Frodo proteins: modulators of Wnt signaling in vertebrate development

The Frodo/dapper (Frd) proteins are recently discovered signaling adaptors, which functionally and physically interact with Wnt and Nodal signaling pathways during vertebrate development. The Frd1 and Frd2 genes are expressed in dynamic patterns in early embryos, frequently in cells undergoing epithelial-mesenchymal transition. The Frd proteins function in multiple developmental processes, including mesoderm and neural tissue specification, early morphogenetic cell movements, and organogenesis. Loss-of-function studies using morpholino antisense oligonucleotides demonstrate that the Frd proteins regulate Wnt signal transduction in a context-dependent manner and may be involved in Nodal signaling. The identification of Frd-associated factors and cellular targets of the Frd proteins should shed light on the molecular mechanisms underlying Frd functions in embryonic development and in cancer.     

Vertebrate head development: Segmentation,novelties, and homology

Vertebrate head development is a classical topic lately invigorated by methodological as well as conceptual advances. In contrast to the classical segmentalist views going back to idealistic morphology, the head is now seen not as simply an extension of the trunk, but as a structure patterned by different mechanisms and tissues. Whereas the trunk paraxial mesoderm imposes its segmental pattern on adjacent tissues such as the neural crest derivatives, in the head the neural crest cells carry pattern information needed for proper morphogenesis of mesodermal derivatives, such as the cranial muscles. Neural crest cells make connective tissue components which attach the muscle fiber to the skeletal elements. These crest cells take their origin from the same visceral arch as the muscle cells, even when the skeletal elements to which the muscle attaches are from another arch. The neural crest itself receives important patterning influences from the pharyngeal endoderm. The origin of jaws can be seen as an exaptation in which a heterotopic shift of the expression domains of regulatory genes was a necessary step that enabled this key innovation. The jaws are patterned by Dlx genes expressed in a nested pattern along the proximo-distal axis, analogous to the anterior–posterior specification governed by Hox genes. Knocking out Dlx 5 and 6 transforms the lower jaw homeotically into an upper jaw. New data indicate that both upper and lower jaw cartilages are derived from one, common anlage traditionally labelled the “mandibular” condensation, and that the “maxillary” condensation gives rise to other structures such as the trabecula. We propose that the main contribution from evolutionary developmental biology to solving homology questions lies in deepening our biological understanding of characters and character states.     

Generation of segment polarity in the paraxial mesoderm of the zebrafish through a T-box-dependent inductive event

The first morphological sign of vertebrate postcranial body segmentation is the sequential production from posterior paraxial mesoderm of blocks of cells termed somites. Each of these embryonic structures is polarized along the anterior/posterior axis, a subdivision first distinguished by marker gene expression restricted to rostral or caudal territories of forming somites. To better understand the generation of segment polarity in vertebrates, we have studied the zebrafish mutant fused somites (fss), because its paraxial mesoderm lacks segment polarity. Previously examined markers of caudal half-segment identity are widely expressed, whereas markers of rostral identity are either missing or dramatically down-regulated, suggesting that the paraxial mesoderm of the fss mutant embryo is profoundly caudalized. These findings gave rise to a model for the formation of segment polarity in the zebrafish in which caudal is the default identity for paraxial mesoderm, upon which is patterned rostral identity in an fss-dependent manner. In contrast to this scheme, the caudal marker gene ephrinA1 was recently shown to be down-regulated in fss embryos. We now show that notch5, another caudal identity marker and a component of the Delta/Notch signaling system, is not expressed in the paraxial mesoderm of early segmentation stage fss embryos. We use cell transplantation to create genetic mosaics between fss and wild-type embryos in order to assay the requirement for fss function in notch5 expression. In contrast to the expression of rostral markers, which have a cell-autonomous requirement for fss, expression of notch5 is induced in fss cells at short range by nearby wild-type cells, indicating a cell-non-autonomous requirement for fss function in this process. These new data suggest that segment polarity is created in a three-step process in which cells that have assumed a rostral identity must subsequently communicate with their partially caudalized neighbors in order to induce the fully caudalized state.     

