Reconstruction and deconstruction of human somitogenesis in vitro.

The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm. Somites then become subdivided into anterior and posterior compartments that are essential for vertebral formation and segmental patterning of the peripheral nervous system1-4. How this key somitic subdivision is established remains poorly understood. Here we introduce three-dimensional culture systems of human pluripotent stem cells called somitoids and segmentoids, which recapitulate the formation of somite-like structures with anteroposterior identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial 'salt and pepper' expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity through an active cell-sorting mechanism. Our research demonstrates that the major patterning modules that are involved in somitogenesis, including the clock and wavefront, anteroposterior polarity patterning and somite epithelialization, can be dissociated and operate independently in our in vitro systems. Together, we define a framework for the symmetry-breaking process that initiates somite polarity patterning. Our work provides a platform for decoding general principles of somitogenesis and advancing knowledge of human development.

A mechanism for gene-environment interaction in the etiology of congenital scoliosis.

Congenital scoliosis, a lateral curvature of the spine caused by vertebral defects, occurs in approximately 1 in 1,000 live births. Here we demonstrate that haploinsufficiency of Notch signaling pathway genes in humans can cause this congenital abnormality. We also show that in a mouse model, the combination of this genetic risk factor with an environmental condition (short-term gestational hypoxia) significantly increases the penetrance and severity of vertebral defects. We demonstrate that hypoxia disrupts FGF signaling, leading to a temporary failure of embryonic somitogenesis. Our results potentially provide a mechanism for the genesis of a host of common sporadic congenital abnormalities through gene-environment interaction.

Vertebrate segmentation: from cyclic gene networks to scoliosis.

One of the most striking features of the human vertebral column is its periodic organization along the anterior-posterior axis. This pattern is established when segments of vertebrates, called somites, bud off at a defined pace from the anterior tip of the embryo's presomitic mesoderm (PSM). To trigger this rhythmic production of somites, three major signaling pathways--Notch, Wnt/ß-catenin, and fibroblast growth factor (FGF)--integrate into a molecular network that generates a traveling wave of gene expression along the embryonic axis, called the "segmentation clock." Recent systems approaches have begun identifying specific signaling circuits within the network that set the pace of the oscillations, synchronize gene expression cycles in neighboring cells, and contribute to the robustness and bilateral symmetry of somite formation. These findings establish a new model for vertebrate segmentation and provide a conceptual framework to explain human diseases of the spine, such as congenital scoliosis.

Zebrafish model for spondylo-megaepiphyseal-metaphyseal dysplasia reveals post-embryonic roles of Nkx3.2 in the skeleton.

The regulated expansion of chondrocytes within growth plates and joints ensures proper skeletal development through adulthood. Mutations in the transcription factor NKX3.2 underlie spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD), which is characterized by skeletal defects including scoliosis, large epiphyses, wide growth plates and supernumerary distal limb joints. Whereas nkx3.2 knockdown zebrafish and mouse Nkx3.2 mutants display embryonic lethal jaw joint fusions and skeletal reductions, respectively, they lack the skeletal overgrowth seen in SMMD patients. Here, we report adult viable nkx3.2 mutant zebrafish displaying cartilage overgrowth in place of a missing jaw joint, as well as severe dysmorphologies of the facial skeleton, skullcap and spine. In contrast, cartilage overgrowth and scoliosis are absent in rare viable nkx3.2 knockdown animals that lack jaw joints, supporting post-embryonic roles for Nkx3.2. Single-cell RNA-sequencing and in vivo validation reveal increased proliferation and upregulation of stress-induced pathways, including prostaglandin synthases, in mutant chondrocytes. By generating a zebrafish model for the skeletal overgrowth defects of SMMD, we reveal post-embryonic roles for Nkx3.2 in dampening proliferation and buffering the stress response in joint-associated chondrocytes.

The antioxidant enzyme Prdx1 controls neuronal differentiation by thiol-redox-dependent activation of GDE2.

