Dstyk mutation leads to congenital scoliosis-like vertebral malformations in zebrafish via dysregulated mTORC1/TFEB pathway.

Congenital scoliosis (CS) is a complex genetic disorder characterized by vertebral malformations. The precise etiology of CS is not fully defined. Here, we identify that mutation in dual serine/threonine and tyrosine protein kinase (dstyk) lead to CS-like vertebral malformations in zebrafish. We demonstrate that the scoliosis in dstyk mutants is related to the wavy and malformed notochord sheath formation and abnormal axial skeleton segmentation due to dysregulated biogenesis of notochord vacuoles and notochord function. Further studies show that DSTYK is located in late endosomal/lysosomal compartments and is involved in the lysosome biogenesis in mammalian cells. Dstyk knockdown inhibits notochord vacuole and lysosome biogenesis through mTORC1-dependent repression of TFEB nuclear translocation. Inhibition of mTORC1 activity can rescue the defect in notochord vacuole biogenesis and scoliosis in dstyk mutants. Together, our findings reveal a key role of DSTYK in notochord vacuole biogenesis, notochord morphogenesis and spine development through mTORC1/TFEB pathway.

On the Enigma of the Human Neurenteric Canal.

Existence and biomedical relevance of the neurenteric canal, a transient midline structure during early neurulation in the human embryo, have been controversially discussed for more than a century by embryologists and clinicians alike. In this study, the authors address the long-standing enigma by high-resolution histology and three-dimensional reconstruction using new and historic histological sections of 5 human 17- to 21-day-old embryos and of 2 marmoset monkey embryos of the species Callithrix jacchus at corresponding stages. The neurenteric canal presents itself as the classical vertical connection between the amniotic cavity and the yolk sac cavity and is lined (a) craniolaterally by a horseshoe-shaped "hinge of involuting notochordal cells" within Hensen's node and (b) caudally by the receding primitive streak epiblast dorsally and by notochordal plate epithelium ventrally, the latter of which covered the (longitudinal) notochordal canal on its ventral side at the preceding stage. Furthermore, asymmetric parachordal nodal expression in Callithrix and morphological asymmetries within the nodes of the other specimens suggest an early non-cilium-dependent left-right symmetry breaking mode previously postulated for other mammals. We conclude that structure and position of the mammalian neurenteric canal support the notion of its homology with the reptilian blastopore as a whole and with a dorsal segment of the blastopore in amphibia. These new features of the neurenteric canal may further clarify the aetiology of foetal malformations such as junctional neurulation defects, neuroendodermal cysts, and the split notochord syndrome.

Large Cytoplasmic Vacuoles within Notochordal Nucleus Pulposus Cells: A Possible Regulator of Intracellular Pressure That Shapes the Cytoskeleton and Controls Proliferation.

Degeneration of the intervertebral disc, which is closely associated with the loss of vacuolated notochordal nucleus pulposus cells (NNPC), remains a major cause of lower-back pain and motor deficiency. Being the most defining characteristic of NNPC, large cytoplasmic vacuoles not only modulate the cytoskeleton and shape cell morphology but they also respond to the disc microenvironment and regulate the biological behavior of vacuolated cells as a potent reporter of the histocytological changes that occur at the beginning of disc aging and degeneration. Here we hypothesize a model in which large cytoplasmic vacuoles primarily function to maintain a reasonable intracellular pressure (Pv) that facilitates NNPC in resisting the extracellular mechanical loading (Pe), part of which is absorbed by the extracellular matrix (Pm), forming the equation Pe = Pm + Pv. By mimicking a situation of contact-induced growth inhibition, the crowded cytoplasmic vacuoles slow down the proliferation of NNPC and restrain the generation of nonvacuolated chondrocytic nucleus pulposus cells (CNPC), whereas increased mechanical loading (↑Pe) alters cytoskeletons and breaches cytoplasmic vacuoles, which in turn weakens the vacuoles-mediated proliferation check, increases the generation of CNPC that accumulates fibrocartilaginous matrix, and rebalances the increased loading with elevated Pm (↑Pm) and lowered Pv (↓Pv), equating to ↑Pe = ↑Pm + ↓Pv. By depicting the biological function and the disappearance of the cytoplasmic vacuoles, our model highlights a mechanical exhaustion of the notochordal cell resources, which might help to elucidate the histocytological changes that initiate disc aging and degeneration.

