On the carapacial ridge in turtle embryos: its developmental origin, function and the chelonian body plan

The chelonian carapace is composed of dorsolaterally expanded ribs; an evolutionary change in the rib-patterning program is assumed to be related to this novelty. Turtle embryos exhibit a longitudinal ridge called the carapacial ridge (CR) on the flank, and its histological resemblance to the apical ectodermal ridge of the limb bud implies its inductive activity in the unique patterning of the ribs. We studied the Chinese soft-shelled turtle, Pelodiscus sinensis, and confirmed by labeling with a lipophilic dye, DiI, that the CR contains the somite-derived dermis and that it is a unique structure among amniotes. Using electroporation of a dominant-negative form of LEF-1, the CR-specific gene, we showed that CR-specific genes function in the growth and maintenance of the CR. Microcauterization or implantation of the CR did not change the dorsoventral pattern of the ribs, and only their fan-shaped pattern was arrested by CR removal. We conclude that the CR is a true embryonic novelty among amniotes and, because of the specific expression of regulatory genes, it functions in the marginal growth of the carapacial primordium, thereby inducing the fan-shaped arrangement of the ribs.     

Development of the turtle carapace: Implications for the evolution of a novel bauplan

The chelonian carapace is composed of the endochondral ribs and vertebrae associated with a specialized dermis. The ribs are found in an aberrant position compared to those of all other tetrapods; they are superficial and dorsal to the limb girdles. This morphological arrangement, which constitutes the unique chelonian Bauplan, is examined from a developmental perspective. Embryos of Chelydra serpentina were studied during stages of carapace development. Tissue morphology, autoradiography, and indirect immunofluorescent localization of adhesion molecules indicate that the outgrowth of the embryonic carapace occurs as the result of an epithelial–mesenchymal interaction in the body wall. A carapacial ridge composed of mesenchyme of the dermis and overlying ectoderm is formed dorsal to the ectodermal boundary between somitic and lateral plate mesoderm. It is the anlage of the carapace margin, in which the ribs will eventually terminate. The ectoderm of the carapacial ridge is thickened into a pseudostratified columnar epithelium, which overlies a condensation in the mesenchyme of the dermis. Patterns of cell proliferation and the distribution of N-CAM and fibronectin in the carapacial ridge are consistent with patterns seen in other structures initiated by epithelial–mesenchymal interactions such as feathers and limb buds. Based on an analogy to this developmental mechanism in the development of the limb skeleton, a further analogy with the evolution of the limbs from lateral fin folds is used to form a hypothesis on the evolution of the carapace from elements of the primitive reptilian integument.     

<Emphasis Type="Italic">Msx</Emphasis> genes are expressed in the carapacial ridge of turtle shell: a study of the European pond turtle,<Emphasis Type="Italic"> Emys orbicularis</Emphasis>

The turtle shell forms by extensive ossification of dermis ventrally and dorsally. The carapacial ridge (CR) controls early dorsal shell formation and is thought to play a similar role in shell growth as the apical ectodermal ridge during limb development. However, the molecular mechanisms underlying carapace development are still unknown. Msx genes are involved in the development of limb mesenchyme and of various skeletal structures. In particular, precocious Msx expression is recorded in skeletal precursors that develop close to the ectoderm, such as vertebral spinous processes or skull. Here, we have studied the embryonic expression of Msx genes in the European pond turtle, Emys orbicularis. The overall Msx expression in head, limb, and trunk is similar to what is observed in other vertebrates. We have focused on the CR area and pre-skeletal shell condensations. The CR expresses Msx genes transiently, in a pattern similar to that of fgf10. In the future carapace domain, the dermis located dorsal to the spinal cord expresses Msx genes, as in other vertebrates, but we did not see expansion of this expression in the dermis located more laterally, on top of the dermomyotomes. In the ventral plastron, although the dermal osseous condensations form in the embryonic Msx-positive somatopleura, we did not observe enhanced Msx expression around these elements. These observations may indicate that common mechanisms participate in limb bud and CR early development, but that pre-differentiation steps differ between shell and other skeletal structures and involve other gene activities than that of Msx genes.Edited by D.A. Weisblat     

Evolutionary developmental perspective for the origin of turtles: the folding theory for the shell based on the developmental nature of the carapacial ridge

