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.

Development of the amniote ventrolateral body wall.

In vertebrates, the trunk consists of the musculoskeletal structures of the back and the ventrolateral body wall, which together enclose the internal organs of the circulatory, digestive, respiratory and urogenital systems. This review gives an overview on the development of the thoracic and abdominal wall during amniote embryogenesis. Specifically, I briefly summarize relevant historical concepts and the present knowledge on the early embryonic development of ribs, sternum, intercostal muscles and abdominal muscles with respect to anatomical bauplan, origin and specification of precursor cells, initial steps of pattern formation, and cellular and molecular regulation of morphogenesis.

Development and patterning of rib primordia are dependent on associated musculature.

The importance of skeletal muscle for rib development and patterning in the mouse embryo has not been resolved, largely because different experimental approaches have yielded disparate results. In this study, we utilize both gene knockouts and muscle cell ablation approaches to re-visit the extent to which rib growth and patterning are dependent on developing musculature. Consistent with previous studies, we show that rib formation is highly dependent on the MYOD family of myogenic regulatory factors (MRFs), and demonstrate that the extent of rib formation is gene-, allele-, and dosage-dependent. In the absence of Myf5 and MyoD, one allele of Mrf4 is sufficient for extensive rib growth, although patterning is abnormal. Under conditions of limiting MRF dosage, MyoD is identified as a positive regulator of rib patterning, presumably due to improved intercostal muscle development. In contrast to previous muscle ablation studies, we show that diphtheria toxin subunit A (DTA)-mediated ablation of muscle progenitors or differentiated muscle, using MyoDiCre or HSA-Cre drivers, respectively, profoundly disrupts rib development. Further, a comparison of three independently derived Rosa26-based DTA knockin alleles demonstrates that the degree of rib perturbations in MyoDiCre/+/DTA embryos is markedly dependent on the DTA allele used, and may in part explain discrepancies with previous findings. The results support the conclusion that the extent and quality of rib formation is largely dependent on the dosage of Myf5 and Mrf4, and that both early myotome-sclerotome interactions, as well as later muscle-rib interactions, are important for proper rib growth and patterning.

Transcriptomic similarities and differences between the limb bud AER and unique carapacial ridge of turtle embryos.

Evolutionary innovation may arise via major departures from an ancestral condition. Turtle shell morphogenesis depends on a unique structure known as the carapacial ridge (CR). This lateral tissue protrusion in turtle embryos exhibits similar properties as the apical ectodermal ridge (AER)-a well-known molecular signaling center involved in limb development. Still, how the CR influences shell morphogenesis is not entirely clear. The present study aimed to describe the CR transcriptome shortly before ribs were halted within its mesenchyme, as required for shell development. Analyses exposed that the mesenchymal marker VIM was one of the most highly co-expressed genes and numerous appendage formation genes were situated within the core of CR and AER co-expression networks. However, there were tissue-specific differences in the activity of these genes. For instance, WNT5A was most frequently assigned to appendage-related annotations of the CR network core, but not in the AER. Several homeobox transcription factors known to regulate limb bud patterning exhibited their highest expression levels in the AER, but were underexpressed in the CR. The results of this study corroborate that novel body plans often originate via alterations of pre-existing genetic networks. Altogether, this exploratory study enhances the groundwork for future experiments on the molecular underpinnings of turtle shell development and evolution.

Prenatal diagnosis of fetal multiple hemivertebrae: the importance of 3D ultrasound assessment.

We herein present a case of fetal multiple hemivertebrae detected at antenatal sonography. The use of the 3 D technology supported by a new contrast enhancement rendering algorithm (Crystal Vue) has allowed the accurate prenatal classification of the defect, confirmed at follow up, that would have been difficult to define by 2 D only.

Role of somite patterning in the formation of Weberian apparatus and pleural rib in zebrafish.

