A Novel Tamoxifen-Inducible Mct8-CreERT2 Mouse Model for Targeted Studies of Mct8-Expressing Cells and Thyroid Hormone Transport and Function

A Novel Tamoxifen-Inducible Mct8-CreERT2 Mouse Model for Targeted Studies of Mct8-Expressing Cells and Thyroid Hormone Transport and Function | 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 A Novel Tamoxifen-Inducible Mct8-CreERT2 Mouse Model for Targeted Studies of Mct8-Expressing Cells and Thyroid Hormone Transport and Function Anna Molenaar, Noémi Mallet, Marin Bralo, Luciano Jan Hoeher, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6796634/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Deficiency of the Monocarboxylate Transporter 8 (MCT8) severely impairs thyroid hormone (TH) transport into the brain, disrupting brain development as well as peripheral TH homeostasis. Studies assessing MCT8 expression patterns and tissue-specific pathologies induced by local TH-deficiency are often inconclusive due to unreliable antibody staining and the lack of functional tools to specifically target MCT8-expressing cells. For this purpose, we generated non-inducible Mct8-Cre and tamoxifen-inducible Mct8-CreERT2 mice. Mct8-Cre;Sun1-sfGFP mice demonstrated ubiquitous Sun1-sfGFP expression, due to early recombination driven by Mct8 gene expression at the stage of trophoblast implantation. Tamoxifen injection in 6-week-old Mct8-CreERT2 mice induced reporter expression specifically in Mct8-expressing cells in the brain and peripherally in liver, kidney, and thyroid, without leaky reporter expression in vehicle controls. Using vDISCO tissue clearing and 3D-imaging of GFP-nanobody-boosted mice, we further identified the sublingual salivary gland and the prostate as prominent Mct8-expressing organs. Nuclei from Mct8-expressing cells could selectively be enriched using fluorescence-activated nuclei sorting on Mct8-CreERT2;Sun1-sfGFP mice and characterized as choroid plexus cells and tanycytes. Our new inducible Mct8-CreERT2 line provides researchers with a tool to reliably mark, enrich, and characterize Mct8-expressing cells and to genetically modify genes specifically in these cells to study thyroid hormone transport and function. Health sciences/Anatomy/Nervous system/Central nervous system Health sciences/Endocrinology/Endocrine system and metabolic diseases/Thyroid diseases Biological sciences/Biochemistry/Hormones/Thyroid hormones Health sciences/Neurology/Neurological disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The monocarboxylate transporter 8 (MCT8) is an influx and efflux transmembrane transporter for thyroid hormones (TH) thyroxine (T 4 ) and 3,3′,5-triiodothyronine (T 3 ), and their metabolites 3,3′,5′-triiodothyronine (rT 3 ) and 3,3′-diiodothyronine [1]. Its role in TH transport is crucial for brain TH availability, evidenced by the devastating neurological impairments of Allan-Herndon-Dudley-Syndrome (AHDS) patients with mutations in the MCT8-encoding solute carrier family member 16a2 ( Slc16a2 ) gene on the X-chromosome [2,3]. Deficient TH transport during crucial stages of brain development causes intellectual disability, motor dysfunctions, and impaired myelination. Reduced TH brain levels are thereby contrasted by elevated T3 plasma levels, causing peripheral thyrotoxicosis [4]. To understand TH transport and its associated diseases, it is important to elucidate the expression pattern of MCT8 in the brain as well as other organs across various developmental stages. Western Blotting found highest levels in the human liver, followed by pituitary and brain, low signals for heart and placenta, and near absence in the lung [5]. MCT8-mapping to the human brain, thyroid, and pituitary was moreover confirmed using immunohistochemistry (IHC) and immunofluorescence (IF) [5-10]. Murine Slc16a2 mRNA levels are high in liver and kidney, low in lung, cerebral cortex, and heart, and barely detectable in testis (Mouse ENCODE transcriptome project PRJNA66167) [11]. Mct8 protein was localized in rodent livers, thyroid glands, kidneys, brains, and to a lesser extent rat hearts [1,9,12-14](Supplemental Table 1). IHC and IF in adult human brains revealed abundant MCT8 levels in barrier cells (endothelial cells (EC), astrocytes, choroid plexus (ChP), tanycytes), clearly marking blood vessels and capillaries, but sparse neuronal staining [6-8]. Neuronal MCT8 signal was readily detectable [5] and human cortical organoids [15]. In murine brains, Mct8 protein was abundant in BV, tanycytes, and ChP, while neuronal expression declined from postnatal day 12 (P12) to P21, until undetectable [13]. Others found Mct8 in the adult mouse hippocampus and Purkinje cells and, diffusely, in cortical neurons [5]. Discrepancies in Mct8 detection are likely due to differences in antibodies and lots used, highlighting the need for alternative visualization methods due to challenges in acquiring effective antibodies and inconsistent neuronal staining [13]. We here explored the use of the Cre-lox system, where expression of Cre recombinase is driven by the endogenous Mct8 promoter, and crossed these with fluorescent reporter mice to identify, visualize and characterize Mct8-expressing cells in vDISCO tissue cleared and 3D imaged whole mice and isolated organs. Results Generation of tamoxifen-inducible Mct8-CreERT2 mice Prompted by discrepant studies on the presence or absence of neural Mct8 protein expression in adult mice (Supplemental Table 1) and our own comparison of commercially available antibodies, which revealed unspecific or insufficient immunofluorescence signals for Mct8 in murine brain slices (Supplemental Fig. 1), we aimed to generate a novel Mct8-CreERT2 mouse line to ultimately assess Mct8 expression patterns in adult mice. Mct8-CreERT2 mice were generated by inserting the sequences of iCreERT2 and “self-cleaving” peptide T2A at the start codon of Mct8-gene Slc16a2 using CRISPR/Cas9 (Fig. 1a) [16-20], generating viable mice at the expected Mendelian ratio and with the correct genotypes (Fig. 1b,c,d). The transgenic mice had comparable levels of Mct8 mRNA in WT male and Mct8-CreERT2 +/y brain areas (Fig. 1e), with highest levels in the ChP of the 4th ventricle (4V ChP) and lateral ventricle (LV ChP), followed by the mediobasal hypothalamus (MBH), then cortex (Ctx) and cerebellum (Cer), consistent with published Mct8 gene expression levels [21]. Likewise, we observed unperturbed endogenous Mct8 protein expression, with comparable fluorescence patterns and intensities in antibody-stained hypothalamic slices of WT and Mct8-CreERT2 mice (Fig. 1f). Ubiquitous recombination in the CNS of non-inducible Mct8-Cre mice Using the same CRISPR-Cas9-based knock-in strategy, we explored germline Mct8 expression patterns using non-inducible Mct8-Cre mice. In the fully viable and fertile Mct8-Cre mice crossed with the fluorescent reporter mouse line Sun1-sfGFP, which expresses the GFP-tagged nuclear lamina protein Sun1, we found ubiquitous reporter expression in adult mouse brains (Supplemental Fig. 2). We next investigated whether this ubiquitous pattern could be due to a very early activation of Mct8-Cre expression and subsequent stop cassette excision in all cells of that lineage. Blastocysts (corresponding to embryonic day E4.5) from the mating of Mct8-Cre with Ai14 mice, known for their strong cytosolic fluorescence, showed no reporter signal, while Mct8-Cre;Sun1-sfGFP embryos at stages E10.5 had reporter expression in virtually all cells (Supplemental Fig. 3 a,b). By analyzing publicly available RNA sequencing data across murine embryonal developmental stages, we identified a peak for the expression of Mct8 at day E5.25, i.e. the stage of implantation directly after the blastocyst stage, to likely drive fluorescent reporter activation (Supplemental Fig. 3c). Validation of CreERT2 recombinase activity in Mct8-expressing cells in the brain To validate the Mct8-CreERT2 mouse line’s specificity, we crossed it with the fluorescent Ai14 and Sun1-sfGFP reporter mouse lines and assessed CreERT2 activity one week after the indicated tamoxifen (TAM) applications. In brains of tamoxifen-injected Mct8-CreERT2;Ai14 and Mct8-CreERT2;Sun1-sfGFP mice, we found profound fluorescence reporter expression in tanycytes and ChP that largely overlapped with Mct8 antibody staining (Fig. 2 a,b; Supplemental Figs. 4 a-c & 5). Fluorescence was moreover found in BV and few neurons spattered across the hippocampus, cortex, and striatum. Neither