Postnatal Development of the Murine Notochord Remnants Quantified by High-resolution Contrast-enhanced MicroCT.

The notochord gives rise to spinal segments during development, and it becomes embedded within the nucleus pulposus of the intervertebral disc (IVD) during maturation. The disruption of the notochord band has been observed with IVD degeneration. Since the mechanical competence of the IVD relies on its structural constituents, defining the structure of the notochord during aging is critical for investigations relating to IVD function and homeostasis. The assessment and imaging of the notochord has classically relied on histological techniques, which introduces sectioning artifacts during preparation and spatial biases. Magnetic resonance imaging (MRI) does not offer sufficient resolution to discriminate the notochord from the surrounding the nucleus pulposus, especially in murine models. Current X-ray based computed tomography systems provide imaging resolutions down to the single- and sub- micron scales, and when coupled with contrast-enhancing agents, enable the high-resolution three-dimensional imaging of relatively small features. Utilizing phosphomolybdic acid to preferentially bind to collagen cationic domains, we describe the structure of the notochord remnants with aging in the lumbar IVDs of BALB/c mice. These results provide a highly quantitative and sensitive approach to monitoring the IVD during postnatal development.

Identification of a new mineralized tissue in the notochord of reared Siberian sturgeon (Acipenser baerii).

In a study aiming to improve knowledge on the mineralization of the axial skeleton in reared Siberian sturgeon (Acipenser baerii Brandt, 1869), we discovered a new mineralized tissue within the notochord. To our knowledge, such a structure has never been reported in any vertebrate species with the exception of the pathological mineralization of the notochord remains in degenerative intervertebral disks of mammals. Here, we describe this enigmatic tissue using X-ray microtomography, histological analyses and solid state NMR-spectroscopy. We also performed a 1-year monitoring of the mineral content (MC) of the notochord in relation with seasonal variations of temperature. In all specimens studied from 2-year-old juveniles onwards, this mineralized structure was found within a particular region of the notochord called funiculus. This feature first appears in the abdominal region then extends posteriorly with ageing, while the notochord MC also increases. The mineral phase is mainly composed of amorphous calcium phosphate, a small amount of which changes into hydroxyapatite with ageing. The putative role of this structure is discussed as either a store of minerals available for the phosphocalcic metabolism, or a mechanical support in a species with a poorly mineralized axial skeleton. A pathological feature putatively related to rearing conditions is also discussed.

Paraxial Nodal Expression Reveals a Novel Conserved Structure of the Left-Right Organizer in Four Mammalian Species.

Nodal activity in the left lateral plate mesoderm is a conserved sign of irreversible left-right asymmetry at early somite stages of the vertebrate embryo. An earlier, paraxial nodal domain accompanies the emergence and initial extension of the notochord and is either left-sided, as in the chick and pig, or symmetrical, as in the mouse and rabbit; intriguingly, this interspecific dichotomy is mirrored by divergent morphological features of the posterior notochord (also known as the left-right organizer), which is ventrally exposed to the yolk sac cavity and carries motile cilia in the latter 2 species only. By introducing the cattle embryo as a new model organism for early left-right patterning, we present data to establish 2 groups of mammals characterized by both the morphology of the left-right organizer and the dynamics of paraxial nodal expression: presence and absence of a ventrally open surface of the early (plate-like) posterior notochord correlates with a symmetrical (in mice and rabbits) versus an asymmetrical (in pigs and cattle) paraxial nodal expression domain next to the notochordal plate. High-resolution histological analysis reveals that the latter domain defines in all 4 mammals a novel 'parachordal' axial mesoderm compartment, the topography of which changes according to the specific regression of the similarly novel subchordal mesoderm during the initial phases of notochord development. In conclusion, the mammalian axial mesoderm compartment (1) shares critical conserved features despite the marked differences in early notochord morphology and early left-right patterning and (2) provides a dynamic topographical framework for nodal activity as part of the mammalian left-right organizer.

