Expression and regulation of eHAND during limb development.

eHAND is a bHLH transcription factor with important functions during embryogenesis. Here, we report that eHAND has a dynamic pattern of expression during limb development. In chick embryos, eHAND expression is first observed in the ventral mesoderm of the emerging limb. Its expression is then restricted to an anteroventral area of mesoderm at mid-level in the proximodistal axis. At later stages, expression is observed in the autopod encompassing the ventral tendons of the digits. In mouse embryos, only the anteroventral domain of expression is conserved, the early ventral expression not being detectable and the late pattern of expression differing clearly from that in the chick. A constant feature of all areas of expression is their ventral and anterior localization. Respecification of the anterior mesoderm as occurs secondarily to Sonic hedgehog (SHH) or retinoic acid application to the anterior border leads to down-regulation of eHAND expression. Accordingly, eHAND expression is not detectable in talpid(2) mutant limbs, which are considered to be posteriorized limbs. However, eHAND expression is little modified in oligozeugodactyly, a chick mutant that lacks Shh signaling in the limb but retains certain anteroposterior polarity. Interestingly, eHAND expression is also linked to the ventral identity of the mesoderm and is repressed by the dorsal ectoderm. It is also positively regulated by bone morphogenetic protein signaling, which is also known to participate in dorsoventral patterning. We suggest that eHAND expression may be related to the anteroventral identity of the mesoderm. However, in overexpression experiments using retroviral vectors, only a low percentage of cases (5%) showed phenotypic alterations, consisting of a duplication of digit 2.

BMP signaling positively regulates Nodal expression during left right specification in the chick embryo.

Exogenous application of BMP to the lateral plate mesoderm (LPM) of chick embryos at the early somite stage had a positive effect on Nodal expression. BMP applications into the right LPM were followed by a rapid activation of Nodal, while applications into the left LPM resulted in expansion of the normal domain of Nodal expression. Conversely, blocking of BMP signaling by Noggin in the left LPM interfered with the activation of Nodal expression. These results support a positive role for endogenous BMP on Nodal expression in the LPM. We also report that BMP positively regulates the expression of Caronte, Snail and Cfc in both the left and right LPM. BMP-treated embryos had molecular impairment of the midline with downregulation of Lefty1, Brachyury and Shh but we also show that the midline defect was not sufficient to induce ectopic Nodal expression. We discuss our findings in the context of the known molecular control of the specification of left-right asymmetry.

c-Myc overexpression increases cell size and impairs cartilage differentiation during chick limb development.

c-Myc is a transcription factor involved in the control of cell proliferation, differentiation, and apoptosis, all basic processes for embryogenesis. To analyze c-Myc roles in limb development, we overexpressed c-myc in chick embryos using a retroviral vector. Forced c-myc expression resulted in enlarged limbs, because of an increase in cell size not accompanied by modifications in cell proliferation. However, at later stages, limbs overexpressing c-myc showed a marked shortening of their skeletal elements, because of the inhibition of chondrocyte maturation. c-Myc interfered with chondrogenesis, independently of the Indian hedgehog/parathyroid hormone-related protein and Wnt5a/Wnt5b regulatory loops. c-myc-infected limbs also exhibited patterning defects, such as extra-phalangeal elements and delayed interdigital apoptosis that occasionally led to interdigital chondrogenesis. In contrast, c-myc overexpression did not interfere with other processes, such as muscle differentiation. Although based on overexpression experiments, our results suggest that endogenous c-Myc may be implicated in the control of cell size and skeletal differentiation during normal limb development.