In the vertebrate body, a metameric structure is present along the anterior-posterior axis. Zebrafish tbx6-/- larvae, in which somite boundaries do not form during embryogenesis, were shown to exhibit abnormal skeletal morphology such as rib, neural arch and hemal arch. In this study, we investigated the role of somite patterning in the formation of anterior vertebrae and ribs in more detail. Using three-dimensional computed tomography scans, we found that anterior vertebrae including the Weberian apparatus were severely affected in tbx6-/- larvae. In addition, pleural ribs of tbx6 mutants exhibited severe defects in the initial ossification, extension of ossification, and formation of parapophyses. Two-colour staining revealed that bifurcation of ribs was caused by fusion or branching of ribs in tbx6-/- . The parapophyses in tbx6-/- juvenile fish showed irregular positioning to centra and abnormal attachment to ribs. Furthermore, we found that the ossification of the distal portion of ribs proceeded along myotome boundaries even in irregularly positioned myotome boundaries. These results provide evidence of the contribution of somite patterning to the formation of the Weberian apparatus and rib in zebrafish.

An in vitro model of region-specific rib formation in chick axial skeleton: Intercellular interaction between somite and lateral plate cells.

The axial skeleton is divided into different regions based on its morphological features. In particular, in birds and mammals, ribs are present only in the thoracic region. The axial skeleton is derived from a series of somites. In the thoracic region of the axial skeleton, descendants of somites coherently penetrate into the somatic mesoderm to form ribs. In regions other than the thoracic, descendants of somites do not penetrate the somatic lateral plate mesoderm. We performed live-cell time-lapse imaging to investigate the difference in the migration of a somite cell after contact with the somatic lateral plate mesoderm obtained from different regions of anterior-posterior axis in vitro on cytophilic narrow paths. We found that a thoracic somite cell continues to migrate after contact with the thoracic somatic lateral plate mesoderm, whereas it ceases migration after contact with the lumbar somatic lateral plate mesoderm. This suggests that cell-cell interaction works as an important guidance cue that regulates migration of somite cells. We surmise that the thoracic somatic lateral plate mesoderm exhibits region-specific competence to allow penetration of somite cells, whereas the lumbosacral somatic lateral plate mesoderm repels somite cells by contact inhibition of locomotion. The differences in the behavior of the somatic lateral plate mesoderm toward somite cells may confirm the distinction between different regions of the axial skeleton.

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.

Short-term foetal immobility temporally and progressively affects chick spinal curvature and anatomy and rib development.

Congenital spine deformities may be influenced by movements in utero, but the effects of foetal immobility on spine and rib development remain unclear. The purpose of the present study was to determine (1) critical time-periods when rigid paralysis caused the most severe disruption in spine and rib development and (2) how the effects of an early, short-term immobilisation were propagated to the different features of spine and rib development. Chick embryos were immobilised once per single embryonic day (E) between E3 and E6 and harvested at E9. To assess the ontogenetic effects following single-day immobilisation, other embryos were immobilised at E4 and harvested daily between E5 and E9. Spinal curvature, vertebral shape and segmentation and rib development were analysed by optical projection tomography and histology. The results demonstrated that periods critical for movement varied for different aspects of spine and rib development. Single-day immobilisation at E3 or E4 resulted in the most pronounced spinal curvature abnormalities, multiple wedged vertebrae and segmentation defects, while single-day immobilisation at E5 led to the most severe rib abnormalities. Assessment of ontogenetic effects following single-day immobilisation at E4 revealed that vertebral segmentation defects were subsequent to earlier vertebral body shape and spinal curvature abnormalities, while rib formation (although delayed) was independent from thoracic vertebral shape or curvature changes. A day-long immobilisation in chicks severely affected spine and rib development, highlighting the importance of abnormal foetal movements at specific time-points and motivating targeted prenatal monitoring for early diagnosis of congenital scoliosis.

A minimally sufficient model for rib proximal-distal patterning based on genetic analysis and agent-based simulations.

