Body orientation affects brain ventricular system development and mechanosensory gene expression in zebrafish embryos

Body orientation affects brain ventricular system development and mechanosensory gene expression in zebrafish embryos | 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 Body orientation affects brain ventricular system development and mechanosensory gene expression in zebrafish embryos Rosa Amini, Ruchi Jain, Vladimir Korzh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8767756/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Most vertebrates maintain a horizontal body orientation. Whereas humans and great apes exhibit postures varying from horizontal to vertical. This exposes the body and its fluid-filled organs to changing gravitational vectors. This study examines how body position influences brain ventricular system development under normogravity. The zebrafish embryos were maintained in distinct orientations for 24 hours. The development of the Reissner fiber, an essential element of the brain ventricular system and proprioception, has been analyzed along with expression of genes associated with mechanotransduction and formation of Reissner fiber. Embryos held in vertical positions exhibited disrupted Reissner fiber, body axis deformities, and altered expression of gravity-responsive genes from the Hippo pathway, particularly yap1a . Among the genes regulating the development of Reissner fiber, the transcript level of chl1a/camel increased dramatically. These findings suggest that body orientation modulates cerebrospinal fluid flow and mechanosensory pathways during development. Understanding these effects provides insight into the mechanotransduction processes underlying proprioception and may inform studies of gravity's impact on vertebrate neurodevelopment. Health sciences/Anatomy Biological sciences/Developmental biology Biological sciences/Neuroscience Biological sciences/Physiology microgravity body orientation cerebrospinal fluid zebrafish development Reissner fiber Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Most vertebrates maintain a predominantly horizontal body orientation, whereas humans and great apes spend much of the day in a vertical posture, shifting to horizontal during sleep. This relatively recent evolutionary adaptation coincided with the transition to bipedalism. Nevertheless, humans frequently change body position throughout daily activities such as working, exercising, walking, and sitting, resulting in continuous exposure of cellular cytoplasm and bodily fluids to varying directions of gravitational force. The ability to tolerate brief periods in atypical positions, such as being upside down, varies depending on individual health and training. Notably, reports from incidents such as prolonged immobilization on roller coasters indicate that most individuals can withstand inverted positions for at least 1.5 hours. Therefore, body orientation relative to the gravitational vector is a critical factor, as gravity is a constant force influencing life on Earth. Living organisms detect gravity through specialized proprioceptive organs containing mechanosensory or gravisensory proteins, which respond to changes in gravitational force and transmit this information via signaling pathways 1 . Astronauts exposed to prolonged microgravity on the International Space Station experience redistribution of body fluids, including cerebrospinal fluid (CSF), increased brain and CSF volumes, altered aqueductal CSF hydrodynamics, impaired balance, and anemia 2 – 4 . However, the effects of microgravity on animal development, both in space and under simulated conditions such as clinostat-induced microgravity, remain poorly understood 5 . A key challenge is to identify the molecular and physiological responses underlying gravity's impact on cell and tissue structure 6 . Studies have shown that mechanotransduction relies on specific sensors (gravisensors), such as Yap1, which are affected by changes in gravity 1 , 7 , 8 . In developing zebrafish, yap1 is expressed in regions of active cell proliferation, including the ventricular zone of the neural tube and otic vesicles 9 , and is associated with the development of the retinal pigmented epithelium, posterior body, and hematopoietic stem cells 10 – 13 . Yap1 regulates several genes, such as lats2 and nf2a 14 and acts upstream of prox1 a to modulate Wnt signaling during development of the otic placode and the lateral line, both involved in proprioception 9 , 15 , 16 . To systematically mimic the space-flight conditions during controlled clinical studies, healthy adult volunteers have been subjected to the 6 0 head-down tilt (HDT) in bed 17 . These studies have shown that HDT can reasonably replicate some physiological responses observed in microgravity 18 , although actual spaceflight conditions are likely to be more extreme. Research on snakes has highlighted the significance of hydrostatic pressure gradients in vertical blood columns 19 , and similar gradients are likely present in the CSF column. These gradients may vary depending on the body's orientation relative to the gravitational vector 20 – 22 . The development of the brain ventricular system (BVS) relies on the activity of the heart and motile cilia, both of which are relatively inefficient in 24 hpf zebrafish embryos due to the lack of a mature cardiac conduction system, heart valves, and established CSF flow in the brain 23 – 26 . The Reissner fiber (RF), a hallmark of the BVS in animals with horizontal body posture such as zebrafish, serves as a marker for BVS development 27 – 32 . The posterior (primordial) RF is formed by the floor plate and its derivative, the flexural organ (FO), while the anterior (definitive) RF is produced by the subcommissural organ (SCO) beginning at 30 hpf and is complete by 48 hpf 33 . This process depends on CSF flow generated by motile cilia 34 , 35 . The RF has been proposed to act as a proprioceptive organ 36 – 38 . Its development is regulated by genes expressed in the roof and floor plates and their derivatives, SCO and FO, respectively. Among these, chl1a/camel , encoding a divergent member of the L1-CAM superfamily of cell adhesion molecules, is notable; its deficiency in zebrafish leads to hydrocephalus similar to that observed in kcng4b mutants 39 , 40 . In hu 33 mans CHL1 deficiency has been associated with mental impairment and schizophrenia 41 – 44 , and Chl1 is required for the development and maintenance of inhibitory neuronal subpopulations 45 . We hypothesized that prolonged changes in body position could alter the distribution of bodily fluids during development. To test this, we examined the development of the BVS and RF in zebrafish embryos maintained in various fixed orientations. Our results show that maintaining 24 hpf embryos in specific orientations for 24 hours leads to defects in RF development. Such defects are reflected at the level of transcription as shown for several genes associated with RF development and mechanosensory responses to gravity. These were affected by vertical body postures, with chl1a/camel transcript levels showing the most significant changes. These findings suggest that chl1a/camel transcript levels may serve as a BVS-specific indicator of CSF flow. Results The abnormal distribution of bodily fluids and in particular the CSF is one of the immediate effects of microgravity. Such changes have been associated with the developmental defects of the BVS mechanism 28 , 29 , 40 . RF is a constitutive element of BVS. The development of the anterior RF is sensitive to changing CSF flow 46 , 47 . Thus, we decided to study the effect of the body position on the RF development, which was studied in detail 33 . With this aim, the 24 hpf embryos were mounted in the low melting point agarose in transparent tubing and left to develop for 24 hpf in different horizontal and vertical positions (Fig. 1). Such experimental setup fixed the body axis in desired position and restrained body movements. At 24 hpf the head remains tilted more than 90 o in respect to the body axis. The head-to-tail angle (HTA) has been used as one of markers for the staging of developing embryos 48 . HTA decreases from 120 o at 24 hpf to 45 o at 48 hpf due to straightening of the body axis that changes its position in respect of the vector of gravity (Fig. 1). RF was detected by immunohistochemistry (Fig. 2A) or in vivo in transgenics (Fig. 2B, C). The embryos held in the horizontal “head down” position formed the anterior RF in the majority of embryos as detected at 48 hpf (Fig. 2A, D, H; n = 17/25) or 72 hpf (Fig. 2C, F, H; n = 23/25). When the embryos were placed in the “head up” “horizontal” position, the outcome was similar. The anterior RF was present in 20 (absent in 5) out of 25 embryos at 48 hpf (Fig. 2G, I). At 72 hpf the anterior RF was present in 14 (absent in 11) out 25 embryos (Fig. 2H, I). When the embryos developed in the “vertical” (erect) position, small deformations to the body axis were noted at 48 hpf (Fig. 3A-C). The Reissner fiber failed to develop normally in the majority of embryos (N = 48/55; Fig. 3B’. B’’, C’, C’’, G). The RF became defasciculated or displaced (Fig. 3B’, B”). Upon release from agarose for “compensation” only 23 out of 30 embryos had RF (Fig. 3A, C, E). An even more dramatic effect on the anterior RF development was detected in the embryos that developed in the “reversed vertical” (reversed erect) position. Here almost all embryos developed curled-down the body axis and cardiac edema (N = 52/55; Fig. 3D-F, G) and failed to develop the anterior RF (Fig. 3D’, D”, E’, E”. F’, F”). The studies in mammals suggested that the developmental