blood vessels nor sparse neurons were labeled using antibody staining. Vehicle-injected Mct8-CreERT2 + and Mct8-CreERT2 - mice that received TAM showed minimal Cre-activity, confirming the highly specific, inducible expression without background activity for both reporter lines. Last, we found comparable recombination efficacies in response to a single i.p. or oral gavage (o.g.) application of tamoxifen. However, reporter expression was profoundly diminished compared to the mice injected with three i.p. doses of TAM (Supplemental Fig. 4 a,b). Overall, these data show that the new Mct8-CreERT2 line is functional and faithfully induces reporter expression in cells known to express Mct8 in adult mice after three doses of 50 mg/kg TAM. Mct8-CreERT2-driven reporter expression in mice After validating the correct reporter expression in mouse brains, we aimed to systematically assess all other Mct8-expressing tissues. We subjected Mct8-CreERT2;Sun1-sfGFP mice to vDISCO, the solvent-clearing of organs and nanobody-based immune staining of GFP-marked nuclei, followed by LSFM. The whole-body scan is visualized in Fig. 3a, videos of the head, torso and whole-body are provided as Supplemental Material (Supplemental Videos 1-3). Strongest fluorescent signals could be localized to the choroid plexus (Fig. 3b), liver and kidney (Fig. 3c). Figure 3d (upper panel) depicts the distinctly labeled sublingual and parotid salivary glands and the thyroid gland. We further observed strong fluorescence in the prostate, as well as the cauda of the epididymis (Fig. 3e) . Intrigued by the detection of reporter signal in the salivary glands and prostate via vDISCO, we examined those tissues in more detail using Mct8-CreERT2;Ai14 mice and fluorescence imaging of cryo-slices. We also included organs that were reported to have Slc16a2 transcripts but showed no or weak reporter expression in the whole mouse scan, namely heart, lung, testis, and skeletal muscle. Of note, we also assessed whether 5 doses of TAM could augment Cre-activity to maximize reporter expression compared to the 3x TAM-treated mice but found no differences in fluorescence intensities (Supplemental Fig. 4c). In the prostate, we found strong fluorescence in cells of the anterior prostate and weak fluorescence in seminal vesicles (Fig. 4 top). In the salivary glands, reporter expression was strongest in the sublingual, modest in the parotid, and near-absent in the submandibular gland (Fig. 4 bottom). Heart, lung, testis, and quadriceps demonstrated sporadic reporter activation, including signals from the femur and caput epididymis (Fig. 5). However, exposure times had to be approximately 30x higher than those necessary for the salivary gland and prostate, indicating a sparsely distributed and comparably lower Mct8 expression in those tissues. Enriching nuclei of Mct8-expressing cells from Mct8-CreERT2;Sun1-sfGFP mice using FANS Finally, we assessed whether the line can be utilized for the enrichment of MCT8-expressing cells from brain tissues of Mct8-CreERT2;Sun1-sfGFP mice by fluorescence-activated nuclei sorting (FANS) of Mct8-positive nuclei tagged with Sun1-sfGFP. 1000 nuclei of the MBH, Ctx including the ChP of the lateral ventricle (LV ChP), and Cer – including the ChP of the 4 th ventricle (4V ChP) were sorted into GFP-positive and -negative fractions, respectively (Fig. 6a). An exemplary gating for GFP + nuclei is shown in Supplemental Fig. 6. All GFP + fractions showed strong Mct8 mRNA enrichment compared to the GFP - fractions (Fig. 6b). qPCR for ChP marker transthyretin( Ttr ) and tanycyte marker µ-crystallin ( Crym ) showed enrichment of ChP- and tanycyte-derived nuclei in the GFP + fractions of the Ctx + LV ChP, Cer + 4V ChP, and MBH, respectively (Fig. 6c). Discussion Our new tamoxifen-inducible Mct8-CreERT2 mouse line allows specific recombination in all Mct8-expressing cells throughout the body with high specificity and minimal leakiness. By inserting the iCreERT2 cassette directly into the endogenous Mct8 locus, we preserve physiological Mct8 expression while enabling controlled iCreERT2 activity. This knock-in strategy offers a clear advantage over traditional approaches relying on bacterial artificial chromosomes (BACs) transgenesis or random transgene integration, which frequently cause unpredictable expression patterns, position effects, gene silencing, or disruption of endogenous loci [22-24]. Such artifacts can introduce major confounders and compromise the physiological relevance of experimental models. In contrast, our targeted approach faithfully recapitulates endogenous Mct8 regulation, minimizing off-target effects and preserving the native biology of the system. The non-inducible Mct8-Cre line showed ubiquitous reporter expression due to the early Mct8 expression peak at day E5.25 of embryonal development. This indicates an important, vaguely understood role of thyroid hormone transport during nidation when embryonal trophoblast cells invade the mother’s endometrium to form the placenta. Notably, Chan et al. [25] found that MCT8 expression is increased in the placentas of humans with intrauterine growth restriction, to potentially increase T3 uptake, and embryos of Mct8-KO rats showed a decreased embryo-to-placental weight ratio. These effects were nonetheless mild, and little evidence pointed toward an impaired trophoblast invasion capability in the KO rats [26]. This may be due to organic anion transporting polypeptide 1c1 (OATP1C1), a transporter for T3 and T4 co-expressed in the rat placental barrier that can partially compensate for the lack of Mct8 in rats and mice but not in humans [27]. Future studies are thus warranted to elucidate the specific roles of Mct8 and OATP1C1 in trophoblast invasion and placental development. In the brains of tamoxifen-induced Mct8-CreERT2 fluorescent reporter mice, we found strong and specific fluorescence in tanycytes and ChP one week after induction, confirmed by Mct8 antibody staining. Additionally, we found reporter signal in blood vessels and, scarcely, in neurons of the hippocampus, cortex, and striatum. Using vDISCO, we could further detect strong reporter signal in liver, thyroid gland, and kidney[1,12,28]. The Encyclopedia of DNA Elements (ENCODE) database reports very low Slc16a2 transcripts in mouse lung, heart, and testes, compared to strongly expressing tissues [11]. Similarly, Slc16a2 expression was reported for various muscle cells using microarrays, but protein staining was only reported for satellite cells [29]. In our Mct8-CreERT2 reporter mice, fluorescent signals in such tissues and organs with low gene expression were weak, but detectable at very high exposure time. Our mouse model is thus a suitable tool to target both organs with a high number of Mct8-expressing cells as well as organs with a low abundance of Mct8-expressing cells. In addition, we found profound reporter signal in organs where a role for Mct8 was not previously described, the sublingual salivary gland and the prostate. Salivation is influenced by TH both in rodents and in humans [30-32], and TH is found in saliva [33]. Our data are consistent with those reports, and highlight that the sublingual and, to a lesser extent, the parotid salivary glands are involved in that salivary TH transport. The prostate secretes fluids that are added to the seminal fluid to facilitate sperm fertility [34]. Prostate fluids also contribute to the generation of copulatory plugs, which is essential for the successful fertilization in mice [35]. The role of Mct8 in male reproduction was already studied in Mct8-KO rats, where Mct8-staining was reported for the rat epididymis. That study explored the role of Mct8 in testes and epididymis for sperm viability and reported a decrease in fertility in male KO rats [36]. Our data highlight that such changes in fertility could also potentially be attributed to the lack of Mct8 in the prostate. Last, the strong enrichment of Mct8, and the selective enrichment of ChP and tanycytes markers in the GFP + fraction of spiked Ctx and Cer samples ultimately confirm that our Mct8-CreERT2 line combined with Sun1-sfGFP reporter mice is a useful model to enrich Mct8-expressing nuclei using FANS. Combined with downstream applications such as single nucleus RNA sequencing, this may one day help to fully elucidate the identity and function of Mct8-expressing cells in the brain and peripheral organs. Likewise, Mct8-CreERT2 mice can be used to generate