Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

Centre Universitaire

The vertebrate skull vault forms almost entirely by the direct mineralisation of mesenchyme, without the formation of a cartilaginous template, a mechanism called membranous ossification. Dlx5 gene mutation leads to cranial dismorphogenesis which differs from the previously studied craniosynostosis syndromes [Development 126 (1999), 3795; Development 126 (1999), 3831]. In avians, little is known about the genetic regulation of cranial vault development. In this study, we analyze Dlx5 expression and regulation during skull formation in the chick embryo. We compare Dlx5 expression pattern with that of several genes involved in mouse cranial suture regulation. This provides an initial description of the expression in the developing skull of the genes encoding the secreted molecules BMP 2, BMP 4, BMP 7, the transmembrane FGF receptors FGFR 1, FGFR 2, FGFR 4, the transcription factors Msx1, Msx2, and Twist, as well as Goosecoid and the early membranous bone differentiation marker osteopontin. We show that Dlx5 is activated in proliferating osteoblast precursors, before osteoblast differentiation. High levels of Dlx5 transcripts are observed at the osteogenic fronts (OFs) and at the edges of the suture mesenchyme, but not in the suture itself. Dlx5 expression is initiated in areas where Bmp4 and Bmp7 genes become coexpressed. In a calvarial explant culture system, Dlx5 transcription is upregulated by BMPs and inhibited by the BMP-antagonist Noggin. In addition, FGF4 activates Bmp4 but not Bmp7 gene transcription and is not sufficient to induce ectopic Dlx5 expression in the immature calvarial mesenchyme. From these data, we propose a model for the regulatory network implicated in early steps of chick calvarial development.

BMP signals regulate Dlx5 during early avian skull development.

At the border of the neural plate, the induction of the neural crest can be achieved by interactions with the epidermis, or with the underlying mesoderm. Wnt signals are required for the inducing activity of the epidermis in chick and amphibian embryos. Here, we analyze the molecular mechanisms of neural crest induction by the mesoderm in Xenopus embryos. Using a recombination assay, we show that prospective paraxial mesoderm induces a panel of neural crest markers (Slug, FoxD3, Zic5 and Sox9), whereas the future axial mesoderm only induces a subset of these genes. This induction is blocked by a dominant negative (dn) form of FGFR1. However, neither dnFGFR4a nor inhibition of Wnt signaling prevents neural crest induction in this system. Among the FGFs, FGF8 is strongly expressed by the paraxial mesoderm. FGF8 is sufficient to induce the neural crest markers FoxD3, Sox9 and Zic5 transiently in the animal cap assay. In vivo, FGF8 injections also expand the Slug expression domain. This suggests that FGF8 can initiate neural crest formation and cooperates with other DLMZ-derived factors to maintain and complete neural crest induction. In contrast to Wnts, eFGF or bFGF, FGF8 elicits neural crest induction in the absence of mesoderm induction and without a requirement for BMP antagonists. In vivo, it is difficult to dissociate the roles of FGF and WNT factors in mesoderm induction and neural patterning. We show that, in most cases, effects on neural crest formation were parallel to altered mesoderm or neural development. However, neural and neural crest patterning can be dissociated experimentally using different dominant-negative manipulations: while Nfz8 blocks both posterior neural plate formation and neural crest formation, dnFGFR4a blocks neural patterning without blocking neural crest formation. These results suggest that different signal transduction mechanisms may be used in neural crest induction, and anteroposterior neural patterning.

Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals.

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.

Msx genes are expressed in the carapacial ridge of turtle shell: a study of the European pond turtle, Emys orbicularis.

FGF, WNT, and BMP signaling promote neural crest formation at the neural plate boundary in vertebrate embryos. To understand how these signals are integrated, we have analyzed the role of the transcription factors Msx1 and Pax3. Using a combination of overexpression and morpholino-mediated knockdown strategies in Xenopus, we show that Msx1 and Pax3 are both required for neural crest formation, display overlapping but nonidentical activities, and that Pax3 acts downstream of Msx1. In neuralized ectoderm, Msx1 is sufficient to induce multiple early neural crest genes. Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. Upstream of this, WNTs initiate Slug induction through Pax3 activity, whereas FGF8 induces neural crest through both Msx1 and Pax3 activities. Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction.

Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals during Xenopus neural crest induction.

The vertebra develops from the ventral part of the somite, the sclerotome. Sclerotome progenitors are subject to multiple signaling molecules secreted by the adjacent tissues that control their fate. The aim of this article is to discuss the mechanisms of sclerotome induction, chondrogenesis and morphogenesis. By integrating the results from classical studies and recent molecular advances, this will illustrate how the powerful combination of experimental embryology and genetic approaches has recently illuminated the multiple steps of vertebra formation.

Sclerotome development and morphogenesis: when experimental embryology meets genetics.

Craniofacial bones derive from cephalic neural crest, by endochondral or intramembranous ossification. Here, we address the role of the homeobox transcription factor Dlx5 during the initial steps of calvaria membranous differentiation and we show that Dlx5 elicits Runx2 induction and full osteoblast differentiation in embryonic suture mesenchyme grown "in vitro". First, we compare Dlx5 expression to bone-related gene expression in the developing skull and mandibular bones. We classify genes into three groups related to consecutive steps of ossification. Secondly, we study Dlx5 activity in osteoblast precursors, by transfecting Dlx5 into skull mesenchyme dissected prior to the onset of either Dlx5 and Runx2 expression or osteogenesis. We find that Dlx5 does not modify the proliferation rate or the expression of suture markers in the immature calvaria cells. Rather, Dlx5 initiates a complete osteogenic differentiation in these early primary cells, by triggering Runx2, osteopontin, alkaline phosphatase, and other gene expression according to the sequential temporal sequence observed during skull osteogenesis "in vivo". Thirdly, we show that BMP signaling activates Dlx5, Runx2, and alkaline phosphatase in those primary cultures and that a dominant-negative Dlx factor interferes with the ability of the BMP pathway to activate Runx2 expression. Together, these data suggest a pivotal role of Dlx5 and related Dlx factors in the onset of differentiation of chick calvaria osteoblasts.

Dlx5 drives Runx2 expression and osteogenic differentiation in developing cranial suture mesenchyme.

Defining which key factors control commitment of an embryonic lineage among a myriad of candidates is a longstanding challenge in developmental biology and an essential prerequisite for developing stem cell-based therapies. Commitment implies that the induced cells not only express early lineage markers but further undergo an autonomous differentiation into the lineage. The embryonic neural crest generates a highly diverse array of derivatives, including melanocytes, neurons, glia, cartilage, mesenchyme, and bone. A complex gene regulatory network has recently classified genes involved in the many steps of neural crest induction, specification, migration, and differentiation. However, which factor or combination of factors is sufficient to trigger full commitment of this multipotent lineage remains unknown. Here, we show that, in contrast to other potential combinations of candidate factors, coactivating transcription factors Pax3 and Zic1 not only initiate neural crest specification from various early embryonic lineages in Xenopus and chicken embryos but also trigger full neural crest determination. These two factors are sufficient to drive migration and differentiation of several neural crest derivatives in minimal culture conditions in vitro or ectopic locations in vivo. After transplantation, the induced cells migrate to and integrate into normal neural crest craniofacial target territories, indicating an efficient spatial recognition in vivo. Thus, Pax3 and Zic1 cooperate and execute a transcriptional switch sufficient to activate full multipotent neural crest development and differentiation.

Anne Helene Monsoro-Burq

Institut Curie,
Université Paris Sud,
CNRS UMR 3347,
INSERM U1021

Centre Universitaire

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

Anne Helene Monsoro-Burq

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Centre Universitaire

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

Institut Curie,
Université Paris Sud,
CNRS UMR 3347,
INSERM U1021