window of the SCO-derived (anterior) RF formation could be relatively short compared to that in birds. When an initiation of the anterior RF is disrupted, it fails to develop during later period 49 , 50 . This posed a question whether similar developmental window for the anterior RF formation exists in zebrafish. To check whether anterior RF could be restored in embryos after its cultivation in “vertical” positions the 48 hpf scospondin-GFP ut24 embryos after incubation in fixed position were released from agarose to free swim in a Petri dish until 72 hpf (“compensation” experiment, Fig. 4). Some embryos held in “vertical” position, where “no” anterior RF was found at 48 hpf, after being “free” in Petri dish from 48 hpf to 72 hpf, formed the anterior RF (Fig. 4A, B). In contrast, all embryos held in the “reversed vertical” position, where at 48 hpf no anterior RF was found, failed to form the anterior RF by 72 hpf after 24 hpf period in a Petri dish (Fig. 5 − 2). Thus, not only the number of embryos held in the reverse vertical position that fail to form the anterior RF was higher compared to embryos in “vertical” position. After “reversed vertical” position embryos were unable to restore anterior RF unlike some embryos held in “vertical” position. Upon release from agarose for “compensation” only 13 out of 30 embryos had RF (Fig. 4B, D, E). In fact, in both vertical positions the number of embryos having RF was slightly reduced between 48 hpf and 72 hpf (Fig. 4E). Previously, several genes were linked with the development of the anterior RF 33,39,47,51 . In the embryos kept in the “vertical” position, the transcript level of sspo, lgals2a, lgals2b was reduced whereas chl1a, spon1a , and chl1a/camel increased with the latter transcript level increasing most dramatically (Fig. 4). In the “reversed vertical” group of embryos, the level of chl1a transcript increased to a value much higher than that in embryos kept in the “vertical” position. In addition, the levels of clu, spon1a, lgals2a transcripts also increased, whereas those of sspo and lgals2b decreased more significantly compared to controls or embryos kept in “vertical” position (Fig. 5; Supplemental Table 1). We hypothesized that the changes of body position may affect the mechanical properties of cells involved in the RF’s development. Previously, several genes of the Hippo pathway such as yap1 have been linked to mechano-elastic properties of cells in medaka, and their expression was affected by changes in gravity 7 , 8 . The developmental defects of the RF were reminiscent of the kcnb1 -/- mutant where the activity of the voltage-gated potassium channel Kv2.1 was affected 33 . Thus, the candidate mechanosensory genes were cross-referenced against the results of the whole-body RNAseq of Kv2.1 gain-of-function zebrafish embryos 52 . The genes with highest level of changes of expression level were selected for analysis of the embryos of “vertical” groups by qRT-PCR. Here the levels of at least two transcripts equally increased in both “vertical” positions – yap1 and actn3b (Fig. 6; Supplemental Table 2). The level of these transcripts seems to be “very sensitive” to changes in body position. In the embryos held in the “reversed vertical” position much more significant increase of the level of lats1, itgb1b.1, ctnna2 transcripts was detected compared to the “vertical” group of embryos (Fig. 6; Supplemental Table 2). The levels of these transcripts may represent the “sensitive” category, whereas itgb2, itga3b, nf2a, mob1 , which levels increased much less formed the “less sensitive” category. Thus, the genes associated with Yap1 signaling are very sensitive to changes in body position. The development of anterior RF was easily affected by both vertical orientations of zebrafish embryos during the second day of development. Such changes correlate well not only with changes in the level of transcripts linked to development of RF like chl1a/camel , but also with changes in the level of transcripts ( yap1 , actn3b, etc. ) associated with mechano-elastic properties of cells. This suggests that the relatively simple developmental tests in vivo at normal gravity using small vertebrates such as zebrafish could be efficiently applied to model in vivo the developmental processes depending on gravity. Here we restricted our analysis to the ventricular system and Reissner fiber. Yet the cardiac edema observed in embryos that developed in vertical positions suggested that the other systems of organs (cardiovascular, excretory, etc.), which development depends on fluid flow could be studied using this approach too. Discussion The development of anterior RF has been affected by changes in orientation of zebrafish embryos at normogravity. The experimental setup to mimic the effect of microgravity is based on a notion that changing body position in adult animals and humans will induce an abnormal CSF flow 17 – 22 . Here we applied the same concept to study the effect of the changing body position on Reissner fiber development, the proprioceptive element of animals with the horizontal body posture 36 – 38 . Such changes result in the deformation of the anterior RF and affect the levels of transcripts associated with the mechano-elastic properties of the RF-associated BVS elements connected to the RF (SCO, FO) or interacting with it (ependyma, CSF-cNs,). Given the suggested role of RF as the component of proprioceptive system, its deficiency may contribute to changes in space orientation and potentially influence structural changes in the neural tube. For example, the midbrain floor plate (or FO) acts as a source of many cell lineages in the brain, including CSF-cNs, dopaminergic and serotoninergic neurons, etc. 53 , whereas SCO has been implicated in the development of posterior commissure 54 . The RF defects may affect these CVOs and related cell lineages. It seems that some embryos held in horizontal position were affected (Fig. 2). In such positions at 24 hpf the anterior neural tube which forms the BVS is oriented almost vertically and only the central canal in the spinal cord is horizontal (Fig. 1). This may cause some disturbance in CSF distribution. As development progresses and the head-to-tail angle decreases by 48 hpf from 120 o to 45 o 48 , the effect of CSF redistribution in horizontal position will likely decrease. In contrast, the straightening of the body axis in vertical positions most likely will progressively affect the CSF flow. No wonder that the cultivation of embryos in both “vertical” orientations which are way more severe comparing to the 6 0 HDT in bed 17 , affects the development of the body axis and not only causes the defect or failure of the RF, but scoliosis and cardiac edema also (Fig. 3D, F). This is in line with previous studies of the developmental role of the RF 33,34,39,46,53 . The change of the body axis of developing zebrafish impacts genes expression linked to the mechano-elastic properties of cells (Fig. 6; Supplemental Table 2). The transfer of mechanical cues depends on conformational changes of the mechanosensory molecules or graviceptors able to connect to mechanically stable cellular elements like cytoskeleton and modify its conformation depending on mechanical input 1 , 55 . Within the group of mechanosensory genes, yap1 seems to be the most sensitive. Despite more severe morphological changes in embryos cultivated in the reversed vertical position, its expression has changed equally in both vertical positions indicating that the detected values may represent the maximal level of expression possible under experimental conditions during this developmental period (Fig. 6; Supplemental Table 2). Given the role of Yap1 in carcinogenesis such increase associated with significant morphological changes in the BVS-associated structures may be a worrying sign 56 , 57 . Similar trend was demonstrated by actn3b (Fig. 6; Supplemental Table 2). Notably an increase in expression of ACTN3 in humans has been associated with acute myeloid leukemia 58 . chl1a was the most affected among the genes linked to development of RF (Fig. 5; Supplemental Table 1). Furthermore, its transcription level reacted to the changing body position much more significantly compared to yap1 , which is well established as a gravisensor. Previously, we reported the regulatory role of chl1a in the RF’s development. Its deficiency caused the deficiency of the RF and scoliosis 39 . As the transmembrane protein interacting with various signaling pathways and cytoskeleton, Chl1a may act as the developmental regulator of the mechano-elastic properties of BVS. In mammals Chl1 is expressed in small to medium size sensory neurons and involved in development of the ventral midbrain (VM) dopaminergic (DA) pathways, a process critical for motor and cognitive function 59 , 60 . Chl1 interacts with extracellular matrix and potassium channels of the plasma membrane 61 . The mammalian Chl1 was linked to regulation of axonogenesis, and its mutations in humans cause neurodegenerative diseases 41 , 42 , 60 , 62 , 63 , and has been linked to scoliosis in different species 39 , 64 . Since the lipid bilayer of plasma membrane behaves as the mechanoreceptor 65 , 66 , perhaps, chl1a/camel functions as a sensitive gravisensor. This suggestion should be validated under microgravity conditions. Materials and Methods Animals Zebrafish ( Danio rerio ) were maintained according to established protocols 67 in the Zebrafish Core Facility at the International Institute of Molecular and Cell Biology in Warsaw. This facility is licensed for breeding and research (PL14656251, registry of the District Veterinary Inspectorate in Warsaw; 064 and 051: registry of the Ministry of Science and Higher Education). The experiments involving zebrafish embryos, larvae, and adults were conducted in accordance with the European Communities Council Directive (63/2010/EEC). The developmental stages in hours post-fertilization (hpf) are based on 48 . The scospondin-GFP ut24 transgenic line is a knock-in allele, where the C-terminal portion of the scospondin gene was precisely tagged with the GFP coding sequence 47 . Embryo mounting in capillaries under defined body orientations Zebrafish embryos were mounted in fluorinated ethylene propylene (FEP) capillaries (BOLA, Germany) using a low-melting agarose–based multilayer mounting approach adapted from a published protocol 68 . FEP tubing was cleaned by sequential washes with NaOH, double-distilled water (ddH₂O), and 70% ethanol with ultrasonic treatment, cut into ~ 3 cm segments, and stored in ddH₂O until use. Low-melting agarose was prepared in E3 medium, melted, and maintained at 38°C; tricaine (0.04%) and PTU (0.003%) were added immediately before mounting to anesthetize embryos and suppress pigmentation. At 24 hpf, embryos were manually dechorionated, transferred individually into molten agarose, and aspirated into FEP capillaries using a syringe-based loading system. Embryos were positioned within the capillary in predefined body orientations (horizontal or vertical), with orientation verified visually prior to agarose solidification. The horizontal positions “head-down” and “head-up” do not recapitulate the prone and supine positions of adult human body. This is due to curvature of the body, where the head-to-tail angle (HTA) at 24 hpf is 120 o . It decreases to 45 o by 48 hpf due to straightening of the body axis 48 . For the same reason, the “vertical” and “reversed vertical” positions are not equivalent to “erect” and “upside-down” positions of adult human body, but mimic those as close as possible under experimental conditions. Capillaries were sealed by insertion into a solid agarose layer and maintained in E3 medium at 28.5°C. Embryos were kept in capillaries from 24 hpf to 48 or 72 hpf and subsequently processed for live imaging, fixed for antibody staining, collected for gene expression analysis, or recovered from agarose and returned to E3 medium for further development. Microscopy Live imaging: Zebrafish embryos were raised in E3 medium (2.5 mM NaCl, 0.1 mM KCl, 0.16 mM CaCl 2 , and 0.43 mM MgCl 2 ) with the addition of 0.2 mM 1-phenyl-2-thiourea (PTU, Merck, Germany) to block pigmentation. At selected developmental stages, the embryos were manually dechorionated and anesthetized with 0.02% tricaine (Sigma-Aldrich, USA). Then, they were oriented after embedding in 2% methylcellulose (Merck, Germany) on the glass slides. A research stereomicroscope SMZ25 (Nikon, Japan) was used for imaging. Light-sheet fluorescent microscopy: In vivo imaging and imaging of fixed specimens were performed as previously described 69 . For imaging of fixed specimen, 0.8% low-melting agarose in phosphate-buffered saline (PBS) was used instead of the E3 0.02% tricaine medium. Zeiss Lightsheet Z.1 microscope with W Plan-Apochromat objectives (20x/1.0 UV-VIS (for in vivo imaging and 40x/1.0 UV-VIS or 63x/1.0 UV-VIS for fixed embryos) were used. Transmitted LED light was used to obtain high-resolution bright-field images. The data were saved in the LSM or CZI format and processed using ZEN (Zeiss) or ImageJ 1.51n (Fiji) software. Maximum intensity or sum slice projections were generated for each z-stack. Brightness and contrast adjustments, as well as resizing were performed using FastStone viewer 7.4 (FastStone Soft). Immunohistochemistry: Embryos were stained using two-color immunohistochemistry for RF with the polyclonal rabbit AFRU antibody (1:1000), a gift of Drs. J. Grondona [Malaga, Spain], E. Rodriguez, and M. Guerra [Valdivia, Chile]), and secondary Alexa Fluor 594 tagged donkey anti-rabbit antibody (1:1000; Invitrogen, USA) according to the described protocol 70 . The detected GFP was expressed in transgenic embryos. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) qRT-PCR analysis was performed to assess the expression of selected genes at 48 hpf in embryos cultured under vertical and reverse-vertical conditions (Supplemental Table 3). Total RNA was extracted from pooled zebrafish embryos using the TRIzol–chloroform method (Sigma-Aldrich, USA), quantified by NanoDrop™ 2000 (Thermo Scientific, USA), and reverse-transcribed from 1 µg RNA using the iScript™ Reverse Transcription kit (Bio-Rad, USA). qPCR reactions were carried out using SsoAdvanced™ Universal SYBR Green Supermix on a CFX Connect™ Real-Time PCR Detection System (Bio-Rad, USA). Gene-specific primers were designed based on ZFIN annotations, and eef1a1l1 was used as the reference (housekeeping) gene. Threshold cycle (Ct) values were obtained using Bio-Rad CFX Maestro software. Relative gene expression was calculated using the ΔΔCt method, with wild-type embryos serving as the reference condition. Data are presented as log₂ fold change relative to wild-type, with the dotted line indicating no change in expression (log₂ = 0). Error bars represent the standard deviation (SD) of biological replicates. Statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test, with comparisons made relative to wild-type controls (** p < 0.001, ** p < 0.01, * p < 0.05; n.s., not significant). Primer efficiency (90–110%) and amplification specificity were confirmed by standard curve analysis, melting-curve profiling, and agarose gel electrophoresis. Declarations Funding declaration: Korzh VK acknowledges support from the Opus grant of the National Science Centre (NCN), Poland (2020/39/B/NZ3/02729). Consent to publish: not applicable. Consent to participate: not applicable. Ethics declaration: Zebrafish ( Danio rerio ) were maintained according to established 67 in the Zebrafish Core Facility at the International Institute of Molecular and Cell Biology in Warsaw (licensed breeding and research facility, PL14656251, registry of the District Veterinary Inspectorate in Warsaw; 064 and 051: registry of the Ministry of Science and Higher Education). All the experiments with zebrafish embryos, larvae and adults were performed in accordance with the European Communities Council Directive (63/2010/EEC). Data availability declaration: the materials and reagents described in the paper are available from authors upon request. Author contribution declaration : R. Rosa Amini: Methodology, Investigation. Ruchi P. Jain: Methodology, Investigation. Vladimir Korzh: Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization. Declaration of generative AI and AI-assisted technologies in the writing process. During the preparation of this work the author(s) used DeepL Write and Nature Research Assistant to improve language and readability. After using this tool/ service, the authors reviewed and edited the content as needed and took full responsibility for the content of the publication. Acknowledgements The authors are thankful to Dr. Ryan Grey (Austin, USA), who kindly shared the scospondin-GFP ut24 transgenic line, Drs. J. Grondona (Malaga, Spain), E. Rodriguez, and M. Guerra (Valdivia, Chile) for AFRU antibody, Prof. Jacek Kuznicki and all members of the Laboratory of Neurodegeneration (IIMCB in Warsaw) for fruitful communication, the Microscopy and Zebrafish Core Facilities (IIMCB in Warsaw) for expert technical help and fish maintenance. Competing Interest declaration . Authors declare no competing interests. References Aventaggiato, M. et al. 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Motile cilia spin the Reissner fiber, a tensioned and anchored extracellular thread essential for body morphogenesis. (2025). doi: 10.1101/2025.09.25.678623 Aboitiz, F. & Montiel, J. F. The Enigmatic Reissner’s Fiber and the Origin of Chordates. Front. Neuroanat. 15, (2021). Kolmer, W. Das Sagittalorgan der wirbeltiere. Zeitschrift Anat. Entwicklungsgesch. 60, 652–717 (1921). Nicholls, G. E. The structure and development of Reissner’s fibre and subcommissural organ. Q. J. Microsc. Sci. 58, 1–116 (1913). Yang, S., Emelyanov, A., You, M.-S., Sin, M. & Korzh, V. Camel regulates development of the brain ventricular system. Cell Tissue Res. 383, 835–852 (2021). Shen, H., Bocksteins, E., Kondrychyn, I., Snyders, D. & Korzh, V. Functional antagonism of voltage-gated K + channel α-subunits in the developing brain ventricular system. Development 143, 4249–4260 (2016). Angeloni, D. et al. CALL gene is haploinsufficient in a 3p- syndrome patient. Am. J. Med. Genet. (1999). doi:10.1002/(SICI)1096-8628(19991029)86:53.0.CO;2-L Frints, S. G. M. et al. CALL interrupted in a patient with non-specific mental retardation: Gene dosage-dependent alteration of murine brain development and behavior. Human Molecular Genetics (2003). doi: 10.1093/hmg/ddg165 Sakurai, K., Migita, O., Toru, M. & Arinami, T. An association between a missense polymorphism in the close homologue of L1 (CHL1, CALL) gene and schizophrenia. Mol. Psychiatry 7, 412–415 (2002). Chu, T. T. & Liu, Y. An integrated genomic analysis of gene-function correlation on schizophrenia susceptibility genes. J. Hum. Genet. 55, 285–292 (2010). Schmalbach, B. et al. Age-dependent loss of parvalbumin‐expressing hippocampal interneurons in mice deficient in CHL 1, a mental retardation and schizophrenia susceptibility gene. J. Neurochem. 135, 830–844 (2015). Rose, C. D. et al. SCO-Spondin Defects and Neuroinflammation Are Conserved Mechanisms Driving Spinal Deformity across Genetic Models of Idiopathic Scoliosis. Curr. Biol. 30, 2363–2373.e6 (2020). Troutwine, B. R. et al. The Reissner Fiber Is Highly Dynamic In Vivo and Controls Morphogenesis of the Spine. Curr. Biol. 30, 2353–2362.e3 (2020). Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. & Schilling, T. F. Stages of embryonic development of the zebrafish. Dev. Dyn. (1995). doi: 10.1002/aja.1002030302 Lang, B. et al. Expression of the human PAC1 receptor leads to dose-dependent hydrocephalus-related abnormalities in mice. J. Clin. Invest. 116, 1924–1934 (2006). Picketts, D. J. Neuropeptide signaling and hydrocephalus: SCO with the flow. J. Clin. Invest. 116, 1828–1832 (2006). Muñoz, R. I. et al. The subcommissural organ and the Reissner fiber: old friends revisited. Cell Tissue Res. (2019). doi: 10.1007/s00441-018-2917-8 Jędrychowska, J. et al. Mutant analysis of Kcng4b reveals how the different functional states of the voltage-gated potassium channel regulate ear development. Dev. Biol. 513, 50–62 (2024). Wyart, C., Carbo-Tano, M., Cantaut-Belarif, Y., Orts-Del’Immagine, A. & Böhm, U. L. Cerebrospinal fluid-contacting neurons: multimodal cells with diverse roles in the CNS. Nat. Rev. Neurosci. 24, 540–556 (2023). Stanic, K., Montecinos, H. & Caprile, T. Subdivisions of chick diencephalic roof plate: Implication in the formation of the posterior commissure. Dev. Dyn. 239, 2584–2593 (2010). Lim, C.-G., Jang, J. & Kim, C. Cellular machinery for sensing mechanical force. BMB Rep. 51, 623–629 (2018). Low, B. C. et al. YAP/TAZ as mechanosensors and mechanotransducers in regulating organ size and tumor growth. FEBS Lett. 588, 2663–2670 (2014). Thomas, A. G., Chattopadhyay, A. & Dineen, R. A. Ependymal Tumors. Neuroimaging Clin. N. Am. 36, 69–83 (2026). Yang, X. et al. High Expression Levels of ACTN1 and ACTN3 Indicate Unfavorable Prognosis in Acute Myeloid Leukemia. J. Cancer 10, 4286–4292 (2019). Zhang, Y. et al. Expression of CHL1 and L1 by Neurons and Glia Following Sciatic Nerve and Dorsal Root Injury. Mol. Cell. Neurosci. 16, 71–86 (2000). Alsanie, W. F., Penna, V., Schachner, M., Thompson, L. H. & Parish, C. L. Homophilic binding of the neural cell adhesion molecule CHL1 regulates development of ventral midbrain dopaminergic pathways. Sci. Rep. (2017). doi: 10.1038/s41598-017-09599-y Kung, C. A possible unifying principle for mechanosensation. Nature 436, 647–654 (2005). Schlatter, M. C., Buhusi, M., Wright, A. G. & Maness, P. F. CHL1 promotes Sema3A-induced growth cone collapse and neurite elaboration through a motif required for recruitment of ERM proteins to the plasma membrane. J. Neurochem. (2008). doi: 10.1111/j.1471-4159.2007.05013.x Senchenko, V. N. et al. Differential expression of CHL1 Gene during development of major human cancers. PLoS One (2011). doi: 10.1371/journal.pone.0015612 Sharma, S. et al. Genome-wide association studies of adolescent idiopathic scoliosis suggest candidate susceptibility genes. Hum. Mol. Genet. (2011). doi: 10.1093/hmg/ddq571 Butler, P. J. & Chien, S. Role of the Plasma Membrane in Endothelial Cell Mechanosensation of Shear Stress. in Cellular Mechanotransduction (eds. Mofrad, M. R. K. & Kamm, R. D.) 61–88 (Cambridge University Press, 2009). Mukherjee, A. et al. Membrane potential mediates the cellular response to mechanical pressure. Cell 189, 143–160.e22 (2026). Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 5th Edition. Univ. Oregon Press. Eugene (2007). Kaufmann, A., Mickoleit, M., Weber, M. & Huisken, J. Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope. Development 139, 3242–3247 (2012). Jedrychowska, J., Gasanov, E. V. & Korzh, V. Kcnb1 plays a role in development of the inner ear. Dev. Biol. (2021). doi: 10.1016/j.ydbio.2020.12.007 Korzh, V., Sleptsova, I., Liao, J., He, J. & Gong, Z. Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation. Dev. Dyn. 213, 92–104 (1998). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 17 Feb, 2026 Editor assigned by journal 06 Feb, 2026 Submission checks completed at journal 06 Feb, 2026 First submitted to journal 02 Feb, 2026 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. 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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-8767756","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":592749391,"identity":"141fb2e1-1c24-4a8d-939c-da8cb2406b88","order_by":0,"name":"Rosa Amini","email":"","orcid":"","institution":"International Institute of Molecular and Cell Biology","correspondingAuthor":false,"prefix":"","firstName":"Rosa","middleName":"","lastName":"Amini","suffix":""},{"id":592749393,"identity":"895d5657-0c27-4e29-bce9-95c6ba454b3b","order_by":1,"name":"Ruchi Jain","email":"","orcid":"","institution":"International Institute of Molecular and Cell Biology","correspondingAuthor":false,"prefix":"","firstName":"Ruchi","middleName":"","lastName":"Jain","suffix":""},{"id":592749399,"identity":"d9c6bf7b-4a18-43c4-9a35-305a75e3693c","order_by":2,"name":"Vladimir Korzh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYDACHiBmbLDhYWNgYIMKMTcQoyUNWQsjUVoOg5hEajE4c/jYx587zsvwiR1+9oDhl01ig3QjAS1n25JnSJ65zcMmnWZuwNiXltggcxC/Fsl+HmMGwzaQlhw2Ccaew8YMEolEaElsO0eCFn7eHmOGg20HIFoYfhyWI6yF51gyY2NbMsQviQ1pcmyE/MLGk3yY8Webnb387ORnDz78seHhl24+gFcLKkhsAxoiQYIGIPgDxCRqGQWjYBSMguEPAGXDPU75iURyAAAAAElFTkSuQmCC","orcid":"","institution":"International Institute of Molecular and Cell Biology","correspondingAuthor":true,"prefix":"","firstName":"Vladimir","middleName":"","lastName":"Korzh","suffix":""}],"badges":[],"createdAt":"2026-02-02 17:24:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8767756/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8767756/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102996171,"identity":"75ddfdc2-d803-4423-8f94-5d448b6c93da","added_by":"auto","created_at":"2026-02-19 12:17:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":710987,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental setup. The 24 hpf embryos were mounted in low-melting point agarose in capillaries in four horizontal positions (head-down, A; head-up, B; lateral left hand-side down, not shown; lateral right hand-side down, not shown) and two vertical positions (vertical upright, C; reversed vertical, D). During development from 24 hpf to 48 hpf the body axis straightens. The different regions of the hollow neural tube (line divided into three sections) are differentially exposed to the vector of gravity (g, arrow).\u003c/p\u003e","description":"","filename":"Figure1Amini.png","url":"https://assets-eu.researchsquare.com/files/rs-8767756/v1/bd0a1709ca0e418c28b1166d.png"},{"id":103049877,"identity":"b009cabf-6f1a-487c-9756-3fb22da87d89","added_by":"auto","created_at":"2026-02-20 07:47:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1592199,"visible":true,"origin":"","legend":"\u003cp\u003eThe formation of the anterior RF in zebrafish embryos. A-C, Representative images of embryos demonstrate the anterior RF formed in embryos mounted in horizontal position and detected by AFRU antibody (magenta, A) or seen in scospondin-GFP\u003csup\u003eut24\u003c/sup\u003e transgenics (B, C). The small whole-body schematics illustrate orientation of the embryos cultivated from 24 hpf to 48 hpf (A, B) or to 72 hpf (C). The position of the images is shown by the green box on the scheme of the brain at corresponding stage. Most embryos developing in horizontal positions form the anterior RF by 72 hpf. The embryos reliably form the anterior RF when developed in the horizontal head–down position, except for the “head-up” position, where the anterior RF was not observed in some embryos at 48 hpf, but was detected at 72 hpf. Scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"Figure2Amini.png","url":"https://assets-eu.researchsquare.com/files/rs-8767756/v1/92c33f56de8a2c6e6ea6518e.png"},{"id":103049373,"identity":"f91fd568-98ec-499b-a804-117809dd6bcf","added_by":"auto","created_at":"2026-02-20 07:40:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2375918,"visible":true,"origin":"","legend":"\u003cp\u003eMost embryos fail to form the anterior RF when developing in the vertical position.\u003c/p\u003e\n\u003cp\u003eA – horizontal position, control. B – C, embryos cultivated in upright vertical position. The mild group (B, B’, B’’) developed the defasciculated anterior RF and FO staining was weak. The severe group (C, C’, C’’) developed mild body deformation and cardiac edema. In a minority of embryos, the anterior RF formed (N = 9/55; B’, B’’). The anterior RF failed to connect to the weakly stained FO (N=46/55; C’, C’’ ).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;When developed in reversed vertical position (D-F), most embryos failed to develop the anterior RF (N = 52/55; D’, E’, F’, D’’, F’’) and the AFRU+ material was found accumulated ectopically in the ventral part of the IIIrd ventricle (F’). whereas in the minority of embryos (N= 3/55; E”) the much reduced anterior RF formed. Anterior RF was visualized either by AFRU immunostaining in fixed embryos or by live imaging of scospondin-GFP\u003csup\u003eut24\u003c/sup\u003e transgenic embryos, as indicated in the corresponding panels. Quantification of anterior RF formation under different body orientations (G).\u003c/p\u003e\n\u003cp\u003eBars represent the fraction of embryos with or without anterior RF. The number of embryos was shown on bars. Scale bar, A-F = 100 μm; B’- F’, B”- F” = 20 μm.