Xenopus in Space and Time: Fossils, Node Calibrations, Tip-Dating, and Paleobiogeography.

Published data from DNA sequences, morphology of 11 extant and 15 extinct frog taxa, and stratigraphic ranges of fossils were integrated to open a window into the deep-time evolution of Xenopus. The ages and morphological characters of fossils were used as independent datasets to calibrate a chronogram. We found that DNA sequences, either alone or in combination with morphological data and fossils, tended to support a close relationship between Xenopus and Hymenochirus, although in some analyses this topology was not significantly better than the Pipa + Hymenochirus topology. Analyses that excluded DNA data found strong support for the Pipa + Hymenochirus tree. The criterion for selecting the maximum age of the calibration prior influenced the age estimates, and our age estimates of early divergences in the tree of frogs are substantially younger than those of published studies. Node-dating and tip-dating calibrations, either alone or in combination, yielded older dates for nodes than did a root calibration alone. Our estimates of divergence times indicate that overwater dispersal, rather than vicariance due to the splitting of Africa and South America, may explain the presence of Xenopus in Africa and its closest fossil relatives in South America.

Extended ultrastructural characterization of chordoma cells: the link to new therapeutic options.

Chordomas are rare bone tumors, developed from the notochord and largely resistant to chemotherapy. A special feature of this tumor is the heterogeneity of its cells. By combining high pressure freezing (HPF) with electron tomography we were able to illustrate the connections within the cells, the cell-cell interface, and the mitochondria-associated endoplasmic reticulum membrane complex that appears to play a special role among the characteristics of chordoma. These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling. Compared to other tumor cells, chordoma cells show a close connection of rough endoplasmic reticulum and mitochondria, which may influence the sphingolipid metabolism and calcium release. We quantified levels of ceramide and glycosylceramide species by the methyl tert-butyl ether extraction method and we assessed the intracellular calcium concentration with the ratiometric fluorescent dye Fura-2AM. Measurements of the changes in the intracellular calcium concentration revealed an increase in calcium due to the application of acetylcholine. With regard to lipid synthesis, glucosylceramide levels in the chordoma cell line were significantly higher than those in normal healthy cells. The accumulation of glycosylceramide in drug resistant cancer cells has been confirmed in many types of cancer and may also account for drug resistance in chordoma. This study aimed to provide a deep morphological description of chordoma cells, it demonstrated that HPF analysis is useful in elucidating detailed structural information. Furthermore we demonstrate how an accumulation of glycosylceramide in chordoma provides links to drug resistance and opens up the field for new research options.

Transient infection of the zebrafish notochord with E. coli induces chronic inflammation.

Zebrafish embryos and larvae are now well-established models in which to study infectious diseases. Infections with non-pathogenic Gram-negative Escherichia coli induce a strong and reproducible inflammatory response. Here, we study the cellular response of zebrafish larvae when E. coli bacteria are injected into the notochord and describe the effects. First, we provide direct evidence that the notochord is a unique organ that is inaccessible to leukocytes (macrophages and neutrophils) during the early stages of inflammation. Second, we show that notochord infection induces a host response that is characterised by rapid clearance of the bacteria, strong leukocyte recruitment around the notochord and prolonged inflammation that lasts several days after bacteria clearance. During this inflammatory response, il1b is first expressed in macrophages and subsequently at high levels in neutrophils. Moreover, knock down of il1b alters the recruitment of neutrophils to the notochord, demonstrating the important role of this cytokine in the maintenance of inflammation in the notochord. Eventually, infection of the notochord induces severe defects of the notochord that correlate with neutrophil degranulation occurring around this tissue. This is the first in vivo evidence that neutrophils can degranulate in the absence of a direct encounter with a pathogen. Persistent inflammation, neutrophil infiltration and restructuring of the extracellular matrix are defects that resemble those seen in bone infection and in some chondropathies. As the notochord is a transient embryonic structure that is closely related to cartilage and bone and that contributes to vertebral column formation, we propose infection of the notochord in zebrafish larvae as a new model to study the cellular and molecular mechanisms underlying cartilage and bone inflammation.

An equatorial contractile mechanism drives cell elongation but not cell division.