conditional gene knock-out or overexpression models specifically in Mct8-expressing cells, to study their physiological relevance in TH biology in those cells. To conclude, our new mouse model is a valuable genetic tool that can facilitate the study of thyroid hormone biology and its transport mechanisms, ultimately providing crucial insights into the physiological roles of Mct8-expressing cells. Methods Animals Animal experiments were conducted in compliance with European Union Directive 2010/63/EU and local regulations for the care and use of laboratory animals and approved by the animal ethics committee of the State of Bavaria, Germany; the study is reported in accordance with ARRIVE guidelines. SPF-mice were housed in IVC cages and maintained on a 12h-dark-light cycle with free access to chow diet and water. Only male mice were assessed in this study, as MCT8-deficiency only affects male patients. Reporter lines were acquired from The Jackson Laboratory (Sun1-sfGFP: JAX #021039, Ai14: JAX #007914). Mct8-Cre and Mct8-CreERT2 mice were generated in house on a C57BL/6N background and are available upon request. Mouse line generation Zygotes were injected with Cas9 protein, guide RNA targeting the Slc16a2 start codon (Supplemental Table 2), and the DNA repair template with iCre-T2A or iCreERT2-T2A sequences. Micro-injected zygotes were transplanted into pseudo-pregnant females. Correct insertions were tested by PCR and Sanger sequencing in F0 offspring, and germline transmission confirmed in F1 mice (Supplemental File 2). Primers used for genotyping are indicated in Fig. 1a and listed in Supplemental Table 2, and the resulting bands are indicated in Figure 1b. Tamoxifen application Mice received 100 µL tamoxifen (TAM) in sunflower seed oil at 10 mg/mL, or oil as control, intraperitoneally (i.p.) for 1, 3, or 5 consecutive days, respectively. Mice that received TAM or VEH were housed in cages separated by treatment to prevent unintended induction. For oral gavage, mice received 200 µL of 10 mg/mL TAM in oil. All mice dosed with TAM or VEH received 5 mg/kg of the non-steroidal anti-inflammatory meloxicam (subcutaneously). Tissues extraction Mice were sacrificed by cervical dislocation, brains excised, and the MBH, 4V ChP, cerebellum, LV ChP, and cortex dissected and frozen in liquid nitrogen. For fluorescence imaging, mice were anesthetized using ketamine/xylazine and perfused through the heart with ice-cold PBS followed by 4% paraformaldehyde (PFA; 4% w/v, pH 7.4, Morphisto, 11762.01000). Organs were postfixed in 4% PFA for 24h and stored in PBS with 0.05% sodium azide at 4°C. For vDISCO, animals were perfused with PBS containing heparin (25 U/ml, Ratiopharm, N68542.03) for 5-10 min and 4% PFA. After skinning, gut cleaning and rinsing in PBS, mice were post-fixed for 24h in 4% PFA and stored in PBS with 0.05% sodium azide at 4°C. Fluorescence-activated nuclei sorting (FANS) MBH, Ctx with LV ChP, or Cer with 4V ChP were transferred to a Dounce-homogenizer containing 700 µL (MBH) or 3 mL of ice-cold nuclei isolation buffer (25 mM sucrose, 25 mM KCl, 5 mM MgCl 2 , 20 mM Tris pH 8.0, 0.4% IGEPAL 630, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, 1x phosphatase & protease inhibitor tablet, 0.4 units RNasin Plus RNase Inhibitor, 0.2 units SuperAsin RNase inhibitor) [37]. Samples were homogenized by 10 pestle strokes with a looser pestle, 5 min incubation on ice, and 20 strokes with a tighter pestle. After filtering through a 20 µm cell strainer and pelleting by centrifugation at 1000g for 10 min at 4°C, 500 µL of staining buffer (RNAse-free PBS pH 7.4, 0.15 mM spermine, 0.5 mM spermidine, 0.4 units RNasin Plus RNase Inhibitor, 1.5% RNAse-free BSA, 1 µg/µL DAPI) were used for resuspension and samples were subjected to sorting on a FACS-Aria III (BD Biosciences) into 350 µL RLT buffer + DTT, then frozen. RNA extraction and qPCR Tissues were homogenized in 500 µL (MBH, ChP) or 1 mL (Ctx, Cer) Quiazol using a Tissue Lyser II for 3 min at 30/sec. 100 µL or 200 µL chloroform were added after 5 min at RT, followed by shaking, 3 min of incubation and centrifugation at 12000 x g for 15 min at 4°C. RNA was isolated from supernatants using the RNeasy Micro Kit (QIAGEN GmbH 74004; MBH, ChP) or NucleoSpin RNA isolation kit (740955, Machery-Nagel; Ctx, Cer) following the manufacturer’s instructions. Reverse transcriptions were performed using the QuantiTect® Reverse Transcription Kit (205311, QIAGEN). Sorted nuclei were mixed with 350 µl of 70% EtOH and transferred to columns of the RNeasy Micro Kit for RNA extraction, following the manufacturer’s instructions. cDNA was synthesized using the SMART-Seq V4 Ultra® Low Input RNA kit (634888, Takara Bio; 11 cycles). qPCRs were performed using the SYBR® Green PCR Master Mix (Applied Biosystems™) and specific primers (Supplemental Table 2) in a QuantStudio 7 Flex Real Time PCR System (Applied Biosystems™). Immunofluorescent staining Perfused and post-fixed organs, saturated with 30% sucrose in Tris-buffered saline (TBS, pH 7.2) for 48h, were sectioned into 16-20 μm slices using a cryostat and mounted on glass slides. 30 µm brain slices were stained free-floating. After washing with TBS, permeabilization and blocking using SUMI (0.25% (w/v) porcine gelatin and 0.5% (v/v) TritonX100 in TBS) at RT for 1-2h, primary antibodies (Supplemental Table 3) in SUMI were incubated overnight at 4°C. After washing with TBS, secondary antibodies (Supplemental Table 3) in SUMI with DAPI were incubated for 2h at RT. After washing and mounting, slides were sealed using Elvanol (150 mM Tris, 12% Mowiol 4-88, 2% DABCO) and imaged on a Leica SP5 confocal microscope or Zeiss Axio Scan 7 slidescanner. vDISCO Perfused and fixed mice were placed in a glass chamber for washing, decolorization, decalcification, permeabilization, staining, and clearing according to the published methods [38-40]. Sun1-sfGFP reporter detection was enhanced using the GFP-Booster Alexa-Fluor®-647 nanobody by ChromoTek (gb2AF647). Nuclei were stained with propidium iodide (PI; Sigma-Aldrich, P4864). Light Sheet Fluorescence Microscopy (LSFM) images were acquired using an UltraMicroscope Blaze Imaging System and ImSpector imaging software from Miltenyi. Images were analyzed and processed using Imaris (Oxford Instruments) and syGlass Inc. virtual-reality software. Statistical analysis Values in bar graphs are plotted as mean with SD if not stated differently. Statistical analysis was done using Graphpad Prism 8. No values were excluded. Assessments were made unblinded in regards to genotype and treatment, but were reported in full faith. Declarations Acknowledgments We thank Miriam Krekel, Balma García Colomer, and Alina Blenninger for technical assistance and assistance with animal studies. Author contributions A.M., N.M., S.C.S., and M.Be. conducted the murine studies; N.M. additionally performed FANS. M.Br., L.J.H., and A.E. carried out tissue clearing and imaging using vDISCO and LSFM. A.C.-S. generated the Mct8-CreERT2 mouse line. E.P. conducted the re-analysis of publicly available single-nucleus RNA sequencing data. The study was conceptualized and designed by A.M., S.C.S., T.D.M., and P.T.P. The manuscript was written by A.M. and P.T.P., with contributions and critical revisions from all authors. Data availability statement The data collected from this study are available from the corresponding authors upon request. Additional information The authors declare that they have no competing interests. Funding Anna Molenaar was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-ID 424957847 – TRR 296. Noémi Mallet declares no funding. Marin Bralo was funded by the European Research Council Consolidator grant (no. GA 865323) and a Nomis Heart Atlas project grant (Nomis Foundation). Luciano Jan Hoeher was funded by the European Research Council Consolidator grant (no. GA 865323) and a Nomis Heart Atlas project grant (Nomis Foundation). Sonja Charlotte Schriever declares no funding. Ekta Pathak declares no funding. Miriam Bernecker received funding from the Helmholtz International Research School for Diabetes graduate program. Timo Dirk Müller was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-ID 424957847 – TRR 296. Ali Ertürk was funded by the European Research Council Consolidator grant (no. GA 865323) and a Nomis Heart Atlas project grant (Nomis Foundation). Alberto Cebrian-Serrano declares no funding. Paul Thomas Pfluger was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-ID 424957847 – TRR 296, and by the European Research Council ERC-CoG grant Yoyo-LepReSens (no. 101002247). References E. C. H. Friesema, S. Ganguly, A. Abdalla, J. E. Manning Fox, A. P. Halestrap, and T. J. 