The six-transmembrane protein GDE2 controls the onset and progression of spinal motor neuron differentiation through extracellular glycerophosphodiester phosphodiesterase metabolism. Although this process is likely to be tightly regulated, the relevant mechanisms that modulate its activity are unknown. Here we show that the antioxidant scavenger peroxiredoxin1 (Prdx1) interacts with GDE2, and that loss of Prdx1 causes motor neuron deficits analogous to GDE2 ablation. Prdx1 cooperates with GDE2 to drive motor neuron differentiation, and this synergy requires Prdx1 thiol-dependent catalysis. Prdx1 activates GDE2 through reduction of an intramolecular disulfide bond that bridges its intracellular N- and C-terminal domains. GDE2 variants incapable of disulfide bond formation acquire independence from Prdx1 and are potent inducers of motor neuron differentiation. These findings define Prdx1 as a pivotal regulator of GDE2 activity and suggest roles for coupled thiol-redox-dependent cascades in controlling neuronal differentiation in the spinal cord.

A Spatio-Temporal-Dependent Requirement of Sonic Hedgehog in the Early Development of Sclerotome-Derived Vertebrae and Ribs.

Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome differentiation. Through conditional loss-of-function in quail embryos, we investigate the timing and impact of Shh activity during early formation of sclerotome-derived vertebrae and ribs, and of lateral mesoderm-derived sternum. To this end, Hedgehog interacting protein (Hhip) was electroporated at various times between days 2 and 5. While the vertebral body and rib primordium showed consistent size reduction, rib expansion into the somatopleura remained unaffected, and the sternal bud developed normally. Additionally, we compared these effects with those of locally inhibiting BMP activity. Transfection of Noggin in the lateral mesoderm hindered sternal bud formation. Unlike Hhip, BMP inhibition via Noggin or Smad6 induced myogenic differentiation of the lateral dermomyotome lip, while impeding the growth of the myotome/rib complex into the somatic mesoderm, thus affirming the role of the lateral dermomyotome epithelium in rib guidance. Overall, these findings underscore the continuous requirement for opposing gradients of Shh and BMP activity in the morphogenesis of proximal and distal flank skeletal structures, respectively. Future research should address the implications of these early interactions to the later morphogenesis and function of the musculo-skeletal system and of possible associated malformations.

The axonemal dynein heavy chain 10 gene is essential for monocilia motility and spine alignment in zebrafish.

Adolescent idiopathic scoliosis (AIS) is a common pediatric musculoskeletal disorder worldwide, characterized by atypical spine curvatures in otherwise healthy children. Human genetic studies have identified candidate genes associated with AIS, however, only a few of these have been shown to recapitulate adult-viable scoliosis in animal models. Using an F0 CRISPR screening approach in zebrafish, we demonstrate that disruption of the dynein axonemal heavy chain 10 (dnah10) gene results in recessive adult-viable scoliosis in zebrafish. Using a stably segregating dnah10 mutant zebrafish, we showed that the ependymal monocilia lining the hindbrain and spinal canal displayed reduced beat frequency, which was correlated with the disassembly of the Reissner fiber and the onset of body curvatures. Taken together, these results suggest that monocilia function in larval zebrafish contributes to the polymerization of the Reissner fiber and straightening of the body axis.

Development of a straight vertebrate body axis.

The vertebrate body plan is characterized by the presence of a segmented spine along its main axis. Here, we examine the current understanding of how the axial tissues that are formed during embryonic development give rise to the adult spine and summarize recent advances in the field, largely focused on recent studies in zebrafish, with comparisons to amniotes where appropriate. We discuss recent work illuminating the genetics and biological mechanisms mediating extension and straightening of the body axis during development, and highlight open questions. We specifically focus on the processes of notochord development and cerebrospinal fluid physiology, and how defects in those processes may lead to scoliosis.