Cell shape changes and proliferation are two fundamental strategies for morphogenesis in animal development. During embryogenesis of the simple chordate Ciona intestinalis, elongation of individual notochord cells constitutes a crucial stage of notochord growth, which contributes to the establishment of the larval body plan. The mechanism of cell elongation is elusive. Here we show that although notochord cells do not divide, they use a cytokinesis-like actomyosin mechanism to drive cell elongation. The actomyosin network forming at the equator of each notochord cell includes phosphorylated myosin regulatory light chain, α-actinin, cofilin, tropomyosin, and talin. We demonstrate that cofilin and α-actinin are two crucial components for cell elongation. Cortical flow contributes to the assembly of the actomyosin ring. Similar to cytokinetic cells, membrane blebs that cause local contractions form at the basal cortex next to the equator and participate in force generation. We present a model in which the cooperation of equatorial actomyosin ring-based constriction and bleb-associated contractions at the basal cortex promotes cell elongation. Our results demonstrate that a cytokinesis-like contractile mechanism is co-opted in a completely different developmental scenario to achieve cell shape change instead of cell division. We discuss the occurrences of actomyosin rings aside from cell division, suggesting that circumferential contraction is an evolutionally conserved mechanism to drive cell or tissue elongation.

A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12.

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer.

Anion translocation through an Slc26 transporter mediates lumen expansion during tubulogenesis.

Lumen formation is a critical event in biological tube formation, yet its molecular mechanisms remain poorly understood. Specifically, how lumen expansion is coordinated with other processes of tubulogenesis is not well known, and the role of membrane transporters in tubulogenesis during development has not been adequately addressed. Here we identify a solute carrier 26 (Slc26) family protein as an essential regulator of tubulogenesis using the notochord of the invertebrate chordate Ciona intestinalis as a model. Ci-Slc26aα is indispensable for lumen formation and expansion, but not for apical/luminal membrane formation and lumen connection. Ci-Slc26aα acts as an anion transporter, mediating the electrogenic exchange of sulfate or oxalate for chloride or bicarbonate and electroneutral chloride:bicarbonate exchange. Mutant rescue assays show that this transport activity is essential for Ci-Slc26aα's in vivo function. Our work reveals the consequences and relationships of several key processes in lumen formation, and establishes an in vivo assay for studying the molecular basis of the transport properties of SLC26 family transporters and their related diseases.

Variation in the schedules of somite and neural development in frogs.

The timing of notochord, somite, and neural development was analyzed in the embryos of six different frog species, which have been divided into two groups, according to their developmental speed. Rapid developing species investigated were Xenopus laevis (Pipidae), Engystomops coloradorum, and Engystomops randi (Leiuperidae). The slow developers were Epipedobates machalilla and Epipedobates tricolor (Dendrobatidae) and Gastrotheca riobambae (Hemiphractidae). Blastopore closure, notochord formation, somite development, neural tube closure, and the formation of cranial neural crest cell-streams were detected by light and scanning electron microscopy and by immuno-histochemical detection of somite and neural crest marker proteins. The data were analyzed using event pairing to determine common developmental aspects and their relationship to life-history traits. In embryos of rapidly developing frogs, elongation of the notochord occurred earlier relative to the time point of blastopore closure in comparison with slowly developing species. The development of cranial neural crest cell-streams relative to somite formation is accelerated in rapidly developing frogs, and it is delayed in slowly developing frogs. The timing of neural tube closure seemed to be temporally uncoupled with somite formation. We propose that these changes are achieved through differential timing of developmental modules that begin with the elongation of the notochord during gastrulation in the rapidly developing species. The differences might be related to the necessity of developing a free-living tadpole quickly in rapid developers.

Muscle development and differentiation in the urodele Ambystoma mexicanum.

Muscle differentiation has been widely described in zebrafish and Xenopus, but nothing is known about this process in amphibian urodeles. Both anatomical features and locomotor activity in urodeles are known to show intermediate features between fish and anurans. Therefore, a better understanding of myogenesis in urodeles could be useful to clarify the evolutionary changes that led to the formation of skeletal muscle in the trunk of land vertebrates. We report here a detailed morphological and molecular investigation on several embryonic stages of Ambystoma mexicanum and show that the first differentiating muscle fibers are the slow ones, originating from a myoblast population initially localized close to the notochord that forms a superficial layer on the somitic surface afterwards. Subsequently, fast fibers differentiation ensues. We also identified and cloned A. mexicanum Myf5 as a muscle-specific transcriptional factor likely involved in urodele muscle differentiation.