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Krishnaswami et al. , “Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons,” Nature Protocols 2016 11:3 , vol. 11, no. 3, pp. 499–524, Feb. 2016, doi: 10.1038/nprot.2016.015. R. Cai et al. , “Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull–meninges connections,” Nature Neuroscience 2018 22:2 , vol. 22, no. 2, pp. 317–327, Dec. 2018, doi: 10.1038/s41593-018-0301-3. R. Cai et al. , “Whole-mouse clearing and imaging at the cellular level with vDISCO,” Nature Protocols 2023 18:4 , vol. 18, no. 4, pp. 1197–1242, Jan. 2023, doi: 10.1038/s41596-022-00788-2. C. Pan et al. , “Shrinkage-mediated imaging of entire organs and organisms using uDISCO,” Nature Methods 2016 13:10 , vol. 13, no. 10, pp. 859–867, Aug. 2016, doi: 10.1038/nmeth.3964. Additional Declarations No competing interests reported. Supplementary Files Mct8CreERT2supplementalfiguresandtables.docx Cite Share Download PDF Status: Posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6796634","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":478486702,"identity":"698ceaf0-84dc-43c5-96f5-8d55fa594c44","order_by":0,"name":"Anna Molenaar","email":"","orcid":"","institution":"Helmholtz Munich","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Molenaar","suffix":""},{"id":478486703,"identity":"d7421a1c-04de-40cd-9178-8571abf5df1b","order_by":1,"name":"Noémi Mallet","email":"","orcid":"","institution":"Helmholtz Munich","correspondingAuthor":false,"prefix":"","firstName":"Noémi","middleName":"","lastName":"Mallet","suffix":""},{"id":478486704,"identity":"5615e55c-4c54-4606-af27-fe213511f3a0","order_by":2,"name":"Marin Bralo","email":"","orcid":"","institution":"Helmholtz Munich","correspondingAuthor":false,"prefix":"","firstName":"Marin","middleName":"","lastName":"Bralo","suffix":""},{"id":478486705,"identity":"ab818910-e0ca-4bd5-837d-0e91edc7fe92","order_by":3,"name":"Luciano Jan Hoeher","email":"","orcid":"","institution":"Helmholtz Munich","correspondingAuthor":false,"prefix":"","firstName":"Luciano","middleName":"Jan","lastName":"Hoeher","suffix":""},{"id":478486706,"identity":"58586f74-41ed-4b68-ba39-cc3328cd2c74","order_by":4,"name":"Sonja Charlotte Schriever","email":"","orcid":"","institution":"Helmholtz Munich","correspondingAuthor":false,"prefix":"","firstName":"Sonja","middleName":"Charlotte","lastName":"Schriever","suffix":""},{"id":478486707,"identity":"ffa162ee-8a33-435c-974a-4a4e75abf477","order_by":5,"name":"Ekta Pathak","email":"","orcid":"","institution":"Helmholtz Munich","correspondingAuthor":false,"prefix":"","firstName":"Ekta","middleName":"","lastName":"Pathak","suffix":""},{"id":478486709,"identity":"57c02536-c043-40d8-8026-26026b214050","order_by":6,"name":"Miriam Bernecker","email":"","orcid":"","institution":"Helmholtz Munich","correspondingAuthor":false,"prefix":"","firstName":"Miriam","middleName":"","lastName":"Bernecker","suffix":""},{"id":478486710,"identity":"ef40f308-ae4a-4f42-b109-be6685fb5af6","order_by":7,"name":"Timo D. Müller","email":"","orcid":"","institution":"Helmholtz Munich","correspondingAuthor":false,"prefix":"","firstName":"Timo","middleName":"D.","lastName":"Müller","suffix":""},{"id":478486711,"identity":"1838cd22-9e19-4099-86aa-6e4e0959b0d7","order_by":8,"name":"Ali Ertürk","email":"","orcid":"","institution":"Helmholtz Munich","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Ertürk","suffix":""},{"id":478486712,"identity":"4715762d-cf60-4043-a297-539370afa598","order_by":9,"name":"Alberto Cebrian-Serrano","email":"","orcid":"","institution":"Helmholtz Munich","correspondingAuthor":false,"prefix":"","firstName":"Alberto","middleName":"","lastName":"Cebrian-Serrano","suffix":""},{"id":478486713,"identity":"db715ca3-688c-427d-b94e-4d445221749a","order_by":10,"name":"Paul Thomas Pfluger","email":"data:image/png;base64,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","orcid":"","institution":"Helmholtz Munich","correspondingAuthor":true,"prefix":"","firstName":"Paul","middleName":"Thomas","lastName":"Pfluger","suffix":""}],"badges":[],"createdAt":"2025-06-01 15:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6796634/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6796634/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85819572,"identity":"16d3bb5c-53cb-472a-80e0-25e9eace4571","added_by":"auto","created_at":"2025-07-02 06:20:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2947328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration and characterization of transgenic Mct8-CreERT2 knock-in mice. \u003c/strong\u003e(a)\u003cstrong\u003e \u003c/strong\u003eCRISPR-Cas9 guided insertion of \u003cem\u003eiCreERT2\u003c/em\u003e and \u003cem\u003eT2A\u003c/em\u003e sequences at the start codon of the Mct8 gene. (b) Representative gel of genotyping results using primers (see Supplemental Table 2) to discriminate the hemizygous (Hemi) male or homozygous (Hom) female mice from heterozygous (Het) females and from the WT mice, using primers indicated in (a) in blue. (c) Ratios of offspring from the mating of hemizygous (Hemi, +/y) males with heterozygous (Het, +/-) females (left) or (d) Hemi males with homozygous (Hom, +/+) females (right). Litters of 7 females for each type of mating were assessed, with a total of 60 and 89 pups, respectively. “+” indicates the mutant allele, “-” the WT allele on the X-chromosome. (e) Relative gene expression levels of Mct8 in the mediobasal hypothalamus (MBH), ChP of the 4\u003csup\u003eth\u003c/sup\u003e ventricle (4V ChP) and lateral ventricle (LV ChP), cerebellum (Cer) and cortex (Ctx) of WT and Mct8-CreERT2 mice, quantified using qPCR and the ddCT method relative to \u003cem\u003eMalat1\u003c/em\u003e. (f) Immunofluorescent staining for Mct8 using the Novus antibody in hypothalamic slices of WT and Mct8-CreERT2 mice. (e) Statistical analysis using multiple t-tests with Holm-Sidak correction, N = 5 per genotype. (f) Scalebar = 100 µm.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6796634/v1/a39e939775d63f798005b955.png"},{"id":85819577,"identity":"a56c3e65-678e-4dfd-9a47-4702761e6ce7","added_by":"auto","created_at":"2025-07-02 06:20:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12953032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMct8-CreERT2 driven tdTomato signal in adult mouse brains. \u003c/strong\u003e(a) Comparison of the tdTomato pattern (left) and Mct8 antibody staining (right; Novus) in an Mct8-CreERT2;Ai14\u003csup\u003e \u003c/sup\u003emouse brain slice. Areas magnified in (b) are marked with boxes; lines included for anatomical guidance. (b) Magnifications of the reporter expression (top), the Mct8 antibody staining (middle) and merge of both (bottom) for the hippocampus, ChP, cortex, and tanycytes. (a) Scalebar = 500 µm. (b) Scalebar = 100\u0026nbsp;µm.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6796634/v1/378031dafd6685381bc874aa.png"},{"id":85819575,"identity":"7ec08c57-da3a-4919-b7d4-08a6b12b1df2","added_by":"auto","created_at":"2025-07-02 06:20:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9399283,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWhole body imaging of tamoxifen-induced Mct8-CreERT2;Sun1-sfGFP mouse using vDISCO and nanobodies directed against GFP.\u003c/strong\u003e (a) Light Sheet Fluorescence Microscopy (LSFM) of an entire mouse at 4x magnification. LSFM of the (b) head region, showing strong fluorescence in the ventricular ChP, and (c) in a section through liver and kidney. (d) LSFM of the neck region showing strong fluorescence in the sublingual and parotid but not submandibular salivary gland, and in the thyroid gland. (e) LSFM of the lower body showing strong fluorescence in the anterior prostate and cauda epididymis. ChP: choroid plexus, Li: liver, Ki: kidney, SL: sublingual salivary gland, TG: thyroid gland, Pa: parotid gland, SM: submandibular salivary gland, AP: anterior prostate, caE: cauda epididymis. Purple: propidium iodide (PI), white: autofluorescence. (a-c) combination of all three channels (nanobody-enhanced Sun1-sfGFP, PI, autofluorescence), (d,e) top: combination of all three channels, bottom: channel for nanobody-enhanced Sun1-sfGFP reporter detection.