ß1 Integrin regulates convergent extension in mouse notogenesis, ensures notochord integrity and the morphogenesis of vertebrae and intervertebral discs.

The notochord drives longitudinal growth of the body axis by convergent extension, a highly conserved developmental process that depends on non-canonical Wnt/planar cell polarity (PCP) signaling. However, the role of cell-matrix interactions mediated by integrins in the development of the notochord is unclear. We developed transgenic Cre mice, in which the ß1 integrin gene (Itgb1) is ablated at E8.0 in the notochord only or in the notochord and tail bud. These Itgb1 conditional mutants display misaligned, malformed vertebral bodies, hemi-vertebrae and truncated tails. From early somite stages, the notochord was interrupted and displaced in these mutants. Convergent extension of the notochord was impaired with defective cell movement. Treatment of E7.25 wild-type embryos with anti-ß1 integrin blocking antibodies, to target node pit cells, disrupted asymmetric localization of VANGL2. Our study implicates pivotal roles of ß1 integrin for the establishment of PCP and convergent extension of the developing notochord, its structural integrity and positioning, thereby ensuring development of the nucleus pulposus and the proper alignment of vertebral bodies and intervertebral discs. Failure of this control may contribute to human congenital spine malformations.

Why a Constant Number of Vertebrae? Digital Control of Segmental Identity during Vertebrate Development: The Somite Cycle Controls a Digital, Chromatin-Based Counter That Defines Segmental Identity and Body Plans in Vertebrate Animals.

It is not understood how the numbers and identities of vertebrae are controlled during mammalian development. The remarkable robustness and conservation of segmental numbers may suggest the digital nature of the underlying process. The study proposes a mechanism that allows cells to obtain and store the segmental information in digital form, and to produce a pattern of chromatin accessibility that in turn regulates Hox gene expression specific to the metameric segment. The model requires that a regulatory element be present such that the number of occurrences of the motif between two consecutive Hox genes equals the number of segments under the control of the anterior gene. This is true for the recently discovered hydroxyl radical cleavage 3bp-periodic (HRC3) motif, associated with histone modifications and developmental genes. The finding not only allows the correct prediction of the numbers of segments using only sequence information, but also resolves the 40-year-old enigma of the function of temporal and spatial collinearity of Hox genes. The logic of the mechanism is illustrated in the attached animated video. How different aspects of the proposed mechanism can be tested experimentally is also discussed.

Development and regeneration dynamics of the Medaka notochord.

The notochord is an embryonic tissue that acts as a hydrostatic skeleton until ossification begins in vertebrates. It is composed of outer sheath cells and inner vacuolated cells, which are generated from a common pool of disc-shaped precursors. Notochord extension during early embryogenesis is driven by the growth of vacuolated cells, reflecting in turn the expansion of their inner vacuole. Here we use desmogon, a novel desmosomal cadherin, to follow notochord development and regeneration in medaka (Oryzias latipes). We trace desmogon â€‹+ disc-shaped precursors at the single cell level to demonstrate that they operate as unipotent progenitors, giving rise to either sheath or vacuolated cells. We reveal that once specified, vacuolated cells grow asynchronously and drive notochord expansion bi-directionally. Additionally, we uncover distinct regenerative responses in the notochord, which depend on the nature of the injury sustained. By generating a desmogon CRISPR mutant we demonstrate that this cadherin is essential for proper vacuolated cell shape and therefore correct notochord and spine morphology. Our work expands the repertoire of model systems to study dynamic aspects of the notochord in vivo, and provides new insights in its development and regeneration properties.

Direct activation of chordoblasts by retinoic acid is required for segmented centra mineralization during zebrafish spine development.

