data Figure 4 \u003c/strong\u003e\u003cem\u003eMathematical prediction of human craniofacial changes upon protein levels in maternal diet, projected from mouse observations\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A-H) Application of the average craniofacial values for embryos whose dams consumed diets containing low or high levels of protein to a model of the human skull resulted in several structural changes in the craniofacial skeleton. (E-H) depict schematic outlines of the different skeletal elements shown in (A-D), respectively. The algorithm utilized for this conversion is described in the Methods.\u003c/p\u003e","description":"","filename":"FigureS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2542333/v1/13085abbc697cd6b1e491649.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The level of protein in the maternal murine diet modulates the facial appearance of the offspring via mTORC1 signaling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA key aspect of most social communication between humans is facial recognition\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Accordingly, congenital craniofacial malformations, including cleft palate, craniosynostosis, and craniofacial skeletal hypoplasia, which together account for more than one-third of all congenital birth defects, can have a profound influence on social interactions\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. From an evolutionary perspective, the viscerocranium harbors vital structures such as the feeding apparatus and supports sensory organs. Its precise sculpting and inheritable reproducibility are of unequivocal importance for survival. At the same time, adaptability of viscerocranium and particularly of the feeding apparatus to the environmental cues allows adjusting to environmental changes, e.g., feeding sources, or even concurring novel ecological niches. The latter type of adaptation is less common in mammals than in several classes of Gnathostomata, e.g., Actinopterygii\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe craniofacial skeleton, one of the most complex and sophisticated part of the skeletal system, is composed of many different parts and formed via the interplay of a variety of genetic, epigenetic, and environmental factors\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Studies involving families with monozygotic and dizygotic twins indicate that the genetic inheritability score for craniofacial morphology in humans varies widely among different facial features, e.g., from 0.8 for the distance between the inner corners of the eyes to approximately 0.5 for the position of the point midway between the nose and upper lip, as well as for nasal protrusion\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. One well-known environmental influence on facial morphogenesis in humans is alcohol consumption during pregnancy\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn all \u003cem\u003eGnathostomata\u003c/em\u003e, including zebrafish, mice, and humans, the viscerocranium develops from descendants of the neural crest cells (NCCs), a transient and multipotent population of embryonic progenitors\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Multiple subpopulations of the mesenchyme derived from NCCs condense and then differentiate further into chondrocytes and osteoblasts, the major types of skeletal cells in the craniofacial region\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Other developmental subpopulations derived from NCCs, such as Schwann cell precursors, also contribute to the formation of chondrocytes and osteoblasts, but to a relatively limited extent\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The shapes of the craniofacial skeletal elements are determined primarily by the shape of these mesenchymal chondrogenic condensations during development, with subsequent fine-tuning by localized intercalation of new chondrogenic clones arising from the surrounding mesenchyme\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA complex interplay between NC cells, the facial ectoderm, placodes, endoderm and neuroepithelium orchestrates accurate sculpturing of the viscerocranium. Not surprisingly, this process involves continuous changes in the expression of thousands of different genes\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Genome-wide association studies (GWAS) have implicated more than 100 loci in the formation of facial morphology within the normal range and more than two hundred single nucleotide polymorphisms (SNPs) that exert a significant impact on this formation\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In association with abnormal morphology of the human facial skeleton, the HPO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://hpo.jax.org\u003c/span\u003e\u003cspan address=\"http://hpo.jax.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) lists 1165 genes\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Although many of these are associated primarily with other systems, such as hematopoiesis and neurogenesis, their large number reflects the complexity of facial morphogenesis.\u003c/p\u003e \u003cp\u003eAt the same time, the proper migration and differentiation of NCCs, as well as their interaction with surrounding tissues during facial development involves limited number of developmentally and evolutionarily conserved signaling pathways. Among those are hedgehog (HH), fibroblast growth factors (FGF), bone morphogenic proteins (BMPs), WNT, retinoic acid (RA) and platelet-derived growth factor (PDGF) pathways\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. These pathways and associated morphogens form a hardware system, genetically responsible for sculpting of viscerocranium. However, the signaling pathways, capable to sense environmental clues and integrate these signals into genetical hardware of facial morphogenesis are rather unknown. One proposed system is HH signaling pathway, which may sense mechanical forces via cilium\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and modify craniofacial formation as it has been shown in bony fish\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eClearly, a major factors underlying natural selection has been the availability of nutrition\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and the feeding apparatus, part of the viscerocranium, is of particular importance in this context. Nutritional sensing by the Mechanistic Target of Rapamycin Complex 1 (mTORC1) signaling pathway has been highly conserved evolutionarily\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Budding yeasts sense the availability of amino acids via mTORC1 and, in response to this information, shift towards the synthesis of proteins or autophagy\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Although this pathway plays a similar role in multicellular organisms\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, in this case levels of oxygen, energy and growth factors (primarily those transducing via P13 kinase and Akt\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e) also exert an influence.