Pattern and morphogenesis of presumptive superficial mesoderm in two closely related species, Xenopus laevis and Xenopus tropicalis

The mesoderm, comprising the tissues that come to lie entirely in the deep layer, originates in both the superficial epithelial and the deep mesenchymal layers of the early amphibian embryo. Here, we characterize the mechanisms by which the superficial component of the presumptive mesoderm ingresses into the underlying deep mesenchymal layer in Xenopus tropicalis and extend our previous findings for Xenopus laevis. Fate mapping the superficial epithelium of pregastrula stage embryos demonstrates ingression of surface cells into both paraxial and axial mesoderm (including hypochord), in similar patterns and amounts in both species. Superficial presumptive notochord lies medially, flanked by presumptive hypochord and both overlie the deep region of the presumptive notochord. These tissues are flanked laterally by superficial presumptive somitic mesoderm, the anterior tip of which also appears to overlay the presumptive deep notochord. Time-lapse recordings show that presumptive somitic and notochordal cells move out of the roof of the gastrocoel and into the deep region during neurulation, whereas hypochordal cells ingress after neurulation. Scanning electron microscopy at the stage and position where ingression occurs suggests that superficial presumptive somitic cells in X. laevis ingress into the deep region as bottle cells whereas those in X. tropicalis ingress by "relamination" (e.g., [Dev. Biol. 174 (1996) 92]). In both species, the superficially derived presumptive somitic cells come to lie in the medial region of the presumptive somites during neurulation. By the early tailbud stages, these cells lie at the horizontal myoseptum of the somites. The morphogenic pathway of these cells strongly resembles that of the primary slow muscle pioneer cells of the zebrafish. We present a revised fate map of Xenopus, and we discuss the conservation of superficial mesoderm within amphibians and across the chordates and its implications for the role of this tissue in patterning the mesoderm.     

Fine-tuning of fgf8a expression through alternative polyadenylation has a selective impact on Fgf-associated developmental processes

The formation of distinct 3′UTRs through alternative polyadenylation is a mechanism of gene expression regulation that has been implicated in many physiological and pathological processes. However, its functions in the context of vertebrate embryonic development have been largely unaddressed, in particular with a gene-specific focus. Here we show that the most abundant 3′UTR for the zebrafish fgf8a gene in the developing embryo mediates a strong translational repression, when compared to a more sparsely used alternative 3′UTR, which supports a higher translation efficiency. By inducing a shift in the selection efficiency of the associated polyadenylation sites, we show a temporally and spatially specific impact of fgf8a 3′UTR usage on embryogenesis, in particular at late stages during sensory system development. In addition, we identified a previously undescribed role for Fgf signalling in the initial stages of superficial retinal vascularization. These results reveal a critical functional importance of gene-specific alternative 3′UTRs in vertebrate embryonic development.     

Msx1 expressing mesoderm is important for the apical ectodermal ridge (AER)-signal transfer in chick limb development

The apical ectodermal ridge (AER) is a specialized thickening of the distal limb ectoderm, and its signals are known to support limb morphogenesis. The expression of a homeobox gene, Msx1 , in the distal limb mesoderm depends on signals from the AER. In the present paper it is reported that Msx1 expression in the distal mesoderm is necessary for the transfer of AER signals in chick limb buds. Interruption of AER-mesoderm interaction by insertion of a thick filter led to the inhibition of pattern specification in the mesoderm just under the filter. In such cases, the expression of Msx1 disappeared in the mesoderm under the filter, suggesting that AER is able to signal over short ranges. In advanced limb buds, Msx1 is also expressed in the proximal mesoderm under the anterior ectoderm. However, it was found that a grafted antero-proximal mesoderm shows no inhibitory effects on pattern specification of the host mesoderm, as is the case with the distal mesoderm. On the other hand, grafted mesoderms without potent Msx1 re-expression, even underneath AER, disturbed normal limb development. In such cases, the expression of Msx1 disappeared in the mesoderm under the grafts, whereas Fgf-8 expression was maintained in the AER above the graft. These results indicate that the expression of Msx1 in the mesoderm is important for the transfer of AER signals.     