Zebrafish mutants with increased retinoic acid (RA) signaling due to the loss of the RA-inactivating enzyme Cyp26b1 develop a hyper-mineralized spine with gradually fusing vertebral body precursors (centra). However, the underlying cellular mechanisms remain incompletely understood. Here, we show that cells of the notochord epithelium named chordoblasts are sensitive to RA signaling. Chordoblasts are uniformly distributed along the anteroposterior axis and initially generate the continuous collagenous notochord sheath. However, subsequently and iteratively, subsets of these cells undergo further RA-dependent differentiation steps, acquire a stellate-like shape, downregulate expression of the collagen gene col2a1a, switch on cyp26b1 expression and trigger metameric sheath mineralization. This mineralization fails to appear upon chordoblast-specific cell ablation or RA signal transduction blockade. Together, our data reveal that, despite their different developmental origins, the activities and regulation of chordoblasts are very similar to those of osteoblasts, including their RA-induced transition from osteoid-producing cells to osteoid-mineralizing ones. Furthermore, our data point to a requirement for locally controlled RA activity within the chordoblast layer in order to generate the segmented vertebral column.

Unveiling the tale of the tail: an illustration of spinal dysraphisms.

Spinal dysraphism is an umbrella term describing herniation of meninges or neural elements through defective neural arch. They can be broadly categorized into open and closed types. MRI is the investigation of choice to study neural abnormalities and to assess the severity of hydrocephalus and Chiari malformation. Knowledge of the embryology of these disorders is valuable in correctly identifying the type of dysraphism. The aim of surgery is untethering and dural reconstruction. Accurate depiction of the abnormal anatomy in cases of spinal dysraphism is of utmost importance for surgical management of these patients. MRI makes this possible due to its excellent soft tissue contrast resolution and multiplanar capability, allowing the radiologist to evaluate the intricate details in small pediatric spinal structures. Imaging enlightens the surgeons about the status of spinal cord and other associated abnormalities and helps detect re-tethering in operated cases. Besides, antenatal surgery to repair myelomeningoceles has made detection of open dysraphisms on fetal MRI and antenatal ultrasound critical. The purpose of this review is to describe the development of spine, illustrate the myriad imaging features of open and closed spinal dysraphisms, and enlist the reporting points the operating surgeon seeks from the radiologist.

Patterns of intracolumnar size variation inform the heterochronic mechanisms underlying extreme body shape divergence in microcephalic sea snakes.

Sea snakes (Hydrophiinae) that specialize on burrowing eel prey have repeatedly evolved tiny heads and reduced forebody relative to hindbody girths. Previous research has found that these "microcephalic" forms have higher counts of precaudal vertebrae, and postnatal ontogenetic changes cause their hindbodies to reach greater girths relative to their forebodies. We examine variation in vertebral size along the precaudal axis of neonates and adults of three species. In the nonmicrocephalic Hydrophis curtus, these intracolumnar patterns take the form of symmetrical curved profiles, with longer vertebrae in the midbody (50% of body length) relative to distal regions. In contrast, intracolumnar profiles in the microcephalic H. macdowelli and H. obscurus are strongly asymmetrical curves (negative skewness) due to the presence of numerous, smaller-sized vertebrate in the forebody (anterior to the heart). Neonate and adult H. macdowelli and H. obscurus specimens all exhibit this pattern, implying an onset of fore- versus hindbody decoupling in the embryo stage. Based on this, we suggest plausible developmental mechanisms involving the presence and positioning of Hox boundaries and heterochronic changes in segmentation. Tests of our hypotheses would give new insights into the drivers of rapid convergent shifts in evolution, but will ultimately require studies of gene expression in the embryos of relevant taxa.

Non-neural surface ectodermal rosette formation and F-actin dynamics drive mammalian neural tube closure.

The mechanisms underlying mammalian neural tube closure remain poorly understood. We report a unique cellular process involving multicellular rosette formation, convergent cellular protrusions, and F-actin cable network of the non-neural surface ectodermal cells encircling the closure site of the posterior neuropore, which are demonstrated by scanning electron microscopy and genetic fate mapping analyses during mouse spinal neurulation. These unique cellular structures are severely disrupted in the surface ectodermal transcription factor Grhl3 mutants that exhibit fully penetrant spina bifida. We propose a novel model of mammalian neural tube closure driven by surface ectodermal dynamics, which is computationally visualized.