\u003c/p\u003e \u003cp\u003eOn the systemic level, mTORC1 can be regarded as belonging to an endocrine network of both up- and downstream of insulin-like growth factors (IGFs) that regulates a variety of processes in response to the availability of nutrition\u003csup\u003e30,32\u0026minus;34\u003c/sup\u003e. Changes in the activity of mTORC1 can alter the shape of craniofacial structures\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and, in addition, the mTORC1 pathway interacts with the HH, BMPs, and Wnts\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e pathways, which are strongly involved in sculpting of the viscerocranium. Accordingly, we hypothesize that the mTORC1 signaling pathway may play a role in mediating interactions between certain environmental factors and the inherited program of craniofacial morphogenesis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003eThe PI3K/mTORC1 pathway is associated with facial appearance in humans\u003c/h2\u003e\n \u003cp\u003eTo identify enhancers actively involved in human facial development, embryonal facial material was CAGE-sequenced (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) and all enhancers actively transcribed during human facial development between week 3 and 12 of gestation were identified (GEO #xxx, supplementary metadata file). All thus identified enhancers were further cross-checked and enriched against enhancers previously identified in ENCODE project\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e (see the Methods). The resulting pool of enhancer coordinates was overlapped with published GWAS hits\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e (see the Methods) identified to be associated with normal-range facial morphology (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Among enhancers thus identified there was a clear enrichment in components of the PI3K/mTORC1/autophagy pathway (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, C, Extended data Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Predictions based on the STRING database (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD) and the specific facial phenotype related to each individual polymorphism (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE) indicated that among the major enhancers active during sculpting of the human face, the mTORC1 pathway was clearly enriched.\u003c/p\u003e\n \u003cp\u003eThus, this approach identified PI3K/mTORC1 pathway as a potentially important player in human facial morphogenesis. To explore the mechanism(s) underlying involvement of this pathway in craniofacial shaping, we manipulated the mTORC1 pathway during facial development in experimental animals.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003emTORC1 modulates the shaping of chondrogenic condensations in the mouse\u003c/h2\u003e\n \u003cp\u003eFirst, we activated the mTORC1 pathway in neural crest cells (NCCs) by crossing \u003cem\u003eTsc1\u003c/em\u003e floxed mice with the \u003cem\u003eSox10\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, in which a pulse of tamoxifen on embryonic day 8.5 (E8.5) causes recombination in NCCs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Reconstruction of the developing craniofacial structures in the offspring utilizing 3D \u0026micro;-CT images with enhanced contrasting of soft tissues revealed alterations in the thickness of skeletal elements, as well as minor developmental abnormalities already on E17.5 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). Overlay of the reconstructed cartilage of \u003cem\u003eTsc1\u003c/em\u003e cKO and control (\u003cem\u003eTsc1\u003c/em\u003e heterozygous) embryos revealed enlargement of a variety of elements of the craniofacial skeleton, as well as enhanced thickness of all components of the nasal cartilage (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). These observations confirmed the involvement of the mTORC1 pathway in craniofacial shaping\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and, in addition, showed that this pathway is involved during early development.\u003c/p\u003e\n \u003cp\u003ePreviously, we demonstrated that in mice craniofacial shape is established at the time of mesenchymal condensation (E12.5-E13.5), with subsequent fine-tuning via intercalation of new clones\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. To reveal the shape of these mesenchymal condensations, KO embryos were stained for Sox9 on E12.5 and, although the overall shape was preserved, their nasal prominence and nasal capsule compartments were thicker (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG-I). We then bred in the \u003cem\u003eR26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e reporter transgene, which allows clonal behavior to be assessed. Analysis of \u003cem\u003eSox10\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eTsc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflfl\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eR26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e embryos on E17.5 (with previous pulsing on E8.5) revealed that in the absence of the \u003cem\u003eTsc1\u003c/em\u003e gene, the clones of nasal chondrocytes appeared as bulky large clusters (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eJ-O), with more extensive dispersion and misalignment (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eP-S ), compared to the individual columns observed in the \u003cem\u003eSox10\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT2\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eTsc1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eR26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e heterozygotes (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eJ-S).\u003c/p\u003e\n \u003cp\u003eThus, activation of the mTORC1 pathway in murine NCCs modulated both chondrogenic condensation and clonal arrangement.