Non-enzymatic decomposition of collagen fibers by a biglycan antibody and a plausible mechanism for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory and destructive joint disorder that affects tens of millions of people worldwide. Normal healthy joints maintain a balance between the synthesis of extracellular matrix (ECM) molecules and the proteolytic degradation of damaged ones. In the case of RA, this balance is shifted toward matrix destruction due to increased production of cleavage enzymes and the presence of (autoimmune) immunoglobulins resulting from an inflammation induced immune response. Herein we demonstrate that a polyclonal antibody against the proteoglycan biglycan (BG) causes tissue destruction that may be analogous to that of RA affected tissues. The effect of the antibody is more potent than harsh chemical and/or enzymatic treatments designed to mimic arthritis-like fibril de-polymerization. In RA cases, the immune response to inflammation causes synovial fibroblasts, monocytes and macrophages to produce cytokines and secrete matrix remodeling enzymes, whereas B cells are stimulated to produce immunoglobulins. The specific antigen that causes the RA immune response has not yet been identified, although possible candidates have been proposed, including collagen types I and II, and proteoglycans (PG's) such as biglycan. We speculate that the initiation of RA associated tissue destruction in vivo may involve a similar non-enzymatic decomposition of collagen fibrils via the immunoglobulins themselves that we observe here ex vivo.

Identification of vimentin- and elastin-like transcripts specifically expressed in developing notochord of Atlantic salmon (Salmo salar L.).

The notochord functions as the midline structural element of all vertebrate embryos, and allows movement and growth at early developmental stages. Moreover, during embryonic development, notochord cells produce secreted factors that provide positional and fate information to a broad variety of cells within adjacent tissues, for instance those of the vertebrae, central nervous system and somites. Due to the large size of the embryo, the salmon notochord is useful to study as a model for exploring notochord development. To investigate factors that might be involved in notochord development, a normalized cDNA library was constructed from a mix of notochords from ∼500 to ∼800 day°. From the 1968 Sanger-sequenced transcripts, 22 genes were identified to be predominantly expressed in the notochord compared to other organs of salmon. Twelve of these genes were found to show expressional regulation around mineralization of the notochord sheath; 11 genes were up-regulated and one gene was down-regulated. Two genes were found to be specifically expressed in the notochord; these genes showed similarity to vimentin (acc. no GT297094) and elastin (acc. no GT297478). In-situ results showed that the vimentin- like transcript was expressed in both chordocytes and chordoblasts, whereas the elastin- like transcript was uniquely expressed in the chordoblasts lining the notochordal sheath. In salmon aquaculture, vertebral deformities are a common problem, and some malformations have been linked to the notochord. The expression of identified transcripts provides further insight into processes taking place in the developing notochord, prior to and during the early mineralization period.

Developmental toxicity of the environmental pollutant 4-nonylphenol in zebrafish.

4-Nonylphenol (4-NP), an estrogen mimicking compound is produced by biodegradation of alkylethoxylates. It is well established that 4-NP can affect the development of aquatic animals by disrupting the endocrine signals. Here we show for the first time in zebrafish that 4-NP does not only target the neuroendocrine system but also the notochord and the muscle. The notochord malformation was first evident as distortions at 24hourspostfertilization (hpf) which within 24h appeared as kinks and herniations. The notochord phenotype was accompanied by reduced motility and impaired swimming behavior. Whole-mount in situ hybridization using chordamesoderm markers and electron microscopic analysis showed failure in the notochord differentiation and disruption of the perinotochordal basement membrane. Late larval stages of 4-NP treated embryos displayed abnormal mineralization, vertebral curvature, fusion of vertebral bodies and abnormal extension of haemal arches. The muscle structure and the maximal active force in isolated muscle preparations were similar between 4-NP exposed and of control embryos, suggesting that 4-NP did not induce major changes in striated muscle function. However, repeated electrical stimulation (>40Hz) of the 4-NP exposed larvae revealed an impaired relaxation between stimuli, possibly reflecting an alteration in the relaxant mechanisms (e.g. in cellular Ca(2+) removal) which could explain the abnormal swimming pattern exhibited by 4-NP exposed larvae. Additionally, we demonstrate that the expression levels of the stress hormone, corticotropin releasing hormonewere elevated in the brain following 4-NP treatment. We also observed a significant decrease in the transcript levels of luteinizing hormone b at early larval stages. Collectively, our results show that 4-NP is able to disrupt the notochord morphogenesis, muscle function and the neuroendocrine system. These data suggest that 4-NP enduringly affects the embryonic development in zebrafish and that this compound might exert these deleterious effects through diverse signaling pathways.