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6796634/v1/646e8d69fa03ed264cb1adb4.png"},{"id":85819573,"identity":"5acfbf1f-a177-4f4b-8a56-7816c4c5d500","added_by":"auto","created_at":"2025-07-02 06:20:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5460790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStrong fluorescent reporter expression in prostate and salivary glands of Mct8-CreERT2;Ai14 mice.\u003c/strong\u003e (a) Cryo-slices of tissues from Mct8-CreERT2;Ai14 mice injected for 5 days with TAM were assessed for fluorescence signals in the prostate (top) including seminal vesicles, and the salivary glands (bottom). (b) Zoom on areas marked with white square in (A). (c) Cryo-slices of Mct8-CreERT2;Ai14 mice injected for 5 days with vehicle (VEH). SV: seminal vesicles, AP: anterior prostate, Ur: urethra, Pa: parotid gland, LN: lymph node, SL: sublingual salivary gland, SM: submandibular salivary gland. Grey: DAPI, red: tdTomato. Scalebar = 1 mm. Exposure time of 555 laser: 0.7 ms.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6796634/v1/3e72dc7eea915830775aa0e5.png"},{"id":85819576,"identity":"3f2694e5-d717-473e-b1f9-50409d0ec097","added_by":"auto","created_at":"2025-07-02 06:20:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11192961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluorescent reporter expression in Mct8-expressing organs of Mct8-CreERT2;Ai14 mice.\u003c/strong\u003e (a) Cryo-slices of tamoxifen-induced Mct8-CreERT2;Ai14 organs, from top to bottom: heart, lung, quadriceps including femur (Fe), testis (Te) including caput epididymis (ctE). (b) Zoom on areas marked with white square in (a). (c) Cryo-slices of Mct8-CreERT2;Ai14 mice injected for 5 days with vehicle (VEH). Scalebar = 1 mm. Exposure time of 555 laser: 20 ms.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6796634/v1/3439fd398d0c64dadbbb7837.png"},{"id":85819574,"identity":"60544468-e7de-4024-8ef6-2292b56c39e4","added_by":"auto","created_at":"2025-07-02 06:20:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1015592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluorescence-activated nuclei sorting (FANS) of GFP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e nuclei from Mct8-CreERT2;Sun1-sfGFP brain areas.\u003c/strong\u003e (a) Scheme of tissue dissection. The whole Cer and MBH and part of the Ctx were extracted along the dashed lines. Before Cer and Ctx were taken, the 4V ChP and LV ChP were isolated with tweezers and added to the Cer and Ctx collection tubes, respectively. The samples were then subjected to FANS to collect 1000 nuclei per GFP\u003csup\u003e+\u003c/sup\u003e and GFP\u003csup\u003e-\u003c/sup\u003e fraction, respectively. (b) qPCR for Mct8 mRNA (\u003cem\u003eSlc16a2\u003c/em\u003e) levels in GFP\u003csup\u003e+\u003c/sup\u003e and GFP\u003csup\u003e-\u003c/sup\u003e fractions of the MBH, Ctx + LV ChP, and Cer + 4V ChP. (c,d) ChP marker transthyretin (\u003cem\u003eTtr\u003c/em\u003e) mRNA levels in the Ctx + LV ChP and Cer + 4V ChP samples. (d) Tanycyte marker µ-crystallin (\u003cem\u003eCrym\u003c/em\u003e) mRNA levels in the MBH. qPCR analyses were done using the ddCT method relative to \u003cem\u003eMalat1\u003c/em\u003e, relative to a GFP\u003csup\u003e-\u003c/sup\u003e reference sample per tissue type. Statistical analyses were done using multiple t-tests with Holm-Sidak correction. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001. N = 3 per genotype.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6796634/v1/34f784a9826bf63c2d2d2898.png"},{"id":88880552,"identity":"f3988aba-25f6-4c56-848a-31489a46bed2","added_by":"auto","created_at":"2025-08-12 11:02:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":39270871,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6796634/v1/6f6b4366-c08b-4f5f-8484-9c8c5c76f63e.pdf"},{"id":85819578,"identity":"d8115bb6-002e-4081-9a20-68bcc3efcfb5","added_by":"auto","created_at":"2025-07-02 06:20:43","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":41594861,"visible":true,"origin":"","legend":"","description":"","filename":"Mct8CreERT2supplementalfiguresandtables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6796634/v1/a9c17d297cd7411e8d366910.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Novel Tamoxifen-Inducible Mct8-CreERT2 Mouse Model for Targeted Studies of Mct8-Expressing Cells and Thyroid Hormone Transport and Function","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe monocarboxylate transporter 8 (MCT8) is an influx and efflux transmembrane transporter for thyroid hormones (TH) thyroxine\u0026nbsp;(T\u003csub\u003e4\u003c/sub\u003e) and 3,3′,5-triiodothyronine (T\u003csub\u003e3\u003c/sub\u003e), and their metabolites 3,3′,5′-triiodothyronine (rT\u003csub\u003e3\u003c/sub\u003e) and 3,3′-diiodothyronine\u0026nbsp;[1].\u0026nbsp;Its role in TH transport is crucial for brain TH availability,\u0026nbsp;evidenced by the devastating neurological impairments of Allan-Herndon-Dudley-Syndrome (AHDS) patients with mutations in the MCT8-encoding \u003cem\u003esolute carrier family member 16a2\u003c/em\u003e (\u003cem\u003eSlc16a2\u003c/em\u003e) gene on the X-chromosome\u0026nbsp;[2,3]. Deficient TH transport during crucial stages of brain development causes intellectual disability, motor dysfunctions, and impaired myelination. Reduced TH brain levels are thereby contrasted by elevated T3 plasma levels, causing peripheral thyrotoxicosis [4].\u003c/p\u003e\n\u003cp\u003eTo understand TH transport and its associated diseases, it is important to elucidate the expression pattern of MCT8 in the brain as well as other organs across various developmental stages. \u0026nbsp;Western Blotting found highest levels in the human liver, followed by pituitary and brain, low signals for heart and placenta, and near absence in the lung [5]. MCT8-mapping to the human brain, thyroid, and pituitary was moreover confirmed using immunohistochemistry (IHC) and immunofluorescence (IF) [5-10].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMurine \u003cem\u003eSlc16a2\u003c/em\u003e mRNA levels are high in liver and kidney, low in lung, cerebral cortex, and heart, and barely detectable in testis (Mouse ENCODE transcriptome project PRJNA66167) [11]. Mct8 protein was localized in rodent livers, thyroid glands, kidneys, brains, and to a lesser extent rat hearts [1,9,12-14](Supplemental Table 1). IHC and IF in adult human brains revealed abundant MCT8 levels in barrier cells (endothelial cells (EC), astrocytes, choroid plexus (ChP), tanycytes), clearly marking blood vessels and capillaries, but sparse neuronal staining [6-8]. Neuronal MCT8 signal was readily detectable [5] and human cortical organoids [15].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn murine brains, Mct8 protein was abundant in BV, tanycytes, and ChP, while neuronal expression declined from postnatal day 12 (P12) to P21, until undetectable [13]. Others found Mct8 in the adult mouse hippocampus and Purkinje cells and, diffusely, in cortical neurons [5]. Discrepancies in Mct8 detection are likely due to differences in antibodies and lots used, highlighting the need for alternative visualization methods due to challenges in acquiring effective antibodies and inconsistent neuronal staining\u0026nbsp;[13]. We here explored the use of the Cre-lox system, where expression of Cre recombinase is driven by the endogenous Mct8 promoter, and crossed these with fluorescent reporter mice to identify, visualize and characterize Mct8-expressing cells in vDISCO tissue cleared and 3D imaged whole mice and isolated organs.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eGeneration of tamoxifen-inducible Mct8-CreERT2 mice\u003c/h2\u003e\n\u003cp\u003ePrompted by discrepant studies on the presence or absence of neural Mct8 protein expression in adult mice (Supplemental Table 1) and our own comparison of commercially available antibodies, which revealed unspecific or insufficient immunofluorescence signals for Mct8 in murine brain slices (Supplemental Fig. 1), we aimed to generate a novel Mct8-CreERT2 mouse line to ultimately assess Mct8 expression patterns in adult mice. Mct8-CreERT2 mice were generated by inserting the sequences of \u003cem\u003eiCreERT2\u0026nbsp;\u003c/em\u003eand “self-cleaving” peptide \u003cem\u003eT2A\u0026nbsp;\u003c/em\u003eat the start codon of Mct8-gene \u003cem\u003eSlc16a2\u003c/em\u003e using CRISPR/Cas9 (Fig. 1a) [16-20], generating viable mice at the expected Mendelian ratio and with the correct genotypes (Fig. 1b,c,d). The transgenic mice had comparable levels of Mct8 mRNA in WT male and Mct8-CreERT2\u003csup\u003e+/y\u003c/sup\u003e brain areas (Fig. 1e), with highest levels in the ChP of the 4th ventricle (4V ChP) and lateral ventricle (LV ChP), followed by the mediobasal hypothalamus (MBH), then cortex (Ctx) and cerebellum (Cer), consistent with published Mct8 gene expression levels [21]. Likewise, we observed unperturbed endogenous Mct8 protein expression, with comparable fluorescence patterns and intensities in antibody-stained hypothalamic slices of WT and Mct8-CreERT2 mice (Fig. 1f).