The body plan of the turtle represents an example of evolutionary novelty for acquisition of the shell. Unlike similar armors in other vertebrate groups, the turtle shell involves the developmental repatterning of the axial skeleton and exhibits an unusual topography of musculoskeletal elements. Thus, the turtle provides an ideal case study for understanding changes in the developmental program associated with the morphological evolution of vertebrates. In this article, the evolution of the turtle-specific body plan is reviewed and discussed. The key to understanding shell patterning lies in the modification of the ribs, for which the carapacial ridge (CR), a turtle-specific embryonic anlage, is assumed to be responsible. The growth of the ribs is arrested in the axial part of the body, allowing dorsal and lateral oriented growth to encapsulate the scapula. Although the CR does not appear to induce this axial arrest per se, it has been shown to support the fan-shaped patterning of the ribs, which occurs concomitant with marginal growth of the carapace along the line of the turtle-specific folding that takes place in the lateral body wall. During the process of the folding, some trunk muscles maintain their ancestral connectivities, whereas the limb muscles establish new attachments specific to the turtle. The turtle body plan can thus be explained with our knowledge of vertebrate anatomy and developmental biology, consistent with the evolutionary origin of the turtle suggested by the recently discovered fossil species, Odontochelys.     

Sclerotomal origin of the ribs

The somites of vertebrate embryos give rise to sclerotomes and dermomyotomes. The sclerotomes form the axial skeleton, whereas the dermomyotomes give rise to all trunk muscles and the dermis of the back. The ribs were thought to be ventral processes of the axial skeleton and therefore to be derived from the sclerotomes; however, recently a dermomyotomal origin of the distal rib (the costal shaft) was suggested, with only the proximal parts (head and neck of the rib) being of sclerotomal origin. We have re-investigated the development of the ribs in quail-chick chimeras and carried out three experimental series. (1) Single dermomyotomes and (2) single sclerotomes were grafted homotopically, and (3) the ectoderm overlying the unsegmented paraxial mesoderm was removed in the prospective thoracic region. We found that the cells of the dermomyotome gave rise to epaxial and hypaxial trunk muscles, dermis of the back and endothelial cells, but not to ribs. Cells of the sclerotome formed the axial skeleton and all parts of the ribs. Ablation of the ectoderm, which affects dermomyotome development, results in severe malformations of the ribs, probably due to disturbed interactions between dermomyotome and sclerotome. Our results strongly confirm the traditional view of the sclerotomal origin of the ribs.     

Skeletal defects in paternal uniparental disomy for chromosome 14 are re-capitulated in the mouse model (paternal uniparental disomy 12)

Human paternal uniparental disomy for chromosome 14 (upd(14)pat) presents with skeletal abnormalities, joint contractures, dysmorphic facial features and developmental delay/mental retardation. Distal human chromosome 14 (HSA14) is homologous to distal mouse chromosome 12 (MMU12) and both regions have been shown to contain imprinted genes. In humans, consistent radiographic findings include a narrow, bell-shaped thorax with caudal bowing of the anterior ribs, cranial bowing of the posterior ribs and flaring of the iliac wings without shortening or dysplasia of the long bones. Mice with upd(12)pat have thin ribs with delayed ossification of the sternum, skull and feet. In both mice and humans, the axial skeleton is predominantly affected. We hypothesize that there is an imprinted gene or genes on HSA14/MMU12 that specifically affects rib/thorax development and the maturation of ossification centers in the sternum, feet and skull with little effect on long bone development.     

FGF signaling is required for initiation of feather placode development

Morphogenesis of hairs and feathers is initiated by an as yet unknown dermal signal that induces placode formation in the overlying ectoderm. To determine whether FGF signals are required for this process we over-expressed soluble versions of FGFR1 or FGFR2 in the skin of chicken embryos. This produced a complete failure of feather formation prior to any morphological or molecular signs of placode development. We further show that Fgf10 is expressed in the dermis of nascent feather primordia, and that anti-FGF10 antibodies block feather placode development in skin explants. In addition we show that FGF10 can induce expression of positive and negative regulators of feather development and can induce its own expression under conditions of low BMP signaling. Together these results demonstrate that FGF signaling is required for the initiation of feather placode development and implicate FGF10 as an early dermal signal involved in this process.     

