Engrailed-1 misexpression in chick embryos prevents apical ridge formation but preserves segregation of dorsal and ventral ectodermal compartments

Using lineage tracers, we recently showed dorsal and ventral ectodermal compartments along the sides of the body in chick embryos. The compartments are formed both in presumptive limb-forming regions where they position the apical ridge and also in presumptive interlimb (flank). Here we show, using a novel technique combining fate mapping and in situ hybridisation, that the ventral compartment coincides with the Engrailed-1 (En-1) domain of expression. This coincidence suggests that En-1 could maintain the ventral compartment and be necessary for apical ridge formation. To test this hypothesis, we ectopically expressed En-1 via retroviral transfer and then examined limb development and cell lineage restriction in the ectoderm. En-1 misexpression can completely prevent formation of both normal limbs and ectopic limbs induced in the flank by application of FGF-2. In both cases, there are no morphological signs of apical ectodermal ridge formation and expression of ridge-associated genes is undetectable. In striking contrast, the lineage restriction between dorsal and ventral ectoderm is not altered. Therefore, En-1 is involved in the regulation of ridge formation but not compartment maintenance.     

Key role of p63 in BMP-4-Induced Epidermal Commitment of Embryonic Stem Cells

In vivo studies, transgenic and knock-out mice have demonstrated that p63 isoforms play pivotal roles in ectodermal and epidermal development but their respective function remains highly controversial. Since embryonic stem (ES) cells can be differentiated into many cell types, they represent an effective tool to recapitulate in vitro the main steps of embryonic development. We recently reported the efficient derivation of ectodermal and epidermal cells from murine ES cells and clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1+ neural precursors to undergo specific apoptosis while inducing epidermal differentiation through ΔNp63 gene activation. ΔNp63 is not required for ectodermal fate but enhances ES-derived ectodermal cell proliferation and epidermal commitment. This unique cellular model should further provide a powerful tool for identifying the molecular mechanisms controlling normal skin development and in p63-ectodermal dysplasia human congenital pathologies.     

Inhibitory control of neural differentiation in mammalian cells

 In Xenopus embryos, a truncated type II activin receptor (Δ1XAR1), capable of blocking signals by several transforming growth factor (TGF)-β family members, can induce neural tissue suggesting neural fate is under inhibitory control. Activin and bone morphogenetic protein 4 (BMP4) can act as neural inhibitors but only BMP4 can induce epidermis in Xenopus ectodermal cells. We have used the pluripotent mouse embryonal carcinoma cell line P19 to examine whether the mechanisms of ectodermal cell fate decisions are conserved among vertebrates. We show that a P19 cell line expressing Δ1XAR1 will differentiate into neurons. In addition, BMP4 inhibits retinoic acid (RA)-induced neural differentiation of P19 cells and induces keratin expression. These results suggest that in mammals as in amphibians neural fate is under inhibitory control and BMP4 can alter ectodermal differentiation. Received: 23 September 1996 / Accepted: 8 January 1997     

The forkhead genes, Foxc1 and Foxc2, regulate paraxial versus intermediate mesoderm cell fate

During vertebrate embryogenesis, the newly formed mesoderm is allocated to the paraxial, intermediate, and lateral domains, each giving rise to different cell and tissue types. Here, we provide evidence that the forkhead genes, Foxc1 and Foxc2, play a role in the specification of mesoderm to paraxial versus intermediate fates. Mouse embryos lacking both Foxc1 and Foxc2 show expansion of intermediate mesoderm markers into the paraxial domain, lateralization of somite patterning, and ectopic and disorganized mesonephric tubules. In gain of function studies in the chick embryo, Foxc1 and Foxc2 negatively regulate intermediate mesoderm formation. By contrast, their misexpression in the prospective intermediate mesoderm appears to drive cells to acquire paraxial fate, as revealed by expression of the somite markers Pax7 and Paraxis. Taken together, the data indicate that Foxc1 and Foxc2 regulate the establishment of paraxial versus intermediate mesoderm cell fates in the vertebrate embryo.     