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eModulation of the activity of the mTORC1 pathway at the stage of intercalation influences shaping of the craniofacial skeleton to a relatively minor extent\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo explore the influence of the mTORC1 pathway during intercalation of new clones into existing mesenchymal condensations\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, we injected tamoxifen on E12.5, the stage at which \u003cem\u003eSox10\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e targets perichondrial cells surrounding cartilage elements and Schwann cell precursors\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Surprisingly, ablation of Tsc1 at this developmental stage did not change the shape of the craniofacial skeleton (not shown) and increased clonal size only slightly (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-J). To further verify this observation, we targeted chondro-progenitors involved in early mesenchymal condensation employing \u003cem\u003eCol2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003c/em\u003e\u003c/sup\u003e mice coupled with both the \u003cem\u003eTsc1\u003c/em\u003e floxed and \u003cem\u003eR26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e strains and pulsed with tamoxifen on E12.5. In line with the previous observation, activation of mTORC1 signaling by \u003cem\u003eCol2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003c/em\u003e\u003c/sup\u003e at this developmental stage did not alter the structure of the craniofacial skeleton and increased clonal size slightly (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eK-O).\u003c/p\u003e\n \u003cp\u003eAt the same time, ablation of mTORC1 signaling in chondro-progenitors by crossing \u003cem\u003eRaptor\u003c/em\u003e floxed mice with the \u003cem\u003eCol2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eR26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e strains and pulsing with tamoxifen on E12.5 augmented facial length on E17.5 (as detected by \u0026micro;CT, see Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eP-R) without affecting any other skeletal parameters (Extended data Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). Ablation of Raptor under these same conditions lowered the number of large clones somewhat and enhanced the number of cells that were single-labeled (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eS-Y). Successful manipulation of mTORC1 activity in these various strains was confirmed by assessment of S6 phosphorylation (Extended data Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD-E and data not shown).\u003c/p\u003e\n \u003cp\u003eThese observations indicate that in mice the mTORC1 pathway is involved in craniofacial shaping predominantly prior to and/or during the stage at which chondrogenic condensations occur.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eInhibition of mTORC1 immediately prior to chondrogenic condensations alters the formation of cartilaginous facial structures in both mice and zebrafish\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo establish the stage of craniofacial skeletogenesis during which the role of mTORC1 signaling is most important, we inhibited mTORC1 with a single injection of rapamycin into pregnant animals on E10.5, when migration of cranial NCCs has been completed, but chondrogenic condensation has not yet begun\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. This resulted in a slightly elongated snout in the embryos on E17.5 in comparison to the controls injected with DMSO (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Moreover, the thickness of chondrogenic mesenchymal condensations on E12.5 was reduced (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). Clonal lineage tracing of chondro-progenitors in these same embryos beginning on E12.5 revealed disorganized clones, with relatively fewer elongated clones containing more than three chondrocytes and a relatively higher number of labeled cells that had not divided (i.e., in which recombination had occurred, but which did not proliferate during the period of tracing) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG-M).\u003c/p\u003e\n \u003cp\u003eTo determine whether this influence of mTORC1 signaling on mesenchymal condensation is conserved among species, we also studied zebrafish, in which shaping of the craniofacial skeleton also occurs via chondrogenic condensation and intercalation of chondro-progenitors into the primary cartilage anlagen\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In these animals the craniofacial skeleton begins to develop between 48 and 72 hours post-fertilization (hpf)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and the first Sox9- and Col2-positive cells appear at 48 and 53 hpf, respectively\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eCol2a1aBAC:mcherry\u003c/em\u003e zebrafish larvae were exposed to rapamycin at various time-points in their development, washed free of this compound, and then allowed to develop until 120 hpf. Exposure prior to (14\u0026ndash;22 hpf, 24\u0026ndash;32 hpf) or during (32\u0026ndash;56 hpf) chondrogenic condensation did not affect the overall size of the facial skeleton, but led to narrowing of cartilaginous structures (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eN-S). Interestingly, significant elongation of the face occurred when the larvae were exposed to rapamycin at 14\u0026ndash;22 hpf or 32\u0026ndash;48 hpf (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eR). Furthermore, exposure prior to chondrogenic condensation resulted in slight curvature of the ethmoid plate (ETH) and re-positioning of several other elements of the cartilage (MC, PQ and CH) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eP,Q).\u003c/p\u003e\n \u003cp\u003eAltogether, these findings indicate that in both mice and zebrafish the mTORC1 pathway modulates the shape of craniofacial structures by regulating the recruitment and clonal expansion of mesenchymal derivatives of neural crest cells. Interestingly, even transient inhibition of mTORC1 activity early during development altered the clonal behavior of NCC progeny, thereby leading to subsequent modulation of the shape of the craniofacial skeleton.