\u003c/p\u003e\n\u003ch2\u003eUbiquitous recombination in the CNS of non-inducible Mct8-Cre mice\u003c/h2\u003e\n\u003cp\u003eUsing the same CRISPR-Cas9-based knock-in strategy, we explored germline Mct8 expression patterns using non-inducible Mct8-Cre mice. In the fully viable and fertile Mct8-Cre mice crossed with the fluorescent reporter mouse line Sun1-sfGFP, which expresses the GFP-tagged nuclear lamina protein Sun1, we found ubiquitous reporter expression in adult mouse brains (Supplemental Fig. 2). We next investigated whether this ubiquitous pattern could be due to a very early activation of Mct8-Cre expression and subsequent stop cassette excision in all cells of that lineage. Blastocysts (corresponding to embryonic day E4.5) from the mating of Mct8-Cre with Ai14 mice, known for their strong cytosolic fluorescence, showed no reporter signal, while Mct8-Cre;Sun1-sfGFP embryos at stages E10.5 had reporter expression in virtually all cells (Supplemental Fig. 3 a,b). By analyzing publicly available RNA sequencing data across murine embryonal developmental stages, we identified a peak for the expression of Mct8 at day E5.25, i.e. the stage of implantation directly after the blastocyst stage, to likely drive fluorescent reporter activation (Supplemental Fig. 3c).\u003c/p\u003e\n\u003ch2\u003eValidation of CreERT2 recombinase activity in Mct8-expressing cells in the brain\u003c/h2\u003e\n\u003cp\u003eTo validate the Mct8-CreERT2 mouse line’s specificity, we crossed it with the fluorescent Ai14 and Sun1-sfGFP reporter mouse lines and assessed CreERT2 activity one week after the indicated tamoxifen (TAM) applications. In brains of tamoxifen-injected Mct8-CreERT2;Ai14 and Mct8-CreERT2;Sun1-sfGFP mice, we found profound fluorescence reporter expression in tanycytes and ChP that largely overlapped with Mct8 antibody staining (Fig. 2 a,b; Supplemental Figs. 4 a-c \u0026amp; 5). Fluorescence was moreover found in BV and few neurons spattered across the hippocampus, cortex, and striatum. Neither blood vessels nor sparse neurons were labeled using antibody staining. Vehicle-injected Mct8-CreERT2\u003csup\u003e+\u003c/sup\u003e and Mct8-CreERT2\u003csup\u003e-\u003c/sup\u003e mice that received TAM showed minimal Cre-activity, confirming the highly specific, inducible expression without background activity for both reporter lines.\u003c/p\u003e\n\u003cp\u003eLast, we found comparable recombination efficacies in response to a single i.p. or oral gavage (o.g.) application of tamoxifen. However, reporter expression was profoundly diminished compared to the mice injected with three i.p. doses of TAM (Supplemental Fig. 4 a,b). Overall, these data show that the new Mct8-CreERT2 line is functional and faithfully induces reporter expression in cells known to express Mct8 in adult mice after three doses of 50 mg/kg TAM.\u003c/p\u003e\n\u003ch2\u003eMct8-CreERT2-driven reporter expression in mice\u003c/h2\u003e\n\u003cp\u003eAfter validating the correct reporter expression in mouse brains, we aimed to systematically assess all other Mct8-expressing tissues. We subjected Mct8-CreERT2;Sun1-sfGFP mice to vDISCO, the solvent-clearing of organs and nanobody-based immune staining of GFP-marked nuclei, followed by LSFM. The whole-body scan is visualized in Fig. 3a, videos of the head, torso and whole-body are provided as Supplemental Material (Supplemental Videos 1-3). Strongest fluorescent signals could be localized to the choroid plexus (Fig. 3b), liver and kidney (Fig. 3c). Figure 3d (upper panel) depicts the distinctly labeled sublingual and parotid salivary glands and the thyroid gland. We further observed strong fluorescence in the prostate, as well as the cauda of the epididymis (Fig. 3e)\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIntrigued by the detection of reporter signal in the salivary glands and prostate via vDISCO, we examined those tissues in more detail using Mct8-CreERT2;Ai14 mice and fluorescence imaging of cryo-slices. We also included organs that were reported to have \u003cem\u003eSlc16a2\u003c/em\u003e transcripts but showed no or weak reporter expression in the whole mouse scan, namely heart, lung, testis, and skeletal muscle. Of note, we also assessed whether 5 doses of TAM could augment Cre-activity to maximize reporter expression compared to the 3x TAM-treated mice but found no differences in fluorescence intensities (Supplemental Fig. 4c). In the prostate, we found strong fluorescence in cells of the anterior prostate and weak fluorescence in seminal vesicles (Fig. 4 top). In the salivary glands, reporter expression was strongest in the sublingual, modest in the parotid, and near-absent in the submandibular gland (Fig. 4 bottom).\u003c/p\u003e\n\u003cp\u003eHeart, lung, testis, and quadriceps demonstrated sporadic reporter activation, including signals from the femur and caput epididymis (Fig. 5). However, exposure times had to be approximately 30x higher than those necessary for the salivary gland and prostate, indicating a sparsely distributed and comparably lower Mct8 expression in those tissues.\u003c/p\u003e\n\u003ch2\u003eEnriching nuclei of Mct8-expressing cells from Mct8-CreERT2;Sun1-sfGFP mice using FANS\u003c/h2\u003e\n\u003cp\u003eFinally, we assessed whether the line can be utilized for the enrichment of MCT8-expressing cells from brain tissues of Mct8-CreERT2;Sun1-sfGFP mice by fluorescence-activated nuclei sorting (FANS) of Mct8-positive nuclei tagged with Sun1-sfGFP. 1000 nuclei of the MBH, Ctx including the ChP of the lateral ventricle (LV ChP), and Cer – including the ChP of the 4\u003csup\u003eth\u003c/sup\u003e ventricle (4V ChP) were sorted into GFP-positive and -negative fractions, respectively (Fig. 6a). An exemplary gating for GFP\u003csup\u003e+\u003c/sup\u003e nuclei is shown in Supplemental Fig. 6. All GFP\u003csup\u003e+\u003c/sup\u003e fractions showed strong Mct8 mRNA enrichment compared to the GFP\u003csup\u003e-\u003c/sup\u003e fractions (Fig. 6b). qPCR for ChP marker transthyretin(\u003cem\u003eTtr\u003c/em\u003e) and tanycyte marker µ-crystallin (\u003cem\u003eCrym\u003c/em\u003e) showed enrichment of ChP- and tanycyte-derived nuclei in the GFP\u003csup\u003e+\u003c/sup\u003e fractions of the Ctx + LV ChP, Cer + 4V ChP, and MBH, respectively (Fig. 6c).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur new tamoxifen-inducible Mct8-CreERT2 mouse line allows specific recombination in all Mct8-expressing cells throughout the body with high specificity and minimal leakiness. By inserting the iCreERT2 cassette directly into the endogenous Mct8 locus, we preserve physiological Mct8 expression while enabling controlled iCreERT2 activity. This knock-in strategy offers a clear advantage over traditional approaches relying on bacterial artificial chromosomes (BACs) transgenesis or random transgene integration, which frequently cause unpredictable expression patterns, position effects, gene silencing, or disruption of endogenous loci [22-24]. Such artifacts can introduce major confounders and compromise the physiological relevance of experimental models. In contrast, our targeted approach faithfully recapitulates endogenous Mct8 regulation, minimizing off-target effects and preserving the native biology of the system.\u003c/p\u003e\n\u003cp\u003eThe non-inducible Mct8-Cre line showed ubiquitous reporter expression due to the early Mct8 expression peak at day E5.25 of embryonal development. This indicates an important, vaguely understood role of thyroid hormone transport during nidation when embryonal trophoblast cells invade the mother’s endometrium to form the placenta. Notably, Chan et al. [25] found that MCT8 expression is increased in the placentas of humans with intrauterine growth restriction, to potentially increase T3 uptake, and embryos of Mct8-KO rats showed a decreased embryo-to-placental weight ratio. These effects were nonetheless mild, and little evidence pointed toward an impaired trophoblast invasion capability in the KO rats [26]. This may be due to organic anion transporting polypeptide 1c1 (OATP1C1), a transporter for T3 and T4 co-expressed in the rat placental barrier that can partially compensate for the lack of Mct8 in rats and mice but not in humans [27]. Future studies are thus warranted to elucidate the specific roles of Mct8 and OATP1C1 in trophoblast invasion and placental development.