\u003c/p\u003e","description":"","filename":"Figure3Amini.png","url":"https://assets-eu.researchsquare.com/files/rs-8767756/v1/f8984eb233ea94fa0522fadc.png"},{"id":102996176,"identity":"c1cebca0-763d-45c8-b345-ef07f444629f","added_by":"auto","created_at":"2026-02-19 12:17:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1684686,"visible":true,"origin":"","legend":"\u003cp\u003eThe development of embryos in vertical positions reduced recovery of Reissner fiber. Most embryos in vertical position failed to form detectable RF at 48 hpf (25/30) (A, G; only very thin microfilaments were detected). Some recovered relatively normally during 48-72 hpf period in Petri dish after release (B) and some did not (C). In this group the SCO retained the shape like that at 48 hpf. The anterior RF defect is even more pronounced in embryos in reversed vertical position, where 27/30 embryos (D, G) failed to form detectable RF at 48 hpf. Some embryos recovered during 48-72 hpf period in Petri dish after release from agarose (E) with others developing the deformed RF (F) or none (not shown). Quantification of anterior RF recovery after release from vertical and reversed vertical positions (G; same graph for both conditions). Scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"Figure4Amini.png","url":"https://assets-eu.researchsquare.com/files/rs-8767756/v1/75bbe1ac30495cebdf566f41.png"},{"id":102996177,"identity":"b3b8050c-ba4d-4ac0-b1d7-e2bed300491d","added_by":"auto","created_at":"2026-02-19 12:17:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":228623,"visible":true,"origin":"","legend":"\u003cp\u003eThe transcript levels of the genes associated with development of the RF in vertical and reversed vertical group at 48 hpf. Data are shown as log\u003csup\u003e2\u003c/sup\u003e-transformed fold change relative to WT. Error bars represent propagated SD.\u003c/p\u003e","description":"","filename":"Figure5Amini.png","url":"https://assets-eu.researchsquare.com/files/rs-8767756/v1/9c6fc286d562f11e9f7dff3f.png"},{"id":102996172,"identity":"3f6c1a1d-4496-43de-9542-8bdf08cb4967","added_by":"auto","created_at":"2026-02-19 12:17:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":268038,"visible":true,"origin":"","legend":"\u003cp\u003eThe transcript levels of the genes linked to mechano-elastic properties of cells changed depending on body position. These changes were more dramatic in embryos held in the reversed vertical position compared to the vertical position. Data are shown as log\u003csup\u003e2\u003c/sup\u003e-transformed fold change relative to WT. Error bars represent propagated SD.\u003c/p\u003e","description":"","filename":"Figure6Amini.png","url":"https://assets-eu.researchsquare.com/files/rs-8767756/v1/773f3bf260a64433a70695bd.png"},{"id":103050687,"identity":"070741b4-991d-4eeb-b471-c7535eb3ffc4","added_by":"auto","created_at":"2026-02-20 07:53:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7030565,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8767756/v1/61073e6d-d6ab-40b1-9683-04ecd38ddcdc.pdf"},{"id":103049292,"identity":"e4cf1e27-8a05-4640-bbfc-3d411977093e","added_by":"auto","created_at":"2026-02-20 07:39:37","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":22959,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8767756/v1/67f3de672b115f6c6aa2371c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Body orientation affects brain ventricular system development and mechanosensory gene expression in zebrafish embryos","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMost vertebrates maintain a predominantly horizontal body orientation, whereas humans and great apes spend much of the day in a vertical posture, shifting to horizontal during sleep. This relatively recent evolutionary adaptation coincided with the transition to bipedalism. Nevertheless, humans frequently change body position throughout daily activities such as working, exercising, walking, and sitting, resulting in continuous exposure of cellular cytoplasm and bodily fluids to varying directions of gravitational force. The ability to tolerate brief periods in atypical positions, such as being upside down, varies depending on individual health and training. Notably, reports from incidents such as prolonged immobilization on roller coasters indicate that most individuals can withstand inverted positions for at least 1.5 hours. Therefore, body orientation relative to the gravitational vector is a critical factor, as gravity is a constant force influencing life on Earth. Living organisms detect gravity through specialized proprioceptive organs containing mechanosensory or gravisensory proteins, which respond to changes in gravitational force and transmit this information via signaling pathways \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAstronauts exposed to prolonged microgravity on the International Space Station experience redistribution of body fluids, including cerebrospinal fluid (CSF), increased brain and CSF volumes, altered aqueductal CSF hydrodynamics, impaired balance, and anemia \u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, the effects of microgravity on animal development, both in space and under simulated conditions such as clinostat-induced microgravity, remain poorly understood \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. A key challenge is to identify the molecular and physiological responses underlying gravity's impact on cell and tissue structure \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Studies have shown that mechanotransduction relies on specific sensors (gravisensors), such as Yap1, which are affected by changes in gravity \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In developing zebrafish, \u003cem\u003eyap1\u003c/em\u003e is expressed in regions of active cell proliferation, including the ventricular zone of the neural tube and otic vesicles \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and is associated with the development of the retinal pigmented epithelium, posterior body, and hematopoietic stem cells \u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Yap1 regulates several genes, such as \u003cem\u003elats2\u003c/em\u003e and \u003cem\u003enf2a\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and acts upstream of \u003cem\u003eprox1\u003c/em\u003ea to modulate Wnt signaling during development of the otic placode and the lateral line, both involved in proprioception \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo systematically mimic the space-flight conditions during controlled clinical studies, healthy adult volunteers have been subjected to the 6\u003csup\u003e0\u003c/sup\u003e head-down tilt (HDT) in bed \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These studies have shown that HDT can reasonably replicate some physiological responses observed in microgravity \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, although actual spaceflight conditions are likely to be more extreme. Research on snakes has highlighted the significance of hydrostatic pressure gradients in vertical blood columns \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, and similar gradients are likely present in the CSF column. These gradients may vary depending on the body's orientation relative to the gravitational vector \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe development of the brain ventricular system (BVS) relies on the activity of the heart and motile cilia, both of which are relatively inefficient in 24 hpf zebrafish embryos due to the lack of a mature cardiac conduction system, heart valves, and established CSF flow in the brain \u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The Reissner fiber (RF), a hallmark of the BVS in animals with horizontal body posture such as zebrafish, serves as a marker for BVS development \u003csup\u003e\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The posterior (primordial) RF is formed by the floor plate and its derivative, the flexural organ (FO), while the anterior (definitive) RF is produced by the subcommissural organ (SCO) beginning at 30 hpf and is complete by 48 hpf \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. This process depends on CSF flow generated by motile cilia \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The RF has been proposed to act as a proprioceptive organ \u003csup\u003e\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Its development is regulated by genes expressed in the roof and floor plates and their derivatives, SCO and FO, respectively. Among these, \u003cem\u003echl1a/camel\u003c/em\u003e, encoding a divergent member of the L1-CAM superfamily of cell adhesion molecules, is notable; its deficiency in zebrafish leads to hydrocephalus similar to that observed in \u003cem\u003ekcng4b\u003c/em\u003e mutants \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In hu\u003csup\u003e33\u003c/sup\u003emans CHL1 deficiency has been associated with mental impairment and schizophrenia \u003csup\u003e\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, and Chl1 is required for the development and maintenance of inhibitory neuronal subpopulations \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe hypothesized that prolonged changes in body position could alter the distribution of bodily fluids during development. To test this, we examined the development of the BVS and RF in zebrafish embryos maintained in various fixed orientations. Our results show that maintaining 24 hpf embryos in specific orientations for 24 hours leads to defects in RF development. Such defects are reflected at the level of transcription as shown for several genes associated with RF development and mechanosensory responses to gravity. These were affected by vertical body postures, with \u003cem\u003echl1a/camel\u003c/em\u003e transcript levels showing the most significant changes. These findings suggest that \u003cem\u003echl1a/camel\u003c/em\u003e transcript levels may serve as a BVS-specific indicator of CSF flow.