For decades, the mechanism of skeletal patterning along a proximal-distal axis has been an area of intense inquiry. Here, we examine the development of the ribs, simple structures that in most terrestrial vertebrates consist of two skeletal elements-a proximal bone and a distal cartilage portion. While the ribs have been shown to arise from the somites, little is known about how the two segments are specified. During our examination of genetically modified mice, we discovered a series of progressively worsening phenotypes that could not be easily explained. Here, we combine genetic analysis of rib development with agent-based simulations to conclude that proximal-distal patterning and outgrowth could occur based on simple rules. In our model, specification occurs during somite stages due to varying Hedgehog protein levels, while later expansion refines the pattern. This framework is broadly applicable for understanding the mechanisms of skeletal patterning along a proximal-distal axis.

Disordered vertebral and rib morphology in pudgy mice. Structural relationships to human scoliosis.

Normal and abnormal vertebral development have been studied over the past 200 years at increasing levels of resolution as techniques for biological investigation have improved. Disordered development of the axial skeleton from the early embryonic period on leads to structurally malformed vertebrae and intervertebral discs and ribs causing the severe deformities of scoliosis, kyphosis, and kyphoscoliosis. Developmental malformation of the axial skeleton therefore has led to considerable biological and clinical interest. This work will detail our studies on the structural deformities of the vertebral column and adjacent ribs in the pudgy mouse [1] caused by mutations in the delta-like 3 (Dll3) gene of the Notch family [2]. While gene abnormalities in the pudgy mouse have been outlined, there has been no in-depth assessment of the histopathology of the pudgy vertebral and rib abnormalities that this study will provide. In addition, although congenital scoliosis has been recognized as a clinical problem since the mid-nineteenth century (1800s) [3] and accurately defined by radiography since the early twentieth century (1900s) [4-6], there have been few detailed histopathologic studies of human cases. We will also relate our histopathologic findings in the pudgy mouse to the histopathology of human vertebral and rib malformations in clinical cases of congenital scoliosis, one of which we defined in detail previously [7].

Tgif1 and Tgif2 Regulate Axial Patterning in Mouse.

Tgif1 and Tgif2 are transcriptional repressors that inhibit the transcriptional response to transforming growth factor ß signaling, and can repress gene expression by direct binding to DNA. Loss of function mutations in TGIF1 are associated with holoprosencephaly (HPE) in humans. In mice, embryos lacking both Tgif1 and Tgif2 fail to complete gastrulation, and conditional double null embryos that survive past gastrulation have HPE and do not survive past mid-gestation. Here we show that in mice of a relatively pure C57BL/6 strain background, loss of Tgif1 alone results in defective axial patterning and altered expression of Hoxc6. The primary defects in Tgif1 null embryos are the presence of extra ribs on the C7 vertebra, consistent with a posterior transformation phenotype. In addition we observed defective cervical vertebrae, primarily C1-C5, in both adult mice and embryos that lacked Tgif1. The combination of Tgif1 and Tgif2 mutations increases the severity and penetrance of the posterior transformation phenotype, without altering the type of defects seen. Similarly, exposure of Tgif1 mutant embryos to retinoic acid at E8.5 increased the severity and penetrance of the Tgif1 phenotype. This suggests that Tgif1 and Tgif2 regulate axial patterning and that reduced TGIF function sensitizes embryos to the effects of retinoic acid.

Mechanism of Developmental Effects in Rats Caused by an N-Phenylimide Herbicide: Transient Fetal Anemia and Sequelae during Mid-to-Late Gestation.