\u003c/p\u003e\n\u003cp\u003eIn the brains of tamoxifen-induced Mct8-CreERT2 fluorescent reporter mice, we found strong and specific fluorescence in tanycytes and ChP one week after induction, confirmed by Mct8 antibody staining. Additionally, we found reporter signal in blood vessels and, scarcely, in neurons of the hippocampus, cortex, and striatum. Using vDISCO, we could further detect strong reporter signal in liver, thyroid gland, and kidney[1,12,28]. The Encyclopedia of DNA Elements (ENCODE) database reports very low \u003cem\u003eSlc16a2\u003c/em\u003e transcripts in mouse lung, heart, and testes, compared to strongly expressing tissues [11]. Similarly, \u003cem\u003eSlc16a2\u0026nbsp;\u003c/em\u003eexpression was reported for various muscle cells using microarrays, but protein staining was only reported for satellite cells [29]. In our Mct8-CreERT2 reporter mice, fluorescent signals in such tissues and organs with low gene expression were weak, but detectable at very high exposure time. Our mouse model is thus a suitable tool to target both organs with a high number of Mct8-expressing cells as well as organs with a low abundance of Mct8-expressing cells.\u003c/p\u003e\n\u003cp\u003eIn addition, we found profound reporter signal in organs where a role for Mct8 was not previously described, the sublingual salivary gland and the prostate. Salivation is influenced by TH both in rodents and in humans [30-32], and TH is found in saliva [33]. Our data are consistent with those reports, and highlight that the sublingual and, to a lesser extent, the parotid salivary glands are involved in that salivary TH transport. The prostate secretes fluids that are added to the seminal fluid to facilitate sperm fertility [34]. Prostate fluids also contribute to the generation of copulatory plugs, which is essential for the successful fertilization in mice [35]. The role of Mct8 in male reproduction was already studied in Mct8-KO rats, where Mct8-staining was reported for the rat epididymis. That study explored the role of Mct8 in testes and epididymis for sperm viability and reported a decrease in fertility in male KO rats [36]. Our data highlight that such changes in fertility could also potentially be attributed to the lack of Mct8 in the prostate.\u003c/p\u003e\n\u003cp\u003eLast, the strong enrichment of Mct8, and the selective enrichment of ChP and tanycytes markers in the GFP\u003csup\u003e+\u003c/sup\u003e fraction of spiked Ctx and Cer samples ultimately confirm that our Mct8-CreERT2 line combined with Sun1-sfGFP reporter mice is a useful model to enrich Mct8-expressing nuclei using FANS. Combined with downstream applications such as single nucleus RNA sequencing, this may one day help to fully elucidate the identity and function of Mct8-expressing cells in the brain and peripheral organs. Likewise, Mct8-CreERT2 mice can be used to generate conditional gene knock-out or overexpression models specifically in Mct8-expressing cells, to study their physiological relevance in TH biology in those cells. To conclude, our new mouse model is a valuable genetic tool that can facilitate the study of thyroid hormone biology and its transport mechanisms, ultimately providing crucial insights into the physiological roles of Mct8-expressing cells.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eAnimals\u003c/h2\u003e\n\u003cp\u003eAnimal experiments were conducted in compliance with European Union Directive 2010/63/EU and local regulations for the care and use of laboratory animals and approved by the animal ethics committee of the State of Bavaria, Germany; the study is reported in accordance with ARRIVE guidelines. SPF-mice were housed in IVC cages and maintained on a 12h-dark-light cycle with free access to chow diet and water. Only male mice were assessed in this study, as MCT8-deficiency only affects male patients. Reporter lines were acquired from The Jackson Laboratory (Sun1-sfGFP: JAX #021039, Ai14: JAX #007914). Mct8-Cre and Mct8-CreERT2 mice were generated in house on a C57BL/6N background and are available upon request.\u003c/p\u003e\n\u003ch2\u003eMouse line generation\u003c/h2\u003e\n\u003cp\u003eZygotes were injected with Cas9 protein, guide RNA targeting the \u003cem\u003eSlc16a2\u0026nbsp;\u003c/em\u003estart codon (Supplemental Table 2), and the DNA repair template with iCre-T2A or iCreERT2-T2A sequences. Micro-injected zygotes were transplanted into pseudo-pregnant females. Correct insertions were tested by PCR and Sanger sequencing in F0 offspring, and germline transmission confirmed in F1 mice (Supplemental File 2). Primers used for genotyping are indicated in Fig. 1a and listed in Supplemental Table 2, and the resulting bands are indicated in Figure 1b.\u003c/p\u003e\n\u003ch2\u003eTamoxifen application\u003c/h2\u003e\n\u003cp\u003eMice received 100 µL tamoxifen (TAM) in sunflower seed oil at 10 mg/mL, or oil as control, intraperitoneally (i.p.) for 1, 3, or 5 consecutive days, respectively. Mice that received TAM or VEH were housed in cages separated by treatment to prevent unintended induction. For oral gavage, mice received 200 µL of 10 mg/mL TAM in oil. All mice dosed with TAM or VEH received 5 mg/kg of the non-steroidal anti-inflammatory meloxicam (subcutaneously).\u003c/p\u003e\n\u003ch2\u003eTissues extraction\u003c/h2\u003e\n\u003cp\u003eMice were sacrificed by cervical dislocation, brains excised, and the MBH, 4V ChP, cerebellum, LV ChP, and cortex dissected and frozen in liquid nitrogen. For fluorescence imaging, mice were anesthetized using ketamine/xylazine and perfused through the heart with ice-cold PBS followed by 4% paraformaldehyde (PFA; 4% w/v, pH 7.4, Morphisto, 11762.01000). Organs were postfixed in 4% PFA for 24h and stored in PBS with 0.05% sodium azide at 4°C. For vDISCO, animals were perfused with PBS containing heparin (25 U/ml, Ratiopharm, N68542.03) for 5-10 min and 4% PFA. After skinning, gut cleaning and rinsing in PBS, mice were post-fixed for 24h in 4% PFA and stored in PBS with 0.05% sodium azide at 4°C.\u003c/p\u003e\n\u003ch2\u003eFluorescence-activated nuclei sorting (FANS)\u003c/h2\u003e\n\u003cp\u003eMBH, Ctx with LV ChP, or Cer with 4V ChP were transferred to a Dounce-homogenizer containing 700 µL (MBH) or 3 mL of ice-cold nuclei isolation buffer (25 mM sucrose, 25 mM KCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 20 mM Tris pH 8.0, 0.4% IGEPAL 630, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, 1x phosphatase \u0026amp; protease inhibitor tablet, 0.4 units RNasin Plus RNase Inhibitor, 0.2 units SuperAsin RNase inhibitor) [37]. Samples were homogenized by 10 pestle strokes with a looser pestle, 5 min incubation on ice, and 20 strokes with a tighter pestle. After filtering through a 20 µm cell strainer and pelleting by centrifugation at 1000g for 10 min at 4°C, 500 µL of staining buffer (RNAse-free PBS pH 7.4, 0.15 mM spermine, 0.5 mM spermidine, 0.4 units RNasin Plus RNase Inhibitor, 1.5% RNAse-free BSA, 1 µg/µL DAPI) were used for resuspension and samples were subjected to sorting on a FACS-Aria III (BD Biosciences) into 350 µL RLT buffer + DTT, then frozen.\u003c/p\u003e\n\u003ch2\u003eRNA extraction and qPCR\u003c/h2\u003e\n\u003cp\u003eTissues were homogenized in 500 µL (MBH, ChP) or 1 mL (Ctx, Cer) Quiazol using a Tissue Lyser II for 3 min at 30/sec. 100 µL or 200 µL chloroform were added after 5 min at RT, followed by shaking, 3 min of incubation and centrifugation at 12000 x g for 15 min at 4°C. RNA was isolated from supernatants using the RNeasy Micro Kit (QIAGEN GmbH 74004; MBH, ChP) or NucleoSpin RNA isolation kit (740955, Machery-Nagel; Ctx, Cer) following the manufacturer’s instructions. Reverse transcriptions were performed using the QuantiTect® Reverse Transcription Kit (205311, QIAGEN).\u003c/p\u003e\n\u003cp\u003eSorted nuclei were mixed with 350 µl of 70% EtOH and transferred to columns of the RNeasy Micro Kit for RNA extraction, following the manufacturer’s instructions. cDNA was synthesized using the SMART-Seq V4 Ultra® Low Input RNA kit (634888, Takara Bio; 11 cycles).