Cilia-like structures anchor the amphioxus notochord to its sheath.

Body stiffness is important during undulatory locomotion in fish. In amphioxus, the myosepta play an important role in transmission of muscular forces to the notochord. In order to define the specific supporting role of the notochord in amphioxus during locomotion, the ultrastructure of 10 adult amphioxus specimens was analyzed using transmission electron microscopy. Numerous cilia-like structures were found on the surface of each notochordal cell at the sites of their attachment to the notochordal sheath. Ultrastructurally, these structures consisted of the characteristic arrangement of peripheral and central microtubular doublets and were anchored to the inner layer of the notochordal sheath. Immunohistochemically, a positive reaction to applied dynein and ß-tubulin antibodies characterized the area of the cilia-like structures. We propose that reduced back-and-forth movements of the cilia-like structures might contribute to the flow of the fluid content inside the notochord, thus modulating the stiffness of the amphioxus body during its undulatory locomotion.

Critical early roles for col27a1a and col27a1b in zebrafish notochord morphogenesis, vertebral mineralization and post-embryonic axial growth.

BACKGROUND: Fibrillar collagens are well known for their links to human diseases, with which all have been associated except for the two most recently identified fibrillar collagens, type XXIV collagen and type XXVII collagen. To assess functions and potential disease phenotypes of type XXVII collagen, we examined its roles in zebrafish embryonic and post-embryonic development. METHODOLOGY/PRINCIPAL FINDINGS: We identified two type XXVII collagen genes in zebrafish, col27a1a and col27a1b. Both col27a1a and col27a1b were expressed in notochord and cartilage in the embryo and early larva. To determine sites of type XXVII collagen function, col27a1a and col27a1b were knocked down using morpholino antisense oligonucleotides. Knockdown of col27a1a singly or in conjunction with col27a1b resulted in curvature of the notochord at early stages and formation of scoliotic curves as well as dysmorphic vertebrae at later stages. These defects were accompanied by abnormal distributions of cells and protein localization in the notochord, as visualized by transmission electron microscopy, as well as delayed vertebral mineralization as detected histologically. CONCLUSIONS/SIGNIFICANCE: Together, our findings indicate a key role for type XXVII collagen in notochord morphogenesis and axial skeletogenesis and suggest a possible human disease phenotype.

Essential role for the alpha 1 chain of type VIII collagen in zebrafish notochord formation.

Several zebrafish mutants identified in large-scale forward genetic screens exhibit notochord distortion. We now report the cloning and further characterization of one such mutant, gulliver(m208) (gul(m208)). The notochord defect in gul(m208) mutants is exacerbated under conditions of copper depletion or lysyl oxidase cuproenzyme inhibition that are without a notochord effect on wild-type embryos. The gul(m208) phenotype results from a missense mutation in the gene encoding Col8a1, a lysyl oxidase substrate, and morpholino knockdown of col8a1 recapitulates the notochord distortion observed in gul(m208) mutants. Of interest, the amino acid mutated in gul(m208) Col8a1 is highly conserved, and the equivalent substitution in a closely related human protein, COL10A1, causes Schmid metaphyseal chondrodysplasia. Taken together, the data identify a new protein essential for notochord morphogenesis, extend our understanding of gene-nutrient interactions in early development, and suggest that human mutations in COL8A1 may cause structural birth defects.