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003eDietary interventions that modulate mTORC1 activity also alter the skeletal structure by impairing clonal dynamics\u003c/h2\u003e\n \u003cp\u003eThe evolutionary conservation and mild variability described above indicate that mTORC1-dependent modulation of craniofacial structures, and particularly those of the feeding apparatus, may be an important adaptive mechanism. As also mentioned above, the activity of the mTORC1 pathway is regulated by nutritional status and, in particular, by dietary levels of amino acids, which act both directly at the cellular level through receptors for arginine and leucine and systemically via pathways involving growth hormone and insulin growth factors (IGFs), which are themselves also controlled by amino acids levels\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Accordingly, we examined whether alteration of mTORC1 activity through feeding diets containing different levels of protein to pregnant dams might modulate craniofacial shaping in the offspring. For this purpose, starting on E6.5 pregnant C57BL/6J mice consumed isocaloric diets containing either 20% protein (a level similar to that in standard mouse chow\u0026thinsp;=\u0026thinsp;the control), 4% (low) protein or 40% (high) protein, with subsequent analysis of at least 4 different litters of embryos from each group.\u003c/p\u003e\n \u003cp\u003eAs expected, mTORC1 activity (as reflected in the level of pS6) was lowest in the control embryos and most pronounced in the high group (Extended data Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA,B), with no differences in body weight (Extended data Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). \u0026micro;CT scans of embryos on E17.5 utilizing phosphotungstic acid (PTA) to augment contrast (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA) revealed that both the length and width of the nasal capsule (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB,C), as well as the length of the Meckel\u0026rsquo;s cartilage (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD) were all influenced by the level of protein in the maternal diet. Thus, comparison of 3D segments of the chondrocranium cartilage showed that both the nasal capsule and mandible were slightly smaller in the embryos whose dams received 4% dietary protein (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). In addition, the thickness of the cartilage of the nasal capsule was elevated by the higher level of dietary protein (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF). To confirm these observations, the same experiment was performed, but utilizing Hexabrix 320 for contrast in connection with the \u0026micro;CT, and similar changes in craniofacial structures were observed (Extended data Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD-M). Lowered proliferation of cells within skeletal elements was observed only in embryos whose dams received the lowest level of dietary protein (Extended data Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eN-T) and there were no differences between the groups with respect to the extent of cell death within cartilaginous elements (Extended data Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eQ-U).\u003c/p\u003e\n \u003cp\u003eWhen the level of protein in the diets was manipulated in this same manner in pregnant \u003cem\u003eSox10\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eTsc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflfl\u003c/em\u003e\u003c/sup\u003e dams (pulsed with tamoxifen on E8.5 and, accordingly, having embryos with constitutively active mTORC1 in all their NCCs-derived cells) and the craniofacial structures of these embryos analyzed on E17.5, again by \u0026micro;CT scans utilizing PTA for contrasting, no differences in any of craniofacial parameters were detected (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG-J). Power analysis showed that at least 320 pups need to be analyzed to detect significant difference in the length of nasal capsule with a power of 0.95 and 55 pups for its width, which is beyond the feasibility in 3D reconstructions of PTA-enhanced embryos. Thus, the lack of any alteration in the craniofacial parameters examined (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH-J), together with the changes in pS6 activity observed above (Extended data Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA,B), indicate that the alterations in craniofacial structures in response to the different levels of dietary protein were mediated by mTORC1 signaling.\u003c/p\u003e\n \u003cp\u003eNext, when the level of protein was manipulated in this same manner in the diets of Col\u003cem\u003e2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eR26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eConfetti\u003c/em\u003e\u003c/sup\u003e mice and these animals injected with tamoxifen on E12.5 and E13.5, both the low and high levels of protein caused remarkable disorganization of the clones within developing cartilaginous elements (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eK-L). This finding is in agreement with the conclusion above that both elevation and attenuation of mTORC1 activity disturbs clonal organization within developing cartilage.\u003c/p\u003e\n \u003cp\u003eFinally, incorporation of the average values obtained in mice with the low and high protein diets to a mathematical model of human skulls for visualization purpose indicated slight, but clear alterations in multiple elements of the craniofacial skeletons (Extended data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, see the Methods for further details).\u003c/p\u003e\n \u003cp\u003eAltogether, these findings indicate that the level of protein in the maternal murine diet during pregnancy influences embryonic shaping of craniofacial cartilage by altering the activity of mTORC1, which in turn changes the clonal dynamics of neural crest progeny.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we have revealed cellular mechanisms underlying mTORC1-dependent shaping of elements of the craniofacial skeleton and demonstrated in both zebrafish and mice that this shaping occurs predominantly in association with mesenchymal chondrogenic condensations, with subsequent fine-tuning to a lesser degree via intercalation. In addition, we have demonstrated that mTORC1 activity in embryos of these species is modulated by the level of protein in the maternal diet, with associated effects on the chondro-cranium and fine-tuning of the shape of the craniofacial skeleton.\u003c/p\u003e \u003cp\u003eIn greater detail, we show here that alterations in the behavior of progeny of NCCs influence skeletal shaping, both at the stage when chondrogenic mesenchymal condensations occur and when the 3D morphology of the cranial skeleton is fine-tuned via clonal intercalation. The finding that the shape of mesenchymal condensations largely determines the subsequent shape of cartilaginous and, later, bony structures\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e provides a link between the expansion of ectomesenchyme derived from murine NCCs lacking Tsc1 and resulting changes in craniofacial shape\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. It is noteworthy that mTORC1 activity influences the shaping of different chondrogenic mesenchymal condensations to different extents, with imperfect preservation of the rough 3D geometry of the entire chondrocranium. For example, constitutively active mTORC1 increases the thickness of the condensations underlying the nasal prominence and nasal capsule, while changing the patterning of the nasal septum to a much more limited degree.