\u003c/p\u003e\n\u003cp\u003eqPCRs were performed using the SYBR® Green PCR Master Mix (Applied Biosystems™) and specific primers (Supplemental Table 2) in a QuantStudio 7 Flex Real Time PCR System (Applied Biosystems™).\u003c/p\u003e\n\u003ch2\u003eImmunofluorescent staining\u003c/h2\u003e\n\u003cp\u003ePerfused and post-fixed organs, saturated with 30% sucrose in Tris-buffered saline (TBS, pH 7.2) for 48h, were sectioned into 16-20 μm slices using a cryostat and mounted on glass slides. 30 µm brain slices were stained free-floating. After washing with TBS, permeabilization and blocking using SUMI (0.25% (w/v) porcine gelatin and 0.5% (v/v) TritonX100 in TBS) at RT for 1-2h, primary antibodies (Supplemental Table 3) in SUMI were incubated overnight at 4°C. After washing with TBS, secondary antibodies (Supplemental Table 3) in SUMI with DAPI were incubated for 2h at RT. After washing and mounting, slides were sealed using Elvanol (150 mM Tris, 12% Mowiol 4-88, 2% DABCO) and imaged on a Leica SP5 confocal microscope or Zeiss Axio Scan 7 slidescanner.\u003c/p\u003e\n\u003ch2\u003evDISCO\u003c/h2\u003e\n\u003cp\u003ePerfused and fixed mice were placed in a glass chamber for washing, decolorization, decalcification, permeabilization, staining, and clearing according to the published methods [38-40]. Sun1-sfGFP reporter detection was enhanced using the GFP-Booster Alexa-Fluor®-647 nanobody by ChromoTek (gb2AF647). Nuclei were stained with propidium iodide (PI; Sigma-Aldrich, P4864). Light Sheet Fluorescence Microscopy (LSFM) images were acquired using an UltraMicroscope Blaze Imaging System and ImSpector imaging software from Miltenyi. Images were analyzed and processed using Imaris (Oxford Instruments) and syGlass Inc. virtual-reality software.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eValues in bar graphs are plotted as mean with SD if not stated differently. Statistical analysis was done using Graphpad Prism 8. No values were excluded.\u003c/p\u003e\n\u003cp\u003eAssessments were made unblinded in regards to genotype and treatment, but were reported in full faith.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe thank Miriam Krekel,\u0026nbsp;Balma Garc\u0026iacute;a Colomer, and Alina Blenninger\u0026nbsp;for technical assistance and assistance with animal studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eA.M., N.M., S.C.S., and M.Be. conducted the murine studies; N.M. additionally performed FANS. M.Br., L.J.H., and A.E. carried out tissue clearing and imaging using vDISCO and LSFM. A.C.-S. generated the Mct8-CreERT2 mouse line. E.P. conducted the re-analysis of publicly available single-nucleus RNA sequencing data. The study was conceptualized and designed by A.M., S.C.S., T.D.M., and P.T.P. The manuscript was written by A.M. and P.T.P., with contributions and critical revisions from all authors.\u003c/p\u003e\n\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eThe data collected from this study are available from the corresponding authors upon request.\u003c/p\u003e\n\u003cp\u003eAdditional information\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eAnna Molenaar was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) \u0026ndash; Project-ID 424957847 \u0026ndash; TRR 296. No\u0026eacute;mi Mallet declares no funding. Marin Bralo was funded by the European Research Council Consolidator grant (no. GA 865323) and a Nomis Heart Atlas project grant (Nomis Foundation). Luciano Jan Hoeher was funded by the European Research Council Consolidator grant (no. GA 865323) and a Nomis Heart Atlas project grant (Nomis Foundation). Sonja Charlotte Schriever declares no funding. Ekta Pathak declares no funding. Miriam Bernecker received funding from the Helmholtz International Research School for Diabetes graduate program. Timo Dirk M\u0026uuml;ller was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) \u0026ndash; Project-ID 424957847 \u0026ndash; TRR 296. Ali Ert\u0026uuml;rk was funded by the European Research Council Consolidator grant (no. GA 865323) and a Nomis Heart Atlas project grant (Nomis Foundation). Alberto Cebrian-Serrano declares no funding. Paul Thomas Pfluger was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) \u0026ndash; Project-ID 424957847 \u0026ndash; TRR 296, and by the European Research Council ERC-CoG grant Yoyo-LepReSens (no. 101002247).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eE. C. H. Friesema, S. Ganguly, A. Abdalla, J. E. Manning Fox, A. P. Halestrap, and T. J. Visser, \u0026ldquo;Identification of Monocarboxylate Transporter 8 as a Specific Thyroid Hormone Transporter,\u0026rdquo; \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e, vol. 278, no. 41, pp. 40128\u0026ndash;40135, Oct. 2003, doi: 10.1074/JBC.M300909200.\u003c/li\u003e\n\u003cli\u003eA. M. Dumitrescu, X. H. Liao, T. B. Best, K. Brockmann, and S. Refetoff, \u0026ldquo;A Novel Syndrome Combining Thyroid and Neurological Abnormalities Is Associated with Mutations in a Monocarboxylate Transporter Gene,\u0026rdquo; \u003cem\u003eThe American Journal of Human Genetics\u003c/em\u003e, vol. 74, no. 1, pp. 168\u0026ndash;175, Jan. 2004, doi: 10.1086/380999.\u003c/li\u003e\n\u003cli\u003eE. C. 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Pan \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Shrinkage-mediated imaging of entire organs and organisms using uDISCO,\u0026rdquo; \u003cem\u003eNature Methods 2016 13:10\u003c/em\u003e, vol. 13, no. 10, pp. 859\u0026ndash;867, Aug. 2016, doi: 10.1038/nmeth.3964.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6796634/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6796634/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDeficiency of the Monocarboxylate Transporter 8 (MCT8) severely impairs thyroid hormone (TH) transport into the brain, disrupting brain development as well as peripheral TH homeostasis. Studies assessing MCT8 expression patterns and tissue-specific pathologies induced by local TH-deficiency are often inconclusive due to unreliable antibody staining and the lack of functional tools to specifically target MCT8-expressing cells.\u003c/p\u003e\n\u003cp\u003eFor this purpose, we generated non-inducible Mct8-Cre and tamoxifen-inducible Mct8-CreERT2 mice. Mct8-Cre;Sun1-sfGFP mice demonstrated ubiquitous Sun1-sfGFP expression, due to early recombination driven by Mct8 gene expression at the stage of trophoblast implantation. Tamoxifen injection in 6-week-old Mct8-CreERT2 mice induced reporter expression specifically in Mct8-expressing cells in the brain and peripherally in liver, kidney, and thyroid, without leaky reporter expression in vehicle controls. Using vDISCO tissue clearing and 3D-imaging of GFP-nanobody-boosted mice, we further identified the sublingual salivary gland and the prostate as prominent Mct8-expressing organs. Nuclei from Mct8-expressing cells could selectively be enriched using fluorescence-activated nuclei sorting on Mct8-CreERT2;Sun1-sfGFP mice and characterized as choroid plexus cells and tanycytes.\u003c/p\u003e\n\u003cp\u003eOur new inducible Mct8-CreERT2 line provides researchers with a tool to reliably mark, enrich, and characterize Mct8-expressing cells and to genetically modify genes specifically in these cells to study thyroid hormone transport and function.\u003c/p\u003e","manuscriptTitle":"A Novel Tamoxifen-Inducible Mct8-CreERT2 Mouse Model for Targeted Studies of Mct8-Expressing Cells and Thyroid Hormone Transport and Function","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-02 06:20:36","doi":"10.21203/rs.3.rs-6796634/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c1ae03ba-44e3-4d8b-941d-0871c22d1d61","owner":[],"postedDate":"July 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50801569,"name":"Health sciences/Anatomy/Nervous system/Central nervous system"},{"id":50801570,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Thyroid diseases"},{"id":50801571,"name":"Biological sciences/Biochemistry/Hormones/Thyroid hormones"},{"id":50801572,"name":"Health sciences/Neurology/Neurological disorders"}],"tags":[],"updatedAt":"2025-08-12T10:53:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-02 06:20:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6796634","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6796634","identity":"rs-6796634","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}