Direct evidence of an essential role for extended involution in the specification of a dorsal marginal mesoderm during Cynops gastrulation

It has been indicated that specification of the dorsal marginal mesoderm of the Cynops gastrula is established by vertical interactions with other layers, which occur during its extended involution. In the present study, when the prospective notochordal area of the early gastrula was almost completely removed together with the dorsal mesoderm-inducing endoderm and most of the bottle cells, the D-less gastrulas still formed the dorsal axis with a well-differentiated notochord; in half of them, where the involution occurred bi-laterally, twin axes were observed. On the other hand, when the wound of a D-less gastrula was repaired by transplanting the ventral marginal zone and ectoderm, the formation of the dorsal axis was inhibited if the involution of the lateral marginal zone was prevented by the transplanted piece. The present study suggests that: (i) cells having dorsal mesoderm-forming potency distribute farther laterally than the fate map; and (ii) the extended involution plays an essential role in the specification of the dorsal marginal mesoderm, especially in notochordal differentiation in normal Cynops embryogenesis.     

Mesoderm induction in amphibians and chick

Induction is a process in which the developmental pathway of one cell is controlled by signals emitted from another. Mesoderm induction is the first inductive interaction in theXenopus enbryo and probably occurs in all vertebrates. It is a very important event as it is implicated in the regulation of morphogenesis. Nieuwkoop first demonstrated the importance of vegetal endoderm in inducing the mesoderm. Slack and co-workers incorporated the information obtained from experimental embryology in a “three signal” model for mesoderm induction in amphibians (signals arising from ventral vegetal hemisphere, dorsal vegetal hemisphere and the organizer). More recent research has resulted in the detection of mesoderm inducing factors which are members of FGF and TGF--β families. Activin, a member of the TGF-β family, has been shown to induce differential gene expression and cell differentiation in a concentration-dependent manner giving credence to the theory of morphogen gradients. Study of mesoderm induction in the chick embryo is much more difficult due to several reasons. Novel experimental approaches, however, have been used which point to the role of activin and FGF in chick mesoderm induction. The demonstration of mesoderm inducing activity of activin and FGF in other groups of vertebrates, particularly the chick embryo brings out the possibility of a universal mechanism of mesoderm induction being operative in all the vertebrates.     

Modulation of Phase Shift between Wnt and Notch Signaling Oscillations Controls Mesoderm Segmentation

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Deciphering the hierarchy of angiohematopoietic progenitors from human pluripotent stem cells

Identification of sequential progenitors leading to blood formation from pluripotent stem cells (PSCs) will be essential for understanding the molecular mechanisms of hematopoietic lineage specification and for development of technologies for in vitro production of hematopoietic stem cells (HSCs). It is well established that during development, blood and endothelial cells in the extraembryonic and embryonic compartments are formed in parallel from precursors with angiogenic and hematopoietic potentials. However, the identity and hierarchy of these precursors in human PSC (hPSC) cultures remain obscure. Using developmental stage-specific mesodermal and endothelial markers and functional assays, we recently identified discrete populations of angiohematopoietic progenitors from hPSCs, including mesodermal precursors and hemogenic endothelial cells with primitive and definitive hematopoietic potentials. In addition, we discovered a novel population of multipotent hematopoietic progenitors with an erythroid phenotype, which retain angiogenic potential. Here we introduce our recent findings and discuss their implication for defining putative HSC precursor and factors required for activation of self-renewal potential in hematopoietic cells emerging from endothelium.