\u003c/p\u003e \u003cp\u003eTherefore, specific mechanisms or processes appear to be localized within distinct regions of the chondrocranium. The potential underlying mechanism(s) may involve the known interactions between the mTORC1 pathway and the major morphogens involved in the shaping of craniofacial structures, including HHs (hedgehogs), FGFs (fibroblast growth factors), BMPs (bone morphogenetic proteins), WNTs (Wingless/Integrated family of morphogens), RA (retinoic acid) and PDGFs (platelet-derived growth factors)\u003csup\u003e25,39,45\u0026minus;49\u003c/sup\u003e. For instance, ablation of mTOR specifically in NCCs reduces the activities of the canonical Wnt and BMP pathways\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. SHH, which is secreted in localized regions by the neuroepithelium and the brain, participates in shaping the anterior chondrocranium in a highly specific manner, e.g., by inducing or permitting formation of the nasal septum\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. At the same time, S6K1, a kinase downstream of mTORC, augments HH signaling by phosphorylating GLl1\u003csup\u003e39\u003c/sup\u003e. Thus, the differential chondrogenic activity of SHH, in combination with its functional interactions with the mTORC1 pathway, may contribute to the difference in the consequences of chondrogenic condensations at different locations in the developing face.\u003c/p\u003e \u003cp\u003eFurthermore, our present findings indicate that the mTORC1 pathway influences facial skeletal shaping by modulating the clonal expansion of committed chondro-progenitors. Previously, we reported that the growth of facial skeletal elements depends on intercalation of chondrocyte clones originating from committed chondro-progenitors within the perichondrium surrounding these elements and oriented transversally into pre-formed cartilage\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This intercalation and subsequent expansion of chondrogenic clones plays a key role in controlling the final thickness and geometry of cartilaginous elements. Here, we show that manipulation of mTORC1 activity prior to formation of the perichondrium and committed chondro-progenitors alters the formation of these oriented clones later in development. With attenuated mTORC1 activity, the intercalated clones in the nasal cartilage of embryos were smaller, whereas elevation of mTORC1 activity in chondro-progenitors via deletion of the \u003cem\u003eTsc1\u003c/em\u003e gene resulted in intercalation of bulky clonal clusters rather than individual clonal columns. Intercalation of these aberrant clones likely underline the altered length and thickness of the nasal cartilage, which eventually influenced the overall craniofacial shape. Thus, during mesenchymal condensation mTORC1 activity regulates the overall geometry of facial cartilage, whereas with respect to committed chondro-progenitors this activity influences individual cartilaginous elements. Therefore, modulation of mTORC1 activity at different time-points may result in a spectrum of somewhat different craniofacial shapes, perhaps thereby also contributing to the variety of defects in patterning observed.\u003c/p\u003e \u003cp\u003eIt is worth pointing out that the mTORC1 pathway is also involved in chondrogenesis in the limbs, with ablation of Raptor in the limb bud mesenchyme resulting in growth impairment\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. However, modulation of mTORC1 activity in mature chondrocytes does not influence limb growth\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. These observations indicate that the appropriate level of mTORC1 activity in chondro-progenitors, rather than in mature chondrocytes, is important for skeletogenesis, in line with our present results.\u003c/p\u003e \u003cp\u003eSince mTORC1 is primarily involved in adjusting cellular responses to the nutrition available\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, either being enhanced directly by amino acids\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e or via insulin and insulin-like growth factors (all of which are tightly regulated by nutritional levels\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e), it is not surprising that we found that modulation of the level of protein in the maternal diet regulates mTORC1 activity resulting in subtle, but distinct changes in the craniofacial shape of the embryos. Availability of nutrition is a major factor in connection with natural selection and such a spectrum of closely related craniofacial shapes may reflect adaptive phenotypic plasticity, and, accordingly, allow various feeding strategies. Phenotypic plasticity in the feeding apparatus of teleost fish has been observed, both in the wild\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e and under experimental conditions\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Recently, it has been reported that the HH pathway mediates plasticity of the feeding apparatus in response to the mechanical properties of the foraging species\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, with mechanical sensing being, at least in theory, mediated by cilia, a mechanical sensor that is a key component of the HH pathway\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Thus, interactions between nutritional sensing by the mTORC1 pathway and mechanical sensing by the HH pathway may mediate phenotypic plasticity of the feeding apparatus in response to external conditions. Interestingly, mTORC1 is also involved in regulating the phenotypic plasticity of skeletal muscles\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, as well as in long-term synaptic plasticity\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn humans, craniofacial plasticity has been described in response to the consistency of the diet and alcohol consumption by the mother during pregnancy, as well as to the climate\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Thus, plasticity of the feeding apparatus, as well as of the entire facial skeleton, may be an evolutionarily conserved characteristic of all gnathostomes, including humans. On the basis of the findings of others and the data documented here, we propose that the mTORC1 pathway is a key part of the molecular machinery that adapts craniofacial structures to nutritional conditions.