A restrictive role for Hedgehog signalling during otic specification in Xenopus

Vertebrate inner ear development is initiated by the specification of the otic placode, an ectodermal structure induced by signals from neighboring tissue. Although several signaling molecules have been identified as candidate otic inducers, many details of the process of inner ear induction remain elusive. Here, we report that otic induction is responsive to the level of Hedgehog (Hh) signaling activity in Xenopus, making use of both gain- and loss-of-function approaches. Ectopic activation of Hedgehog signaling resulted in the development of ectopic vesicular structures expressing the otic marker genes XPax-2, Xdll-3, and Xwnt-3A, thus revealing otic identity. Induction of ectopic otic vesicles was also achieved by misexpression of two different inhibitors of Hh signaling: the putative Hh antagonist mHIP and XPtc1deltaLoop2, a dominant-negative form of the Hh receptor Patched. In addition, misexpression of XPtc1deltaLoop2 as well as treatment of Xenopus embryos with the specific Hh signaling antagonist cyclopamine resulted in the formation of enlarged otic vesicles. In summary, our observations suggest that a defined level of Hh signaling provides a restrictive environment for otic fate in Xenopus embryos.     

The BMP/CHORDIN antagonism controls sensory pigment cell specification and differentiation in the ascidian embryo

We have investigated the role of the bone morphogenetic protein (BMP) pathway during neural tissue formation in the ascidian embryo. The orthologue of the BMP antagonist, chordin, was isolated from the ascidian Halocynthia roretzi. While both the expression pattern and the phenotype observed by overexpressing chordin or BMPb (the dpp-subclass BMP) do not suggest a role for these factors in neural induction, BMP/CHORDIN antagonism was found to affect neural patterning. Overexpression of BMPb induced ectopic sensory pigment cells in the brain lineages that do not normally form pigment cells and suppressed pressure organ formation within the brain. Reciprocally, overexpressing chordin suppressed pigment cell formation and induced ectopic pressure organ. We show that pigment cell formation occurs in three steps. (1) During cleavage stages ectodermal cells are neuralized by a vegetal signal that can be substituted by bFGF. (2) At the early gastrula stage, BMPb secreted from the lateral nerve cord blastomeres induces those neuralized blastomeres in close proximity to adopt a pigment cell fate. (3) At the tailbud stage, among these pigment cell precursors, BMPb induces the differentiation of specifically the anterior type of pigment cell, the otolith; while posteriorly, CHORDIN suppresses BMP activity and allows ocellus differentiation.     

Cranial neural crest-derived mesenchymal proliferation is regulated by Msx1-mediated p19(INK4d) expression during odontogenesis

Neural crest cells are multipotential progenitors that contribute to various cell and tissue types during embryogenesis. Here, we have investigated the molecular and cellular mechanism by which the fate of neural crest cell is regulated during tooth development. Using a two- component genetic system for indelibly marking the progeny of neural crest cells, we provide in vivo evidence of a deficiency of CNC-derived dental mesenchyme in Msx1 null mutant mouse embryos. The deficiency of the CNC results from an elevated CDK inhibitor p19(INK4d) activity and the disruption of cell proliferation. Interestingly, in the absence of Msx1, the CNC-derived dental mesenchyme misdifferentiates and possesses properties consistent with a neuronal fate, possibly through a default mechanism. Attenuation of p19(INK4d) in Msx1 null mutant mandibular explants restores mitotic activity in the dental mesenchyme, demonstrating the functional significance of Msx1-mediated p19(INK4d) expression in regulating CNC cell proliferation during odontogenesis. Collectively, our results demonstrate that homeobox gene Msx1 regulates the fate of CNC cells by controlling the progression of the cell cycle. Genetic mutation of Msx1 may alternatively instruct the fate of these progenitor cells during craniofacial development.     

Density-dependent growth inhibition of fibroblasts ectopically expressing p27(kip1)

The cyclin/cyclin-dependent kinase (cdk) inhibitor p27(kip1) is thought to be responsible for the onset and maintenance of the quiescent state. It is possible, however, that cells respond differently to p27(kip1) in different conditions, and using a BALB/c-3T3 cell line (termed p27-47) that inducibly expresses high levels of this protein, we show that the effect of p27(kip1) on cell cycle traverse is determined by cell density. We found that ectopic expression of p27(kip1) blocked the proliferation of p27-47 cells at high density but had little effect on the growth of cells at low density whether exponentially cycling or stimulated from quiescence. Regardless of cell density, the activities of cdk4 and cdk2 were markedly repressed by p27(kip1) expression, as was the cdk4-dependent dissociation of E2F4/p130 complexes. Infection of cells with SV40, a DNA tumor virus known to abrogate formation of p130- and Rb-containing complexes, allowed dense cultures to proliferate in the presence of supraphysiological amounts of p27(kip1) but did not stimulate cell cycle traverse when cultures were cotreated with the potent cdk2 inhibitor roscovitine. Our data suggest that residual levels of cyclin/cdk activity persist in p27(kip1)-expressing p27-47 cells and are sufficient for the growth of low-density cells and of high-density cells infected with SV40, and that effective disruption of p130 and/or Rb complexes is obligatory for the proliferation of high-density cultures.     

