Véronique Duboc

Nodal and BMP2/4 Signaling Organizes the Oral-Aboral Axis of the Sea Urchin Embryo

In the sea urchin embryo, the oral-aboral axis is specified after fertilization by mechanisms that are largely unknown. We report that early sea urchin embryos express Nodal and Antivin in the presumptive oral ectoderm and demonstrate that these genes control formation of the oral-aboral axis. Overexpression of nodal converted the whole ectoderm into oral ectoderm and induced ectopic expression of the orally expressed genes goosecoid, brachyury, BMP2/4, and antivin. Conversely, when the function of Nodal was blocked, by injection of an antisense Morpholino oligonucleotide or by injection of antivin mRNA, neither the oral nor the aboral ectoderm were specified. Injection of nodal mRNA into Nodal-deficient embryos induced an oral-aboral axis in a largely non-cell-autonomous manner. These observations suggest that the mechanisms responsible for patterning the oral-aboral axis of the sea urchin embryo may share similarities with mechanisms that pattern the dorsoventral axis of other deuterostomes.

FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis and regulate gastrulation during sea urchin development

The sea urchin embryo is emerging as an attractive model to study morphogenetic processes such as directed migration of mesenchyme cells and cell sheet invagination, but surprisingly, few of the genes regulating these processes have yet been characterized. We present evidence that FGFA, the first FGF family member characterized in the sea urchin, regulates directed migration of mesenchyme cells, morphogenesis of the skeleton and gastrulation during early development. We found that at blastula stages, FGFA and a novel putative FGF receptor are expressed in a pattern that prefigures morphogenesis of the skeletogenic mesoderm and that suggests that FGFA is one of the elusive signals that guide migration of primary mesenchyme cells (PMCs). We first show that fgfA expression is correlated with abnormal migration and patterning of the PMCs following treatments that perturb specification of the ectoderm along the oral-aboral and animal-vegetal axes. Specification of the ectoderm initiated by Nodal is required to restrict fgfA to the lateral ectoderm, and in the absence of Nodal, fgfA is expressed ectopically throughout most of the ectoderm. Inhibition of either FGFA, FGFR1 or FGFR2 function severely affects morphogenesis of the skeleton. Furthermore, inhibition of FGFA and FGFR1 signaling dramatically delays invagination of the archenteron, prevents regionalization of the gut and abrogates formation of the stomodeum. We identified several genes acting downstream of fgfA in these processes, including the transcription factors pea3 and pax2/5/8 and the signaling molecule sprouty in the lateral ectoderm and SM30 and SM50 in the primary mesenchyme cells. This study identifies the FGF signaling pathway as an essential regulator of gastrulation and directed cell migration in the sea urchin embryo and as a key player in the gene regulatory network directing morphogenesis of the skeleton.

Ancestral Regulatory Circuits Governing Ectoderm Patterning Downstream of Nodal and BMP2/4 Revealed by Gene Regulatory Network Analysis in an Echinoderm

Echinoderms, which are phylogenetically related to vertebrates and produce large numbers of transparent embryos that can be experimentally manipulated, offer many advantages for the analysis of the gene regulatory networks (GRN) regulating germ layer formation. During development of the sea urchin embryo, the ectoderm is the source of signals that pattern all three germ layers along the dorsal-ventral axis. How this signaling center controls patterning and morphogenesis of the embryo is not understood. Here, we report a large-scale analysis of the GRN deployed in response to the activity of this signaling center in the embryos of the Mediterranean sea urchin Paracentrotus lividus, in which studies with high spatial resolution are possible. By using a combination of in situ hybridization screening, overexpression of mRNA, recombinant ligand treatments, and morpholino-based loss-of-function studies, we identified a cohort of transcription factors and signaling molecules expressed in the ventral ectoderm, dorsal ectoderm, and interposed neurogenic (“ciliary band”) region in response to the known key signaling molecules Nodal and BMP2/4 and defined the epistatic relationships between the most important genes. The resultant GRN showed a number of striking features. First, Nodal was found to be essential for the expression of all ventral and dorsal marker genes, and BMP2/4 for all dorsal genes. Second, goosecoid was identified as a central player in a regulatory sub-circuit controlling mouth formation, while tbx2/3 emerged as a critical factor for differentiation of the dorsal ectoderm. Finally, and unexpectedly, a neurogenic ectoderm regulatory circuit characterized by expression of “ciliary band” genes was triggered in the absence of TGF beta signaling. We propose a novel model for ectoderm regionalization, in which neural ectoderm is the default fate in the absence of TGF beta signaling, and suggest that the stomodeal and neural subcircuits that we uncovered may represent ancient regulatory pathways controlling embryonic patterning.