\u003c/p\u003e \u003cp\u003eIn summary, we have demonstrated here that the mTORC1 pathway modulates the embryonic shaping of craniofacial skeletal elements at the stage of chondrogenic condensations, with subsequent fine-tuning during intercalation of chondro-progenitors. Furthermore, we provide evidence for an impact of maternal protein intake during pregnancy on the shaping of fetal craniofacial cartilage. These findings provide novel and important insights into the mechanisms underlying craniofacial shaping and, potentially, the phenotypic plasticity of this process as well and, in addition, help elucidate the role of material dietary protein during pregnancy in this context.\u003c/p\u003e "},{"header":"Experimental Procedures","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eHuman embryos\u003c/h2\u003e \u003cp\u003eHuman fetal tissue collection was reviewed and approved by the local ethics committee of Institute of Fundamental Medicine and Biology of Kazan Federal University (No. 8, May 2018). Written informed consent was obtained from the patients subjected to medical abortion.\u003c/p\u003e \u003cp\u003eTo identify enhancers actually transcribed in human embryonic faces, human facial material was collected between weeks 3 and 12 of development, time-window that potentially influence human facial individuality. Next, we performed CAGE-sequencing on embryonic human facial material and compared the transcriptional start sites, proximal promoters and distal transcribed enhancers\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e thus identified to loci indicated as being involved in human facial variability by genome-wide sequencing\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://portaldev.sph.umich.edu/docs/api/v1/#introduction\u003c/span\u003e\u003cspan address=\"http://portaldev.sph.umich.edu/docs/api/v1/#introduction\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor our CAGE-sequencing, total RNA (2\u0026ndash;3 mg) was extracted from the facial portion of human embryos, preserved in RNAlate and stored at -80\u003csup\u003eo\u003c/sup\u003e C using the RNeasy Fibrous Tissue Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer\u0026rsquo;s protocol. The concentration and purity of extracted RNA were determined on the basis of absorption employing the NanoDrop\u0026trade; 8000 Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) and quality verified with the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).\u003c/p\u003e \u003cp\u003eLibraries were then prepared utilizing the standard nAnT-iCAGE (non-Amplified non-Tagging Illumina Cap Analysis of Gene Expression) protocol\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, employing 2.5\u0026ndash;3 \u0026micro;g total RNA as a template for synthesis of the first cDNA strand (nAnT-iCAGE Library Preparation kit DNA form, Yokohama, Japan and SuperScript III Reverse Transcriptase, Invitrogen, Waltham, MA, USA). This cDNA was subsequently biotinylated at its 5\u0026acute;- end (nAnT-iCAGE Library Preparation kit, DNA form, Yokohama, Japan), which allowed selection of the 5\u0026acute;-cap containing molecules with streptavidin beads (Dynabeads M-270 Streptavidin, ThermoFisher Scientific, USA). In this manner, rRNA, as well as truncated or not fully transcribed RNA was eliminated.\u003c/p\u003e \u003cp\u003eFor more complete removal of RNA, the cDNA was treated with RNase I and H (nAnT-iCAGE Library Preparation kit, DNA form, Japan) and then purified using RNACleanUP (Beckman Coulter, Brea, CA, USA). Next, linkers were ligated to the 5\u0026prime;and 3\u0026prime; ends (nAnT-iCAGE Library Preparation kit, DNAform, Japan) of the cap-trapped cDNA. The 5\u0026prime;- linker employed contained recognition sites for the XmaJI restriction endonuclease (nAnT-iCAGE Library Preparation kit, DNA form, Japan) and the MmeI class II restriction enzyme (nAnT-iCAGE Library Preparation kit, DNA form, Japan), as well as a barcode for multiplexing. The 3\u0026prime;- linker contained a recognition site an XbaI restriction enzyme (nAnT-iCAGE Library Preparation kit, DNAform, Japan). Treatment with these restriction enzymes yielded short CAGE tags to which a sequencing primer was ligated.\u003c/p\u003e \u003cp\u003eIn the final stage, a second cDNA strand was synthesized from these short CAGE tags??? (nAnT-iCAGE Library Preparation kit, DNAform, Japan). The concentration of the resulting libraries was determined by the PicoGreen Assay in a GloMax\u0026reg; Multi Detection System (Promega, Madison, WI, USA) and their quality assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Finally, the libraries were validated using real-time PCR (KAPA Library Quantification Kits Illumina, KAPA Biosystems, Wilmington, MA, South Africa) and sequencing on a HiSeq 2500 platform (Illumina, San Diego, CA, USA) using the HiSeq v4 reagent kit (HiSeq SR Cluster Kit v4 cBot and HiSeq SBS Kit v4 50 cycles, Illumina, San Diego, CA, USA) in the 50-bp single-end mode.\u003c/p\u003e \u003cp\u003eSingle-read sequences were analyzed for quality and over-represented adapter sequences identified with the FastQC tool. Quality filtering trimming was performed with the fastx_trimmer (FASTX Toolkit 0.0.13.2) and Trimmomatic-0.39 and RNAdust 1.06 utilized as adapters and for removal of rRNA removal. Read mapping on human genome hg38 and mouse genome mm10 was performed with BWA-0.7.10, with unmapped reads being realigned using Hisat2-2.2.1. Aggregation of CAGE tag start sites (CTSS) for each sample, with subsequent peak clustering, were carried out employing the PromoterPipeline script from the C1 CAGE protocol\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Bidirectional enhancers were identified using the pipeline described by Andersson and colleagues (2014)\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. The statistical significance of the differential expression of CAGE peaks was calculated using the edgeR package for R.