BCR-ABL and interleukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2 and p27Kip1 expression

Although it is evident that BCR-ABL can rescue cytokine-deprived hematopoietic progenitor cells from cell cycle arrest and apoptosis, the exact mechanism of action of BCR/ABL and interleukin (IL)-3 to promote proliferation and survival has not been established. Using the pro-B cell line BaF3 and a BaF3 cell line stably overexpressing BCR-ABL (BaF3-p210), we investigated the proliferative signals derived from BCR-ABL and IL-3. The results indicate that both IL-3 and BCR-ABL target the expression of cyclin Ds and down-regulation of p27(Kip1) to mediate pRB-related pocket protein phosphorylation, E2F activation, and thus S phase progression. These findings were further confirmed in a BaF3 cell line (TonB.210) where the BCR-ABL expression is inducible by doxycyclin and by using the drug STI571 to inactivate BCR-ABL activity in BaF3-p210. To establish the functional significance of cyclin D2 and p27(Kip1) expression in response to IL-3 and BCR-ABL expression, we studied the effects of ectopic expression of cyclin D2 and p27(Kip1) on cell proliferation and survival. Our results demonstrate that both cyclin D2 and p27(Kip1) have a role in BaF3 cell proliferation and survival, as ectopic expression of cyclin D2 is sufficient to abolish the cell cycle arrest and apoptosis induced by IL-3 withdrawal or by BCR-ABL inactivation, while overexpression of p27(Kip1) can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27(Kip1), cyclin E, and cyclin D3, and therefore, plays an essential part in integrating the signals from IL-3 and BCR-ABL with the pRB/E2F pathway.     

Neural crest determination by co-activation of Pax3 and Zic1 genes in Xenopus ectoderm

A number of regulatory genes have been implicated in neural crest development. However, the molecular mechanism of how neural crest determination is initiated in the exact ectodermal location still remains elusive. Here, we show that the cooperative function of Pax3 and Zic1 determines the neural crest fate in the amphibian ectoderm. Pax3 and Zic1 are expressed in an overlapping manner in the presumptive neural crest area of the Xenopus gastrula, even prior to the onset of the expression of the early bona fide neural crest marker genes Foxd3 and Slug. Misexpression of both Pax3 and Zic1 together efficiently induces ectopic neural crest differentiation in the ventral ectoderm, whereas overexpression of either one of them only expands the expression of neural crest markers within the dorsolateral ectoderm. The induction of neural crest differentiation by Pax3 and Zic1 requires Wnt signaling. Loss-of-function studies in vivo and in the animal cap show that co-presence of Pax3 and Zic1 is essential for the initiation of neural crest differentiation. Thus, co-activation of Pax3 and Zic1, in concert with Wnt, plays a decisive role for early neural crest determination in the correct place of the Xenopus ectoderm.     

Ectodysplasin A1 promotes placodal cell fate during early morphogenesis of ectodermal appendages

Organs developing as appendages of the ectoderm are initiated from epithelial thickenings called placodes. Their formation is regulated by interactions between the ectoderm and underlying mesenchyme, and several signalling molecules have been implicated as activators or inhibitors of placode formation. Ectodysplasin (Eda) is a unique signalling molecule in the tumour necrosis factor family that, together with its receptor Edar, is necessary for normal development of ectodermal organs both in humans and mice. We have shown previously that overexpression of the Eda-A1 isoform in transgenic mice stimulates the formation of several ectodermal organs. In the present study, we have analysed the formation and morphology of placodes using in vivo and in vitro models in which both the timing and amount of Eda-A1 applied could be varied. The hair and tooth placodes of K14-Eda-A1 transgenic embryos were enlarged, and extra placodes developed from the dental lamina and mammary line. Exposure of embryonic skin to Eda-A1 recombinant protein in vitro stimulated the growth and fusion of placodes. However, it did not accelerate the initiation of the first wave of hair follicles giving rise to the guard hairs. Hence, the function of Eda-A1 appears to be downstream of the primary inductive signal required for placode initiation during skin patterning. Analysis of BrdU incorporation indicated that the formation of the epithelial thickening in early placodes does not involve increased cell proliferation and also that the positive effect of Eda-A1 on placode expansion is not a result of increased cell proliferation. Taken together, our results suggest that Eda-A1 signalling promotes placodal cell fate during early development of ectodermal organs.