Evolutionary mechanisms of rib loss in anurans: a comparative developmental approach

ABSTRACT The presence of free ribs is presumed to be a primitive morphological character observed only in a few families of Recent anurans, whereas the absence of ribs has been considered to be a derived condition that is widespread within this order. A comparative study of rib development based on representatives of several anuran lineages (Alytes, Bombina, Bufo, Discoglossus, Hyla, Pelobates, Pelodytes, Rana, and Xenopus) reveals a previously undetected diversity of developmental features in the formation and interaction between neural arches and ribs. The absence of free ribs at premetamorphic or later stages is verified in some groups, but we present for the first time evidence of the existence of larval rib rudiments in others, both in the anterior (Rana, Hyla) and posterior (Bufo, Discoglossus, Pelobates) presacral regions. Heterochrony seems to have played a major role in the processes underlying rib reduction. The intracolumnar differences between anterior (V(2)-V(4)) and posterior (V(5)-V(8)) regions are based on perturbations in the timing of early differentiation. Furthermore, a clear shift in the relative timing of ossification among evolutionary lineages was detected. In this respect Xenopus has a highly derived condition. The use of the morphological character of "rib loss" in phylogenetic analyses must be reconsidered due to the different convergent developmental paths described here. The phylogenetic analysis of a "sequence units" matrix of rib development is compared with current anuran phylogenies. Some evolutionary information appears to be clearly present in the ontogenetic data of this "missing morphology," but its value for evolutionary inferences is rather limited.     

Quantitative assessment of FGF regulation by cell surface heparan sulfates

Heparin/heparan sulfate-like glycosaminoglycans (HSGAGs) modulate the activity of the fibroblast growth factor (FGF) family of proteins. Through interactions with both FGFs and FGF receptors (FGFRs), HSGAGs mediate FGF-FGFR binding and oligomerization leading to FGFR phosphorylation and initiation of intracellular signaling cascades. We describe a methodology to examine the impact of heparan sulfate fine structure and source on FGF-mediated signaling. Mitogenic assays using BaF3 cells transfected with specific FGFR isoforms allow for the quantification of FGF1 and FGF2 induced responses independent of conflicting influences. As such, this system enables a systematic investigation into the role of cell surface HSGAGs on FGF signaling. We demonstrate this approach using cell surface-derived HSGAGs and find that distinct HSGAGs elicit differential FGF response patterns through FGFR1c and FGFR3c. We conclude that this assay system can be used to probe the ability of distinct HSGAG species to regulate the activity of specific FGF-FGFR pairs.     

Signaling pathways activated by epidermal growth factor receptor or fibroblast growth factor receptor differentially regulate branching morphogenesis in fetal mouse submandibular glands

Although growth factor signaling is required for embryonic development of organs, individual signaling mechanisms regulating these organotypic processes are just beginning to be defined. We compared signaling activated in fetal mouse submandibular glands (SMGs) by three growth factors, epidermal growth factor (EGF), fibroblast growth factor (FGF) 7, or FGF10, and correlated it with specific events of branching morphogenesis. Immunoblotting showed that EGF strongly stimulated phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and weakly stimulated phosphorylation of phospholipase C γ 1 (PLC γ 1) and phosphatidylinositol-3 kinase (PI3K) in cultured E14 SMG. However, FGF7 and FGF10 stimulated phosphorylation of both PLC γ 1 and PI3K, but elicited only minimal phosphorylation of ERK-1/2. Morphological study of mesenchyme-free SMG epithelium cultured in Matrigel revealed that EGF induced cleft formation of endpieces, that FGF7 stimulated both cleft formation and stalk elongation, but that FGF10 induced only stalk elongation. In mesenchyme-free SMG epithelium cultured with EGF, FGF7 and FGF10, U0126 (MEK inhibitor) completely blocked cleft formation, whereas U73122 (PLC γ 1 inhibitor) suppressed stalk elongation. These finding suggest that EGF stimulates cleft formation and drives branch formation via ERK-1/2, and that FGF7 stimulates both cleft formation and stalk elongation via PLC γ 1 and partly via ERK-1/2, but that FGF10 stimulates stalk elongation mainly via PLC γ 1.     