\u003c/p\u003e \u003cp\u003eTriple overlap of GWAS-derived data of coordinates of face shape-affecting loci with already pre-identified and annotated enhancers (from genome annotation\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.ucsc.edu/cgi-bin/hgTrackUi?db=mm10\u0026amp;g=encodeCcreCombined\u003c/span\u003e\u003cspan address=\"https://genome.ucsc.edu/cgi-bin/hgTrackUi?db=mm10\u0026amp;g=encodeCcreCombined\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; ENCODE Project Consortium) and our human CAGE-seq-derived coordinates of active facial human embryonic enhancers was done involving a specific prior filtration step such as: we selected polymorphisms falling within 5 kilo-base pairs distance from the CDS in any direction for GWAS-identified genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003e All animal experiments were pre-approved by the Stockholm North Ethical committee and performed in accordance with the guidelines of the Swedish Animal Agency. The Sox10-CreERT2, Col2-CreERT2, R26Confetti, Tsc1 flox, and Raptor flox strains of mice employed have been described in detail previously\u003csup\u003e41,66\u0026minus;69\u003c/sup\u003e. Embryonic Cre recombination was induced by intraperitoneal (i.p.) injection of 1\u0026ndash;3 mg tamoxifen (Sigma) into each pregnant dam. The day on which the plug was detected was defined as embryonic day 0.5 (E0.5). Rapamycin (0.02 mg, LC Laboratories) was injected i.p. into each pregnant dam.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eZebrafish\u003c/h2\u003e \u003cp\u003eThe Col2a1aBAC:mcherry strain of zebrafish was kindly provided by Prof. Chrissy Hammond (University of Bristol, UK) and has been utilized as described in detail elsewhere\u003csup\u003e\u003cspan additionalcitationids=\"CR71\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Zebrafish larvae were exposed to 400 nM rapamycin at the time-points indicated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003eManipulation of the maternal murine diet\u003c/h2\u003e \u003cp\u003ePregnant dams received standard mouse chow containing 4% protein until E6.5 and thereafter an isocaloric diet containing 4%, 20% or 40% protein (TD. 93032, TD. 91352 and TD. 90018 from Envigo) until the day of sacrifice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eX-ray computed microtomography (\u0026micro;CT) with enhanced contrast achieved with phosphotungstic acid (PTA)\u003c/h2\u003e \u003cp\u003eOn E17.5, the heads of mouse embryos were placed in a 1% PTA/methanol solution to enhance the contrast of soft structures, as described previously\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. \u0026micro;CT scans were performed with the GE phoenix v|tome|x L 240 system equipped with a nanofocus X-ray tube (180 kV/15 W maximal power) and high flat panel (dynamic 41|100 with 4000 \u0026times; 4000 pixels, each 100 \u0026times; 100 \u0026micro;m in size). Acquisition involved the use of a 0.2-mm aluminum filter to soften the beam; 60 kV and 200 \u0026micro;A; exposure for 600 ms; and averaging of 3 projections to reduce noise. 1800 images were acquired over 360\u0026deg;, requiring a scanning time of one hour per sample. The isotropic voxel size was 6.2 \u0026micro;m in all cases. The tomographic reconstructions were performed in the GE phoenix datos|x 2.0 3D computed tomography software. Segmentation of craniofacial structures was performed manually using a combination of the Avizo (Thermo Fisher Scientific, USA) and VG Studio MAX 3.2 software (Volume Graphics GmbH, Germany), as described elsewhere\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHigh-resolution microfocus computed tomography using enhanced contrast with Hexabrix (CE-HR\u0026micro;CT) and subsequent image processing and 3D analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFollowing fixation, samples were stored in PBS at 4\u0026deg; C. Prior to scanning, these samples were transferred to Eppendorf tubes containing 1.5 ml 30% Hexabrix 320 in PBS (Guerbet Nederland B.V.); incubated for two weeks with continuous gentle shaking at 4\u0026deg; C; and then scanned while still inside the same tubes. Hexabrix 320, a negatively charged ioxaglate, is repelled by the anionic sulfated-glycosaminoglycan (sGAG), resulting in negative staining of cartilage, while still providing good contrast between mineralized tissues and the background.\u003c/p\u003e \u003cp\u003eFor acquisition of all images, the NanoTom M (GE Measurement and Control Solutions, Germany) system in combination with a diamond-coated tungsten target was employed with the following conditions: a 0.2-mm aluminum filter to soften the beam; 60 kV and 300 \u0026micro;A; exposure for 500 ms; and averaging of each sample individually and a skip of 0 (\u0026lsquo;fast scan mode\u0026rsquo;). 2400 images were acquired over 360\u0026deg;, requiring a scanning time of 20 minutes per sample. In all cases the isotropic voxel size was 5 \u0026micro;m. Reconstruction was performed using the Phoenix datos|x CT software, applying a correction of 5 for beam hardening and a Gaussian filter (radius 3) to reduce noise.\u003c/p\u003e \u003cp\u003eThe transaxial, coronal and sagittal cross-sections of each sample were visualized with the DataViewer (Bruker MicroCT, Belgium); while 3D visualization of the cartilage and mineralized tissue and quantification of their volumes were performed with the Mimics Innovation suite (Materialise NV, Belgium). Briefly, two threshold values were selected manually to distinguish between non-mineralized cartilage and mineralized tissues (i.e., mineralized cartilage and subchondral bone) and these thresholds then fine-tuned with dynamic region-growing and multi-slice edit. Using these adjusted threshold values, 3D models based on marching-cubes were generated and the volumes of mineralized tissue versus non-mineralized cartilage and the ratio between these volumes calculated. In addition, the length and width of the nasal capsule and the Meckel cartilage were measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eImmunohistochemical analyses\u003c/h2\u003e \u003cp\u003eEmbryos were fixed in 4% paraformaldehyde (PFA) for 6 hours at 4\u0026deg; C and tissues then embedded in OCT (Tissue-Tek) on dry ice for sectioning. Thereafter, the 30-\u0026micro;m frozen sections were blocked in PBST (PBS\u0026thinsp;+\u0026thinsp;0.01% Tween20)\u0026thinsp;+\u0026thinsp;3% normal horse serum (Vector laboratories) for one hour prior to incubation with the primary antibody (anti-pS6 (Cell Signaling), anti-PAR3 (Millipore), anti-acetylated-tubulin (Santa Cruz), anti-PKCζ (Santa Cruz), or anti-β-catenin (Santa Cruz)) overnight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eTUNEL staining\u003c/h2\u003e \u003cp\u003e30-\u0026micro;m tissue sections were treated with 10 \u0026micro;g/ml proteinase K (Ambion) for 40 minutes at 37\u0026deg; C before applying the TUNEL reaction mix (Roche Inc.) for 90 minutes. The cell nuclei were then counterstained with DAPI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eStaining with haematoxylin and eosin\u003c/h2\u003e \u003cp\u003e15-\u0026micro;m frozen sections were stained with haematoxylin for 30 seconds and 0.02% eosin for 2 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003eMicroscopy and image analysis\u003c/h2\u003e \u003cp\u003eImages were acquired with a LSM710 confocal microscope. 