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe abnormal distribution of bodily fluids and in particular the CSF is one of the immediate effects of microgravity. Such changes have been associated with the developmental defects of the BVS mechanism \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. RF is a constitutive element of BVS. The development of the anterior RF is sensitive to changing CSF flow \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Thus, we decided to study the effect of the body position on the RF development, which was studied in detail \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. With this aim, the 24 hpf embryos were mounted in the low melting point agarose in transparent tubing and left to develop for 24 hpf in different horizontal and vertical positions (Fig.\u0026nbsp;1). Such experimental setup fixed the body axis in desired position and restrained body movements. At 24 hpf the head remains tilted more than 90\u003csup\u003eo\u003c/sup\u003e in respect to the body axis. The head-to-tail angle (HTA) has been used as one of markers for the staging of developing embryos \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. HTA decreases from 120\u003csup\u003eo\u003c/sup\u003e at 24 hpf to 45\u003csup\u003eo\u003c/sup\u003e at 48 hpf due to straightening of the body axis that changes its position in respect of the vector of gravity (Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eRF was detected by immunohistochemistry (Fig.\u0026nbsp;2A) or \u003cem\u003ein vivo\u003c/em\u003e in transgenics (Fig.\u0026nbsp;2B, C). The embryos held in the horizontal \u0026ldquo;head down\u0026rdquo; position formed the anterior RF in the majority of embryos as detected at 48 hpf (Fig.\u0026nbsp;2A, D, H; n\u0026thinsp;=\u0026thinsp;17/25) or 72 hpf (Fig.\u0026nbsp;2C, F, H; n\u0026thinsp;=\u0026thinsp;23/25). When the embryos were placed in the \u0026ldquo;head up\u0026rdquo; \u0026ldquo;horizontal\u0026rdquo; position, the outcome was similar. The anterior RF was present in 20 (absent in 5) out of 25 embryos at 48 hpf (Fig.\u0026nbsp;2G, I). At 72 hpf the anterior RF was present in 14 (absent in 11) out 25 embryos (Fig.\u0026nbsp;2H, I).\u003c/p\u003e \u003cp\u003eWhen the embryos developed in the \u0026ldquo;vertical\u0026rdquo; (erect) position, small deformations to the body axis were noted at 48 hpf (Fig.\u0026nbsp;3A-C). The Reissner fiber failed to develop normally in the majority of embryos (N\u0026thinsp;=\u0026thinsp;48/55; Fig.\u0026nbsp;3B\u0026rsquo;. B\u0026rsquo;\u0026rsquo;, C\u0026rsquo;, C\u0026rsquo;\u0026rsquo;, G). The RF became defasciculated or displaced (Fig.\u0026nbsp;3B\u0026rsquo;, B\u0026rdquo;). Upon release from agarose for \u0026ldquo;compensation\u0026rdquo; only 23 out of 30 embryos had RF (Fig.\u0026nbsp;3A, C, E). An even more dramatic effect on the anterior RF development was detected in the embryos that developed in the \u0026ldquo;reversed vertical\u0026rdquo; (reversed erect) position. Here almost all embryos developed curled-down the body axis and cardiac edema (N\u0026thinsp;=\u0026thinsp;52/55; Fig.\u0026nbsp;3D-F, G) and failed to develop the anterior RF (Fig.\u0026nbsp;3D\u0026rsquo;, D\u0026rdquo;, E\u0026rsquo;, E\u0026rdquo;. F\u0026rsquo;, F\u0026rdquo;).\u003c/p\u003e \u003cp\u003eThe studies in mammals suggested that the developmental window of the SCO-derived (anterior) RF formation could be relatively short compared to that in birds. When an initiation of the anterior RF is disrupted, it fails to develop during later period \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. This posed a question whether similar developmental window for the anterior RF formation exists in zebrafish. To check whether anterior RF could be restored in embryos after its cultivation in \u0026ldquo;vertical\u0026rdquo; positions the 48 hpf \u003cem\u003escospondin-GFP\u003c/em\u003e\u003csup\u003e\u003cem\u003eut24\u003c/em\u003e\u003c/sup\u003e embryos after incubation in fixed position were released from agarose to free swim in a Petri dish until 72 hpf (\u0026ldquo;compensation\u0026rdquo; experiment, Fig.\u0026nbsp;4). Some embryos held in \u0026ldquo;vertical\u0026rdquo; position, where \u0026ldquo;no\u0026rdquo; anterior RF was found at 48 hpf, after being \u0026ldquo;free\u0026rdquo; in Petri dish from 48 hpf to 72 hpf, formed the anterior RF (Fig.\u0026nbsp;4A, B). In contrast, all embryos held in the \u0026ldquo;reversed vertical\u0026rdquo; position, where at 48 hpf no anterior RF was found, failed to form the anterior RF by 72 hpf after 24 hpf period in a Petri dish (Fig.\u0026nbsp;5\u0026thinsp;\u0026minus;\u0026thinsp;2). Thus, not only the number of embryos held in the reverse vertical position that fail to form the anterior RF was higher compared to embryos in \u0026ldquo;vertical\u0026rdquo; position. After \u0026ldquo;reversed vertical\u0026rdquo; position embryos were unable to restore anterior RF unlike some embryos held in \u0026ldquo;vertical\u0026rdquo; position. Upon release from agarose for \u0026ldquo;compensation\u0026rdquo; only 13 out of 30 embryos had RF (Fig.\u0026nbsp;4B, D, E). In fact, in both vertical positions the number of embryos having RF was slightly reduced between 48 hpf and 72 hpf (Fig.\u0026nbsp;4E).\u003c/p\u003e \u003cp\u003ePreviously, several genes were linked with the development of the anterior RF \u003csup\u003e33,39,47,51\u003c/sup\u003e. In the embryos kept in the \u0026ldquo;vertical\u0026rdquo; position, the transcript level of \u003cem\u003esspo, lgals2a, lgals2b\u003c/em\u003e was reduced whereas \u003cem\u003echl1a, spon1a\u003c/em\u003e, and \u003cem\u003echl1a/camel\u003c/em\u003e increased with the latter transcript level increasing most dramatically (Fig.\u0026nbsp;4). In the \u0026ldquo;reversed vertical\u0026rdquo; group of embryos, the level of \u003cem\u003echl1a\u003c/em\u003e transcript increased to a value much higher than that in embryos kept in the \u0026ldquo;vertical\u0026rdquo; position. In addition, the levels of \u003cem\u003eclu, spon1a, lgals2a\u003c/em\u003e transcripts also increased, whereas those of \u003cem\u003esspo\u003c/em\u003e and \u003cem\u003elgals2b\u003c/em\u003e decreased more significantly compared to controls or embryos kept in \u0026ldquo;vertical\u0026rdquo; position (Fig.\u0026nbsp;5; Supplemental Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eWe hypothesized that the changes of body position may affect the mechanical properties of cells involved in the RF\u0026rsquo;s development. Previously, several genes of the Hippo pathway such as \u003cem\u003eyap1\u003c/em\u003e have been linked to mechano-elastic properties of cells in medaka, and their expression was affected by changes in gravity \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The developmental defects of the RF were reminiscent of the \u003cem\u003ekcnb1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mutant where the activity of the voltage-gated potassium channel Kv2.1 was affected \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Thus, the candidate mechanosensory genes were cross-referenced against the results of the whole-body RNAseq of Kv2.1 gain-of-function zebrafish embryos \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The genes with highest level of changes of expression level were selected for analysis of the embryos of \u0026ldquo;vertical\u0026rdquo; groups by qRT-PCR. Here the levels of at least two transcripts equally increased in both \u0026ldquo;vertical\u0026rdquo; positions \u0026ndash; \u003cem\u003eyap1\u003c/em\u003e and \u003cem\u003eactn3b\u003c/em\u003e (Fig.\u0026nbsp;6; Supplemental Table\u0026nbsp;2). The level of these transcripts seems to be \u0026ldquo;very sensitive\u0026rdquo; to changes in body position. In the embryos held in the \u0026ldquo;reversed vertical\u0026rdquo; position much more significant increase of the level of \u003cem\u003elats1, itgb1b.1, ctnna2\u003c/em\u003e transcripts was detected compared to the \u0026ldquo;vertical\u0026rdquo; group of embryos (Fig.\u0026nbsp;6; Supplemental Table\u0026nbsp;2). The levels of these transcripts may represent the \u0026ldquo;sensitive\u0026rdquo; category, whereas \u003cem\u003eitgb2, itga3b, nf2a, mob1\u003c/em\u003e, which levels increased much less formed the \u0026ldquo;less sensitive\u0026rdquo; category. Thus, the genes associated with Yap1 signaling are very sensitive to changes in body position.\u003c/p\u003e \u003cp\u003eThe development of anterior RF was easily affected by both vertical orientations of zebrafish embryos during the second day of development. Such changes correlate well not only with changes in the level of transcripts linked to development of RF like \u003cem\u003echl1a/camel\u003c/em\u003e, but also with changes in the level of transcripts (\u003cem\u003eyap1\u003c/em\u003e, \u003cem\u003eactn3b, etc.\u003c/em\u003e) associated with mechano-elastic properties of cells. This suggests that the relatively simple developmental tests \u003cem\u003ein vivo\u003c/em\u003e at normal gravity using small vertebrates such as zebrafish could be efficiently applied to model \u003cem\u003ein vivo\u003c/em\u003e the developmental processes depending on gravity. Here we restricted our analysis to the ventricular system and Reissner fiber. Yet the cardiac edema observed in embryos that developed in vertical positions suggested that the other systems of organs (cardiovascular, excretory, etc.), which development depends on fluid flow could be studied using this approach too.