Nodal and BMP2/4 pattern the mesoderm and endoderm during development of the sea urchin embryo.

Nodal factors play fundamental roles in induction and patterning of the mesoderm and endoderm in vertebrates, but whether this reflects an ancient role or one that evolved recently in vertebrates is not known. Here, we report that in addition to its primary role in patterning the ectoderm, sea urchin Nodal is crucial for patterning of the endoderm and skeletogenic mesoderm through the regulation of the expression of key transcription factors and signalling molecules, including BMP2/4 and FGFA. In addition, we uncovered an essential role for Nodal and BMP2/4 in the formation and patterning of the non-skeletogenic mesoderm. By comparing the effects of misexpressing Nodal or an activated Nodal receptor in clones of cells, we provide evidence that Nodal acts over a long range in the endomesoderm and that its effects on the blastocoelar cell precursors are likely to be direct. The activity of Nodal and BMP2/4 are antagonistic, and although bmp2/4 is transcribed in the ventral ectoderm downstream of Nodal, the BMP2/4 ligand is translocated to the dorsal side, where it activates signalling in the dorsal primary mesenchyme cells, the dorsal endoderm and in pigment cell precursors. Therefore, correct patterning of the endomesoderm depends on a balance between ventralising Nodal signals and dorsalising BMP2/4 signals. These experiments confirm that Nodal is a key regulator of dorsal-ventral polarity in the sea urchin and support the idea that the ventral ectoderm, like the Spemann organiser in vertebrates, is an organising centre that is required for patterning all three germ layers of the embryo.

Lefty acts as an essential modulator of Nodal activity during sea urchin oral-aboral axis formation

Nodal is a key player in the process regulating oral-aboral axis formation in the sea urchin embryo. Expressed early within an oral organizing centre, it is required to specify both the oral and aboral ectoderm territories by driving an oral-aboral gene regulatory network. A model for oral-aboral axis specification has been proposed relying on the self activation of Nodal and the diffusion of the long-range antagonist Lefty resulting in a sharp restriction of Nodal activity within the oral field. Here, we describe the expression pattern of lefty and analyse its function in the process of secondary axis formation. lefty expression starts at the 128-cell stage immediately after that of nodal, is rapidly restricted to the presumptive oral ectoderm then shifted toward the right side after gastrulation. Consistently with previous work, neither the oral nor the aboral ectoderm are specified in embryos in which Lefty is overexpressed. Conversely, when Leftys function is blocked, most of the ectoderm is converted into oral ectoderm through ectopic expression of nodal. Reintroducing lefty mRNA in a restricted territory of Lefty depleted embryos caused a dose-dependent effect on nodal expression. Remarkably, injection of lefty mRNA into one blastomere at the 8-cell stage in Lefty depleted embryos blocked nodal expression in the whole ectoderm consistent with the highly diffusible character of Lefty in other models. Taken together, these results demonstrate that Lefty is essential for oral-aboral axis formation and suggest that Lefty acts as a long-range inhibitor of Nodal signalling in the sea urchin embryo.

Regulation of Limb Bud Initiation and Limb-Type Morphology

While the paired forelimb and hindlimb buds of vertebrates are initially morphologically homogeneous, as the limb progenitors differentiate, each individual tissue element attains a characteristic limb‐type morphology that ultimately defines the constitution of the forelimb or hindlimb. This review focuses on contemporary understanding of the regulation of limb bud initiation and formation of limb‐type specific morphologies and how these regulatory mechanisms evolved in vertebrates. We also attempt to clarify the definition of the terms limb‐type identity and limb‐type morphology that have frequently been used interchangeably. Over the last decade, three genes, Tbx4, Tbx5, and Pitx1, have been extensively studied for their roles in limb initiation and determining limb‐type morphologies. The role of Tbx4 and Tbx5 in limb initiation is clearly established. However, their putative role in the generation of limb‐type morphologies remains controversial. In contrast, all evidence supports a function for Pitx1 in determination of hindlimb morphologies. Developmental Dynamics 240:1017–1027, 2011.