BACKGROUND: Rat developmental toxicity including embryolethality and teratogenicity (mainly ventricular septal defects [VSDs] and wavy ribs) was produced by an N-phenylimide herbicide that inhibits protoporphyrinogen oxidase (PPO) common to chlorophyll and heme biosynthesis. Major characteristics of the developmental toxicity included species difference between rats and rabbits, compound-specific difference among structurally similar herbicides, and sensitive period. Protoporphyrin accumulation in treated fetuses closely correlated with the major characteristics. Iron deposits in erythroblastic mitochondria and degeneration of erythroblasts were observed in treated rat fetuses. In this study we investigated fetal anemia and subsequent developmental effects in rats, and inhibition of PPO in rats, rabbits, and humans by the herbicides in vitro. METHODS: Fetuses were treated on gestational day (GD) 12 and removed on GDs 13 through 20. All litters were examined externally. One half of litters were examined for blood and skeletal development, and the other half for interventricular foramen closure. Effects on PPO were determined in mitochondria from embryos and adult livers. RESULTS: Fetal anemia in rats was evident on GDs 13 through 16. Subsequently, enlarged heart, delayed closure of the foramen, reduced serum protein, and retarded rib ossification were observed. In vitro PPO inhibition exhibited species- and compound-specific differences corresponding to the developmental toxicity. CONCLUSION: We propose that developmental toxicity results from PPO inhibition in primitive erythroblasts, causing transient fetal anemia followed by death. Compensatory enlargement of the fetal heart results in failure of interventricular foramen closure and VSD. Reduced serum protein leads to delayed ossification and wavy ribs.

Developmental Patterning as a Quantitative Trait: Genetic Modulation of the Hoxb6 Mutant Skeletal Phenotype.

The process of patterning along the anterior-posterior axis in vertebrates is highly conserved. The function of Hox genes in the axis patterning process is particularly well documented for bone development in the vertebral column and the limbs. We here show that Hoxb6, in skeletal elements at the cervico-thoracic junction, controls multiple independent aspects of skeletal pattern, implicating discrete developmental pathways as substrates for this transcription factor. In addition, we demonstrate that Hoxb6 function is subject to modulation by genetic factors. These results establish Hox-controlled skeletal pattern as a quantitative trait modulated by gene-gene interactions, and provide evidence that distinct modifiers influence the function of conserved developmental genes in fundamental patterning processes.

Hoxb6 can interfere with somitogenesis in the posterior embryo through a mechanism independent of its rib-promoting activity.

Formation of the vertebrate axial skeleton requires coordinated Hox gene activity. Hox group 6 genes are involved in the formation of the thoracic area owing to their unique rib-promoting properties. Here we show that the linker region (LR) connecting the homeodomain and the hexapeptide is essential for Hoxb6 rib-promoting activity in mice. The LR-defective Hoxb6 protein was still able to bind a target enhancer together with Pax3, producing a dominant-negative effect, indicating that the LR brings additional regulatory factors to target DNA elements. We also found an unexpected association between Hoxb6 and segmentation in the paraxial mesoderm. In particular, Hoxb6 can disturb somitogenesis and anterior-posterior somite patterning by dysregulation of Lfng expression. Interestingly, this interaction occurred differently in thoracic versus more caudal embryonic areas, indicating functional differences in somitogenesis before and after the trunk-to-tail transition. Our results suggest the requirement of precisely regulated Hoxb6 expression for proper segmentation at tailbud stages.

Development of the ventral body wall in the human embryo.

Migratory failure of somitic cells is the commonest explanation for ventral body wall defects. However, the embryo increases ~ 25-fold in volume in the period that the ventral body wall forms, so that differential growth may, instead, account for the observed changes in topography. Human embryos between 4 and 10 weeks of development were studied, using amira reconstruction and cinema 4D remodeling software for visualization. Initially, vertebrae and ribs had formed medially, and primordia of sternum and hypaxial flank muscle primordium laterally in the body wall at Carnegie Stage (CS)15 (5.5 weeks). The next week, ribs and muscle primordium expanded in ventrolateral direction only. At CS18 (6.5 weeks), separate intercostal and abdominal wall muscles differentiated, and ribs, sterna, and muscles began to expand ventromedially and caudally, with the bilateral sternal bars fusing in the midline after CS20 (7 weeks) and the rectus muscles reaching the umbilicus at CS23 (8 weeks). The near-constant absolute distance between both rectus muscles and approximately fivefold decline of this distance relative to body circumference between 6 and 10 weeks identified dorsoventral growth in the dorsal body wall as determinant of the 'closure' of the ventral body wall. Concomitant with the straightening of the embryonic body axis after the 6th week, the abdominal muscles expanded ventrally and caudally to form the infraumbilical body wall. Our data, therefore, show that the ventral body wall is formed by differential dorsoventral growth in the dorsal part of the body.