3D visualization and all quantification were performed utilizing the IMARIS (Bitplane) and ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eMathematical modelling of human craniofacial anatomy\u003c/h2\u003e \u003cp\u003eTo examine how the morphological changes observed in mice might be manifested in humans, we transformed polygon data on human craniofacial anatomy extracted from full-body MRI (BodyParts3D, Database Center for Life Science, Japan, Tokyo). The skull was divided into 53 high-resolution images of teeth, bones and ligaments and transformation carried out in Mathematica 11.0 (Wolfram Research, USA, Illinois) using custom-written code. The non-linear 3D transform was designed as a three-dimensional 'magnifying glass' (adopted from\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e) with a radius of 3 cm and centered on the nasal cavity. The magnification was adjusted so that the width of the nasal cavity increased in the same manner as observed in mice. The algorithm allowed us to selectively magnify defined anatomical regions of the skull, while maintaining the rest of the craniofacial anatomy unchanged.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe wish to thank Olga Kharchenko for the artwork included and Ostap Dregval for technical assistance. This study was supported by the Swedish Research Council (Projects 2020-02298 to A.S.C., 2018-02713 to I.A., 2022-00611 to K.F. and 2021-01805 to M.X.), an ALF-agreement (ALFGBG-966178 to A.S.C.) and the NovoNordisk Foundation (NNF21OC0070314 to A.S.C.). M.X. was supported by a long-term postdoctoral fellowship from the European Molecular Biology Organization and by Stiftelsen Frimurare Barnhuset i Stockholm. E.I. was supported by a grant from the Russian Basic Science Foundation (#19-29-04115 to A.S.C). M.T., T.Z. and J.K. acknowledge financial support in the form of project CEITEC 2020 (LQ1601) from the Ministry of Education, Youth and Sports of the Czech Republic under the National Sustainability Program II and help from the CzechNanoLab Research Infrastructure supported by MEYS CR (LM2018110). M.T. was the recipient of a Ph.D. Talent Scholarship from the Brno City Municipality.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: M.X. and A.S.C.; Methodology and Investigation: M.X., M.T., T.Z., G.K., E.I., H.Z., D.B., R.D., G.G., E.S., and P.T.N.; Resources: O.G., K.F., J.K., I.A., and A.S.C.; Writing – Original Draft, Review \u0026amp; Editing: M.X., I.A. and A.S.C; Funding Acquisition: A.S.C., I.A., K.F., M.X., J.K. All authors have read and agreed to submission of the final manuscript for publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKohn, L. A. P. The Role of Genetics in Craniofacial Morphology and Growth. \u003cem\u003eAnnual Review of Anthropology\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 261-276 (1991). https://doi.org:https://doi.org/10.1146/annurev.an.20.100191.001401\u003c/li\u003e\n\u003cli\u003ePorras-Hurtado, X. S.-G. a. G. L. Characterization of congenital craniofacial anomalies in a specialized hospital of Risaralda, Colombia. 2010-2014. \u003cem\u003eRev. Fac. Med.\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 223-227 (2018). \u003c/li\u003e\n\u003cli\u003eWHO. 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Magnifying Glass. \u003cem\u003eWolfram Demonstrations Project\u003c/em\u003e (2011). https://demonstrations.wolfram.com/MagnifyingGlass/ \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Craniofacial, neural crest, skeleton, mTORC1, protein, shape, mesenchymal condensation","lastPublishedDoi":"10.21203/rs.3.rs-2542333/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2542333/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of craniofacial skeletal structures is fascinatingly complex and elucidation of the underlying mechanisms will not only provide novel scientific insights, but also help develop more effective clinical approaches to the treatment and/or prevention of the numerous congenital craniofacial malformations. To this end, we performed CAGE-sequencing of the facial mesenchyme of human embryos and cross-checked the active enhancers thus identified against genes, identified by GWAS for the normal range human facial appearance. Among the identified active cis-enhancers, several belonged to the components of the mTORC1 (Mechanistic Target of Rapamycin Complex 1) pathway. To assess the functional role of this pathway, we manipulated it both genetically and pharmacologically in mice and zebrafish. These experiments revealed that mTORC1 signaling modulates craniofacial shaping at the stage of skeletal mesenchymal condensations, with subsequent fine-tuning during clonal intercalation. This ability of mTORC1 pathway to modulate facial shaping, along with its evolutionary conservation and ability to sense external stimuli, in particular dietary amino acids, indicate that the mTORC1 pathway may play a role in facial phenotypic plasticity. Indeed, the level of protein in the diet of pregnant female mice influenced the activity of mTORC1 in fetal craniofacial structures and altered the size of skeletogenic clones, thus exerting an impact on the local geometry and craniofacial shaping. Overall, our findings indicate that the mTORC1 signaling pathway is involved in the effect of environmental conditions on the shaping of craniofacial structures.\u003c/p\u003e","manuscriptTitle":"The level of protein in the maternal murine diet modulates the facial appearance of the offspring via mTORC1 signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-15 23:24:08","doi":"10.21203/rs.3.rs-2542333/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9456c08b-572f-4e43-acc0-0d55c224665e","owner":[],"postedDate":"March 15th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":19138438,"name":"Biological sciences/Genetics/Gene regulation"},{"id":19138439,"name":"Biological sciences/Developmental biology/Morphogenesis/Cell lineage"}],"tags":[],"updatedAt":"2024-03-27T07:05:21+00:00","versionOfRecord":{"articleIdentity":"rs-2542333","link":"https://doi.org/10.1038/s41467-024-46030-3","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-03-26 04:00:00","publishedOnDateReadable":"March 26th, 2024"},"versionCreatedAt":"2023-03-15 23:24:08","video":"","vorDoi":"10.1038/s41467-024-46030-3","vorDoiUrl":"https://doi.org/10.1038/s41467-024-46030-3","workflowStages":[]},"version":"v1","identity":"rs-2542333","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2542333","identity":"rs-2542333","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}