Reciprocal Signaling between the Ectoderm and a Mesendodermal Left-Right Organizer Directs Left-Right Determination in the Sea Urchin Embryo

During echinoderm development, expression of nodal on the right side plays a crucial role in positioning of the rudiment on the left side, but the mechanisms that restrict nodal expression to the right side are not known. Here we show that establishment of left-right asymmetry in the sea urchin embryo relies on reciprocal signaling between the ectoderm and a left-right organizer located in the endomesoderm. FGF/ERK and BMP2/4 signaling are required to initiate nodal expression in this organizer, while Delta/Notch signaling is required to suppress formation of this organizer on the left side of the archenteron. Furthermore, we report that the H+/K+-ATPase is critically required in the Notch signaling pathway upstream of the S3 cleavage of Notch. Our results identify several novel players and key early steps responsible for initiation, restriction, and propagation of left-right asymmetry during embryogenesis of a non-chordate deuterostome and uncover a functional link between the H+/K+-ATPase and the Notch signaling pathway.

Building limb morphology through integration of signalling modules

Growth and patterning of the vertebrate limb relies on signals produced by three discrete signalling centres: the Apical Ectodermal Ridge (AER), the Zone of Polarising Activity (ZPA) and the dorsal ectoderm. The molecular identities of these signals and their associated downstream pathways have begun to be uncovered. In this review, we focus on recent work that has highlighted the importance of cross-talk between these signalling centres and how mesenchymal progenitors integrate multiple signalling inputs. We also discuss recent evidence suggesting how modulations of key signalling pathways have been used to generate the morphological diversity seen between different vertebrate limb appendages.

Pitx1 is necessary for normal initiation of hindlimb outgrowth through regulation of Tbx4 expression and shapes hindlimb morphologies via targeted growth control.

The forelimbs and hindlimbs of vertebrates are morphologically distinct. Pitx1, expressed in the hindlimb bud mesenchyme, is required for the formation of hindlimb characteristics and produces hindlimb-like morphologies when misexpressed in forelimbs. Pitx1 is also necessary for normal expression of Tbx4, a transcription factor required for normal hindlimb development. Despite the importance of this protein in these processes, little is known about its mechanism of action. Using a transgenic gene replacement strategy in a Pitx1 mutant mouse, we have uncoupled two discrete functions of Pitx1. We show that, firstly, this protein influences hindlimb outgrowth by regulating Tbx4 expression levels and that, subsequently, it shapes hindlimb bone and soft tissue morphology independently of Tbx4. We provide the first description of how Pitx1 sculpts the forming hindlimb skeleton by localised modulation of the growth rate of discrete elements.

21-P040 Tbx4 does not rescue limb-type specification in Pitx1−/− mouse

Vertebrates have two pairs of morphologically distinct appendages, the forelimbs and the hindlimbs. Although the mechanisms necessary for limb outgrowth and polarity are becoming fairly well-understood little is known about how limb-type identity is regulated. The homeobox containing transcription factor Pitx1 expressed in hindlimb bud mesenchyme and has been shown to be involved in limb-type determination. A complication to the interpretation of the Pitx1 phenotype has arisen following the demonstration that Pitx1 activity is required for normal expression of Tbx4. Since Tbx4 is required for normal limb outgrowth some of the abnormalities observed in the Pitx1 hindlimb could be explained by down regulation of Tbx4. We are using transgenic lines expressing Tbx4, Tbx5 or Pitx1 in combination with Pitx1 mouse to study the activity of Pitx1 independently of outgrowth defects. Both Tbx4 and Tbx5 rescued hindlimbs are closer to a wild-type size but none of the hindlimb characteristics are compensated by the expression of these T-box genes. Our data show that Pitx1 function is necessary for hindlimb patterning and is consistent with Tbx4 and Tbx5 being insufficient to determine limb-type but contributing to limb initiation and outgrowth.