Emerging from the rib: resolving the turtle controversies.

Two of the major controversies in the present study of turtle shell development involve the mechanism by which the carapacial ridge initiates shell formation and the mechanism by which each rib forms the costal bones adjacent to it. This paper claims that both sides of each debate might be correct-but within the species examined. Mechanism is more properly "mechanisms," and there is more than one single way to initiate carapace formation and to form the costal bones. In the initiation of the shell, the rib precursors may be kept dorsal by either "axial displacement" (in the hard-shell turtles) or "axial arrest" (in the soft-shell turtle Pelodiscus), or by a combination of these. The former process would deflect the rib into the dorsal dermis and allow it to continue its growth there, while the latter process would truncate rib growth. In both instances, though, the result is to keep the ribs from extending into the ventral body wall. Our recent work has shown that the properties of the carapacial ridge, a key evolutionary innovation of turtles, differ greatly between these two groups. Similarly, the mechanism of costal bone formation may differ between soft-shell and hard-shell turtles, in that the hard-shell species may have both periosteal flattening as well as dermal bone induction, while the soft-shelled turtles may have only the first of these processes.

The evolutionary origin of the turtle shell and its dependence on the axial arrest of the embryonic rib cage.

Turtles are characterized by their possession of a shell with dorsal and ventral moieties: the carapace and the plastron, respectively. In this review, we try to provide answers to the question of the evolutionary origin of the carapace, by revising morphological, developmental, and paleontological comparative analyses. The turtle carapace is formed through modification of the thoracic ribs and vertebrae, which undergo extensive ossification to form a solid bony structure. Except for peripheral dermal elements, there are no signs of exoskeletal components ontogenetically added to the costal and neural bones, and thus the carapace is predominantly of endoskeletal nature. Due to the axial arrest of turtle rib growth, the axial part of the embryo expands laterally and the shoulder girdle becomes encapsulated in the rib cage, together with the inward folding of the lateral body wall in the late phase of embryogenesis. Along the line of this folding develops a ridge called the carapacial ridge (CR), a turtle-specific embryonic structure. The CR functions in the marginal growth of the carapacial primordium, in which Wnt signaling pathway might play a crucial role. Both paleontological and genomic evidence suggest that the axial arrest is the first step toward acquisition of the turtle body plan, which is estimated to have taken place after the divergence of a clade including turtles from archosaurs. The developmental relationship between the CR and the axial arrest remains a central issue to be solved in future.

Characterization of the skeletal fusion with sterility (sks) mouse showing axial skeleton abnormalities caused by defects of embryonic skeletal development.

The development of the axial skeleton is a complex process, consisting of segmentation and differentiation of somites and ossification of the vertebrae. The autosomal recessive skeletal fusion with sterility (sks) mutation of the mouse causes skeletal malformations due to fusion of the vertebrae and ribs, but the underlying defects of vertebral formation during embryonic development have not yet been elucidated. For the present study, we examined the skeletal phenotypes of sks/sks mice during embryonic development and the chromosomal localization of the sks locus. Multiple defects of the axial skeleton, including fusion of vertebrae and fusion and bifurcation of ribs, were observed in adult and neonatal sks/sks mice. In addition, we also found polydactyly and delayed skull ossification in the sks/sks mice. Morphological defects, including disorganized vertebral arches and fusions and bifurcations of the axial skeletal elements, were observed during embryonic development at embryonic day 12.5 (E12.5) and E14.5. However, no morphological abnormality was observed at E11.5, indicating that defects of the axial skeleton are caused by malformation of the cartilaginous vertebra and ribs at an early developmental stage after formation and segmentation of the somites. By linkage analysis, the sks locus was mapped to an 8-Mb region of chromosome 4 between D4Mit331 and D4Mit199. Since no gene has already been identified as a cause of malformation of the vertebra and ribs in this region, the gene responsible for sks is suggested to be a novel gene essential for the cartilaginous vertebra and ribs.