Signaling by fibroblast growth factors (FGF) and fibroblast growth factor receptor 2 (FGFR2)-activating mutations blocks mineralization and induces apoptosis in osteoblasts

Fibroblast growth factors (FGF) play a critical role in bone growth and development affecting both chondrogenesis and osteogenesis. During the process of intramembranous ossification, which leads to the formation of the flat bones of the skull, unregulated FGF signaling can produce premature suture closure or craniosynostosis and other craniofacial deformities. Indeed, many human craniosynostosis disorders have been linked to activating mutations in FGF receptors (FGFR) 1 and 2, but the precise effects of FGF on the proliferation, maturation and differentiation of the target osteoblastic cells are still unclear. In this report, we studied the effects of FGF treatment on primary murine calvarial osteoblast, and on OB1, a newly established osteoblastic cell line. We show that FGF signaling has a dual effect on osteoblast proliferation and differentiation. FGFs activate the endogenous FGFRs leading to the formation of a Grb2/FRS2/Shp2 complex and activation of MAP kinase. However, immature osteoblasts respond to FGF treatment with increased proliferation, whereas in differentiating cells FGF does not induce DNA synthesis but causes apoptosis. When either primary or OB1 osteoblasts are induced to differentiate, FGF signaling inhibits expression of alkaline phosphatase, and blocks mineralization. To study the effect of craniosynostosis-linked mutations in osteoblasts, we introduced FGFR2 carrying either the C342Y (Crouzon syndrome) or the S252W (Apert syndrome) mutation in OB1 cells. Both mutations inhibited differentiation, while dramatically inducing apoptosis. Furthermore, we could also show that overexpression of FGF2 in transgenic mice leads to increased apoptosis in their calvaria. These data provide the first biochemical analysis of FGF signaling in osteoblasts, and show that FGF can act as a cell death inducer with distinct effects in proliferating and differentiating osteoblasts.     

Thyroid hormone activates fibroblast growth factor receptor-1 in bone

Thyroid hormone (T3) and the T3 receptor (TR) alpha gene are essential for bone development whereas adult hyperthyroidism increases the risk of osteoporotic fracture. We isolated fibroblast growth factor receptor-1 (FGFR1) as a T3-target gene in osteoblasts by subtraction hybridization. FGFR1 mRNA was induced 2- to 3-fold in osteoblasts treated with T3 for 6-48 h, and FGFR1 protein was stimulated 2- to 4-fold. Induction of FGFR1 was independent of mRNA half-life and abolished by actinomycin D and cycloheximide, indicating the involvement of an intermediary protein. Fibroblast growth factor 2 (FGF2) stimulated MAPK in osteoblasts, and pretreatment with T3 for 6 h induced a more rapid response to FGF that was increased in magnitude by 2- to 3-fold. Similarly, T3 enhanced FGF2-activated autophosphorylation of FGFR1, but did not modify FGF2-induced phosphorylation of the docking protein FRS2. These effects were abolished by the FGFR-selective inhibitors PD166866 and PD161570. In situ hybridization analyses of TRalpha-knockout mice, which have impaired ossification and skeletal mineralization, revealed reduced FGFR1 mRNA expression in osteoblasts and osteocytes, whereas T3 failed to stimulate FGFR1 mRNA or enhance FGF2-activated MAPK signaling in TRalpha-null osteoblasts. These findings implicate FGFR1 signaling in T3-dependent bone development and the pathogenesis of skeletal disorders resulting from thyroid disease.     

BMP-FGF Signaling Axis Mediates Wnt-Induced Epidermal Stratification in Developing Mammalian Skin

Epidermal stratification of the mammalian skin requires proliferative basal progenitors to generate intermediate cells that separate from the basal layer and are replaced by post-mitotic cells. Although Wnt signaling has been implicated in this developmental process, the mechanism underlying Wnt-mediated regulation of basal progenitors remains elusive. Here we show that Wnt secreted from proliferative basal cells is not required for their differentiation. However, epidermal production of Wnts is essential for the formation of the spinous layer through modulation of a BMP-FGF signaling cascade in the dermis. The spinous layer defects caused by disruption of Wnt secretion can be restored by transgenically expressed Bmp4. Non-cell autonomous BMP4 promotes activation of FGF7 and FGF10 signaling, leading to an increase in proliferative basal cell population. Our findings identify an essential BMP-FGF signaling axis in the dermis that responds to the epidermal Wnts and feedbacks to regulate basal progenitors during epidermal stratification.