High-resolution diffusion MRI studies of development in pregnant mice visualized by novel spatiotemporal encoding schemes.

This study introduces an MRI approach to map diffusion of water in vivo with high resolution under challenging conditions; the approach's potential is then used in diffusivity characterizations of embryos and fetoplacental units in pregnant mice, as well as of newborn mice in their initial postnatal period. The method relies on performing self-referenced spatiotemporal encoded MRI acquisitions, which can achieve the motional and susceptibility immunities needed to target challenging regions such as a mouse's abdominal cavity in a single shot. When suitably combined with zooming-in and novel interleaving procedures, these scans can overcome the inhomogeneity and sensitivity challenges arising upon targeting ≈100 µm in-plane resolutions, and thereby enable longitudinal development studies of abdominal organs that have hitherto eluded in vivo diffusion-weighted imaging. This is employed here to follow processes related to embryonic implantation and placentation, including the final stages of mouse gastrulation, the development of white matter in fetal brains, the maturation of fetal spines, and the evolution of the different layers making up mouse hemochorial placentas. The protocol's ability to extract diffusivity information in challenging regions as a function of embryonic mouse development is thus demonstrated, and its usefulness as a tool for visualizing pregnancy-related developmental changes in rodents is discussed.

Human embryonic ribs all progress through common morphological forms irrespective of their position on the axis.

BACKGROUND: We aimed to analyze the morphogenesis of all ribs from 1st to 12th rib pairs plus vertebrae to compare their differences and features according to the position along the cranial-caudal axis during the human embryonic period. RESULTS: Rib pair formation was analyzed using high-resolution digitalized imaging data (n = 29) between Carnegie stage (CS) 18 and CS23 (corresponding to ED13-14 in mouse; HH29-35 in chick). A total of 348 rib pairs, from 1st to 12th rib pairs of each sample were subjected to Procrustes and principal component (PC) analyses. PC1 and PC2 accounted for 76.3% and 16.4% (total 92.7%) of the total variance, respectively, indicating that two components mainly accounted for the change in shape. The distribution of PC1 and PC2 values for each rib showed a "fishhook-like shape" upon fitting to a quartic equation. PC1 and PC2 value position for each rib pair moved along the fitted curve according to the development. Thus, the change in PC1 and PC2 could be expressed by a single parameter using a fitted curve as a linear scale for shape. CONCLUSION: Human embryonic ribs all progress through common morphological forms irrespective of their position on the axis.

The role of the notochord in amniote vertebral column segmentation.

The vertebral column is segmented, comprising an alternating series of vertebrae and intervertebral discs along the head-tail axis. The vertebrae and outer portion (annulus fibrosus) of the disc are derived from the sclerotome part of the somites, whereas the inner nucleus pulposus of the disc is derived from the notochord. Here we investigate the role of the notochord in vertebral patterning through a series of microsurgical experiments in chick embryos. Ablation of the notochord causes loss of segmentation of vertebral bodies and discs. However, the notochord cannot segment in the absence of the surrounding sclerotome. To test whether the notochord dictates sclerotome segmentation, we grafted an ectopic notochord. We find that the intrinsic segmentation of the sclerotome is dominant over any segmental information the notochord may possess, and no evidence that the chick notochord is intrinsically segmented. We propose that the segmental pattern of vertebral bodies and discs in chick is dictated by the sclerotome, which first signals to the notochord to ensure that the nucleus pulposus develops in register with the somite-derived annulus fibrosus. Later, the notochord is required for maintenance of sclerotome segmentation as the mature vertebral bodies and intervertebral discs form. These results highlight differences in vertebral development between amniotes and teleosts including zebrafish, where the notochord dictates the segmental pattern. The relative importance of the sclerotome and notochord in vertebral patterning has changed significantly during evolution.