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe development of anterior RF has been affected by changes in orientation of zebrafish embryos at normogravity. The experimental setup to mimic the effect of microgravity is based on a notion that changing body position in adult animals and humans will induce an abnormal CSF flow \u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Here we applied the same concept to study the effect of the changing body position on Reissner fiber development, the proprioceptive element of animals with the horizontal body posture \u003csup\u003e\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSuch changes result in the deformation of the anterior RF and affect the levels of transcripts associated with the mechano-elastic properties of the RF-associated BVS elements connected to the RF (SCO, FO) or interacting with it (ependyma, CSF-cNs,). Given the suggested role of RF as the component of proprioceptive system, its deficiency may contribute to changes in space orientation and potentially influence structural changes in the neural tube. For example, the midbrain floor plate (or FO) acts as a source of many cell lineages in the brain, including CSF-cNs, dopaminergic and serotoninergic neurons, \u003cem\u003eetc.\u003c/em\u003e \u003csup\u003e53\u003c/sup\u003e, whereas SCO has been implicated in the development of posterior commissure \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The RF defects may affect these CVOs and related cell lineages.\u003c/p\u003e \u003cp\u003eIt seems that some embryos held in horizontal position were affected (Fig.\u0026nbsp;2). In such positions at 24 hpf the anterior neural tube which forms the BVS is oriented almost vertically and only the central canal in the spinal cord is horizontal (Fig.\u0026nbsp;1). This may cause some disturbance in CSF distribution. As development progresses and the head-to-tail angle decreases by 48 hpf from 120\u003csup\u003eo\u003c/sup\u003e to 45\u003csup\u003eo \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, the effect of CSF redistribution in horizontal position will likely decrease.\u003c/p\u003e \u003cp\u003eIn contrast, the straightening of the body axis in vertical positions most likely will progressively affect the CSF flow. No wonder that the cultivation of embryos in both \u0026ldquo;vertical\u0026rdquo; orientations which are way more severe comparing to the 6\u003csup\u003e0\u003c/sup\u003e HDT in bed \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, affects the development of the body axis and not only causes the defect or failure of the RF, but scoliosis and cardiac edema also (Fig.\u0026nbsp;3D, F). This is in line with previous studies of the developmental role of the RF \u003csup\u003e33,34,39,46,53\u003c/sup\u003e. The change of the body axis of developing zebrafish impacts genes expression linked to the mechano-elastic properties of cells (Fig.\u0026nbsp;6; Supplemental Table\u0026nbsp;2). The transfer of mechanical cues depends on conformational changes of the mechanosensory molecules or graviceptors able to connect to mechanically stable cellular elements like cytoskeleton and modify its conformation depending on mechanical input \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWithin the group of mechanosensory genes, \u003cem\u003eyap1\u003c/em\u003e seems to be the most sensitive. Despite more severe morphological changes in embryos cultivated in the reversed vertical position, its expression has changed equally in both vertical positions indicating that the detected values may represent the maximal level of expression possible under experimental conditions during this developmental period (Fig.\u0026nbsp;6; Supplemental Table\u0026nbsp;2). Given the role of Yap1 in carcinogenesis such increase associated with significant morphological changes in the BVS-associated structures may be a worrying sign \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Similar trend was demonstrated by \u003cem\u003eactn3b\u003c/em\u003e (Fig.\u0026nbsp;6; Supplemental Table\u0026nbsp;2). Notably an increase in expression of \u003cem\u003eACTN3\u003c/em\u003e in humans has been associated with acute myeloid leukemia \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003echl1a\u003c/em\u003e was the most affected among the genes linked to development of RF (Fig.\u0026nbsp;5; Supplemental Table\u0026nbsp;1). Furthermore, its transcription level reacted to the changing body position much more significantly compared to \u003cem\u003eyap1\u003c/em\u003e, which is well established as a gravisensor. Previously, we reported the regulatory role of \u003cem\u003echl1a\u003c/em\u003e in the RF\u0026rsquo;s development. Its deficiency caused the deficiency of the RF and scoliosis \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. As the transmembrane protein interacting with various signaling pathways and cytoskeleton, Chl1a may act as the developmental regulator of the mechano-elastic properties of BVS. In mammals Chl1 is expressed in small to medium size sensory neurons and involved in development of the ventral midbrain (VM) dopaminergic (DA) pathways, a process critical for motor and cognitive function \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Chl1 interacts with extracellular matrix and potassium channels of the plasma membrane \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. The mammalian Chl1 was linked to regulation of axonogenesis, and its mutations in humans cause neurodegenerative diseases \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, and has been linked to scoliosis in different species \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Since the lipid bilayer of plasma membrane behaves as the mechanoreceptor \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, perhaps, \u003cem\u003echl1a/camel\u003c/em\u003e functions as a sensitive gravisensor. This suggestion should be validated under microgravity conditions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eZebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) were maintained according to established protocols \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e in the Zebrafish Core Facility at the International Institute of Molecular and Cell Biology in Warsaw. This facility is licensed for breeding and research (PL14656251, registry of the District Veterinary Inspectorate in Warsaw; 064 and 051: registry of the Ministry of Science and Higher Education). The experiments involving zebrafish embryos, larvae, and adults were conducted in accordance with the European Communities Council Directive (63/2010/EEC). The developmental stages in hours post-fertilization (hpf) are based on \u003csup\u003e48\u003c/sup\u003e. The \u003cem\u003escospondin-GFP\u003c/em\u003e\u003csup\u003e\u003cem\u003eut24\u003c/em\u003e\u003c/sup\u003e transgenic line is a knock-in allele, where the C-terminal portion of the \u003cem\u003escospondin\u003c/em\u003e gene was precisely tagged with the GFP coding sequence \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEmbryo mounting in capillaries under defined body orientations\u003c/h3\u003e\n\u003cp\u003eZebrafish embryos were mounted in fluorinated ethylene propylene (FEP) capillaries (BOLA, Germany) using a low-melting agarose\u0026ndash;based multilayer mounting approach adapted from a published protocol \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. FEP tubing was cleaned by sequential washes with NaOH, double-distilled water (ddH₂O), and 70% ethanol with ultrasonic treatment, cut into ~\u0026thinsp;3 cm segments, and stored in ddH₂O until use. Low-melting agarose was prepared in E3 medium, melted, and maintained at 38\u0026deg;C; tricaine (0.04%) and PTU (0.003%) were added immediately before mounting to anesthetize embryos and suppress pigmentation. At 24 hpf, embryos were manually dechorionated, transferred individually into molten agarose, and aspirated into FEP capillaries using a syringe-based loading system. Embryos were positioned within the capillary in predefined body orientations (horizontal or vertical), with orientation verified visually prior to agarose solidification. The horizontal positions \u0026ldquo;head-down\u0026rdquo; and \u0026ldquo;head-up\u0026rdquo; do not recapitulate the prone and supine positions of adult human body. This is due to curvature of the body, where the head-to-tail angle (HTA) at 24 hpf is 120\u003csup\u003eo\u003c/sup\u003e. It decreases to 45\u003csup\u003eo\u003c/sup\u003e by 48 hpf due to straightening of the body axis \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. For the same reason, the \u0026ldquo;vertical\u0026rdquo; and \u0026ldquo;reversed vertical\u0026rdquo; positions are not equivalent to \u0026ldquo;erect\u0026rdquo; and \u0026ldquo;upside-down\u0026rdquo; positions of adult human body, but mimic those as close as possible under experimental conditions. Capillaries were sealed by insertion into a solid agarose layer and maintained in E3 medium at 28.5\u0026deg;C. Embryos were kept in capillaries from 24 hpf to 48 or 72 hpf and subsequently processed for live imaging, fixed for antibody staining, collected for gene expression analysis, or recovered from agarose and returned to E3 medium for further development.\u003c/p\u003e\n\u003ch3\u003eMicroscopy\u003c/h3\u003e\n\u003cp\u003eLive imaging: Zebrafish embryos were raised in E3 medium (2.5 mM NaCl, 0.1 mM KCl, 0.16 mM CaCl\u003csub\u003e2\u003c/sub\u003e, and 0.43 mM MgCl\u003csub\u003e2\u003c/sub\u003e) with the addition of 0.2 mM 1-phenyl-2-thiourea (PTU, Merck, Germany) to block pigmentation. At selected developmental stages, the embryos were manually dechorionated and anesthetized with 0.02% tricaine (Sigma-Aldrich, USA). Then, they were oriented after embedding in 2% methylcellulose (Merck, Germany) on the glass slides. A research stereomicroscope SMZ25 (Nikon, Japan) was used for imaging.\u003c/p\u003e \u003cp\u003eLight-sheet fluorescent microscopy: \u003cem\u003eIn vivo\u003c/em\u003e imaging and imaging of fixed specimens were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. For imaging of fixed specimen, 0.8% low-melting agarose in phosphate-buffered saline (PBS) was used instead of the E3 0.02% tricaine medium. Zeiss Lightsheet Z.1 microscope with W Plan-Apochromat objectives (20x/1.0 UV-VIS (for \u003cem\u003ein vivo\u003c/em\u003e imaging and 40x/1.0 UV-VIS or 63x/1.0 UV-VIS for fixed embryos) were used. Transmitted LED light was used to obtain high-resolution bright-field images. The data were saved in the LSM or CZI format and processed using ZEN (Zeiss) or ImageJ 1.51n (Fiji) software. Maximum intensity or sum slice projections were generated for each z-stack. Brightness and contrast adjustments, as well as resizing were performed using FastStone viewer 7.4 (FastStone Soft).\u003c/p\u003e \u003cp\u003eImmunohistochemistry: Embryos were stained using two-color immunohistochemistry for RF with the polyclonal rabbit AFRU antibody (1:1000), a gift of Drs. J. Grondona [Malaga, Spain], E. Rodriguez, and M. Guerra [Valdivia, Chile]), and secondary Alexa Fluor 594 tagged donkey anti-rabbit antibody (1:1000; Invitrogen, USA) according to the described protocol \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. The detected GFP was expressed in transgenic embryos.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real-Time Polymerase Chain Reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eqRT-PCR analysis was performed to assess the expression of selected genes at 48 hpf in embryos cultured under vertical and reverse-vertical conditions (Supplemental Table\u0026nbsp;3). Total RNA was extracted from pooled zebrafish embryos using the TRIzol\u0026ndash;chloroform method (Sigma-Aldrich, USA), quantified by NanoDrop\u0026trade; 2000 (Thermo Scientific, USA), and reverse-transcribed from 1 \u0026micro;g RNA using the iScript\u0026trade; Reverse Transcription kit (Bio-Rad, USA). qPCR reactions were carried out using SsoAdvanced\u0026trade; Universal SYBR Green Supermix on a CFX Connect\u0026trade; Real-Time PCR Detection System (Bio-Rad, USA). Gene-specific primers were designed based on ZFIN annotations, and eef1a1l1 was used as the reference (housekeeping) gene. Threshold cycle (Ct) values were obtained using Bio-Rad CFX Maestro software. Relative gene expression was calculated using the ΔΔCt method, with wild-type embryos serving as the reference condition. Data are presented as log₂ fold change relative to wild-type, with the dotted line indicating no change in expression (log₂ = 0). Error bars represent the standard deviation (SD) of biological replicates. Statistical significance was assessed using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test, with comparisons made relative to wild-type controls (** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; n.s., not significant). Primer efficiency (90\u0026ndash;110%) and amplification specificity were confirmed by standard curve analysis, melting-curve profiling, and agarose gel electrophoresis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding declaration:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKorzh VK acknowledges support from the Opus grant of the National Science Centre (NCN), Poland (2020/39/B/NZ3/02729).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent to publish: \u003c/strong\u003enot applicable.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e not applicable.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics declaration:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) were maintained according to established \u003csup\u003e67\u003c/sup\u003e in the Zebrafish Core Facility at the International Institute of Molecular and Cell Biology in Warsaw (licensed breeding and research facility, PL14656251, registry of the District Veterinary Inspectorate in Warsaw; 064 and 051: registry of the Ministry of Science and Higher Education). All the experiments with zebrafish embryos, larvae and adults were performed in accordance with the European Communities Council Directive (63/2010/EEC).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability declaration:\u003c/strong\u003e the materials and reagents described in the paper are available from authors upon request.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contribution declaration\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR. Rosa Amini: Methodology, Investigation. Ruchi P. Jain: Methodology, Investigation. Vladimir Korzh: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process.\u003c/strong\u003e During the preparation of this work the author(s) used DeepL Write and Nature Research Assistant to improve language and readability. After using this tool/ service, the authors reviewed and edited the content as needed and took full responsibility for the content of the publication.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to Dr. Ryan Grey (Austin, USA), who kindly shared the \u003cem\u003escospondin-GFP\u003c/em\u003e\u003cem\u003e\u003csup\u003eut24\u003c/sup\u003e\u003c/em\u003e transgenic line, Drs. J. Grondona (Malaga, Spain), E. Rodriguez, and M. Guerra (Valdivia, Chile) for AFRU antibody, Prof. Jacek Kuznicki and all members of the Laboratory of Neurodegeneration (IIMCB in Warsaw) for fruitful communication, the Microscopy and Zebrafish Core Facilities (IIMCB in Warsaw) for expert technical help and fish maintenance.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting Interest declaration\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eAuthors declare no competing interests. \u003c/p\u003e\n\n\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAventaggiato, M. \u003cem\u003eet al.\u003c/em\u003e Putative Receptors for Gravity Sensing in Mammalian Cells: The Effects of Microgravity. \u003cem\u003eAppl. Sci.\u003c/em\u003e 10, 2028 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrudel, G., Stratis, D., Rocheleau, L., Pelchat, M. \u0026amp; Laneuville, O. 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Dyn.\u003c/em\u003e 213, 92\u0026ndash;104 (1998).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"npj-microgravity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjmgrav","sideBox":"Learn more about [npj Microgravity](http://www.nature.com/npjmgrav/)","snPcode":"41526","submissionUrl":"https://submission.springernature.com/new-submission/41526/3","title":"npj Microgravity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"microgravity, body orientation, cerebrospinal fluid, zebrafish development, Reissner fiber","lastPublishedDoi":"10.21203/rs.3.rs-8767756/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8767756/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMost vertebrates maintain a horizontal body orientation. Whereas humans and great apes exhibit postures varying from horizontal to vertical. This exposes the body and its fluid-filled organs to changing gravitational vectors. This study examines how body position influences brain ventricular system development under normogravity. The zebrafish embryos were maintained in distinct orientations for 24 hours. The development of the Reissner fiber, an essential element of the brain ventricular system and proprioception, has been analyzed along with expression of genes associated with mechanotransduction and formation of Reissner fiber. Embryos held in vertical positions exhibited disrupted Reissner fiber, body axis deformities, and altered expression of gravity-responsive genes from the Hippo pathway, particularly \u003cem\u003eyap1a\u003c/em\u003e. Among the genes regulating the development of Reissner fiber, the transcript level of \u003cem\u003echl1a/camel\u003c/em\u003e increased dramatically. These findings suggest that body orientation modulates cerebrospinal fluid flow and mechanosensory pathways during development. Understanding these effects provides insight into the mechanotransduction processes underlying proprioception and may inform studies of gravity's impact on vertebrate neurodevelopment.\u003c/p\u003e","manuscriptTitle":"Body orientation affects brain ventricular system development and mechanosensory gene expression in zebrafish embryos","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-19 12:17:22","doi":"10.21203/rs.3.rs-8767756/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-02-17T08:43:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-06T05:21:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-06T05:20:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Microgravity","date":"2026-02-02T17:11:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-microgravity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjmgrav","sideBox":"Learn more about [npj Microgravity](http://www.nature.com/npjmgrav/)","snPcode":"41526","submissionUrl":"https://submission.springernature.com/new-submission/41526/3","title":"npj Microgravity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3a421bba-5402-4544-8816-ea68ebc7adcc","owner":[],"postedDate":"February 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63071902,"name":"Health sciences/Anatomy"},{"id":63071903,"name":"Biological sciences/Developmental biology"},{"id":63071904,"name":"Biological sciences/Neuroscience"},{"id":63071905,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-02-19T12:17:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-19 12:17:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8767756","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8767756","identity":"rs-8767756","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}