LKB1 signaling in cephalic neural crest cells is essential for vertebrate head development.

Head development in vertebrates proceeds through a series of elaborate patterning mechanisms and cell-cell interactions involving cephalic neural crest cells (CNCC). These cells undergo extensive migration along stereotypical paths after their separation from the dorsal margins of the neural tube and they give rise to most of the craniofacial skeleton. Here, we report that the silencing of the LKB1 tumor suppressor affects the delamination of pre-migratory CNCC from the neural primordium as well as their polarization and survival, thus resulting in severe facial and brain defects. We further show that LKB1-mediated effects on the development of CNCC involve the sequential activation of the AMP-activated protein kinase (AMPK), the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Collectively, these results establish that the complex morphogenetic processes governing head formation critically depends on the activation of the LKB1 signaling network in CNCC.

Ventral vs. dorsal chick dermal progenitor specification.

The dorsal and the ventral trunk integuments of the chick differ in their dermal cell lineage (originating from the somatic and somatopleural mesoderm respectively) and in the distribution of their feather fields. The dorsal macropattern has a large spinal pteryla surrounded by semi-apteria, whereas the ventral skin has a true medial apterium surrounded by the ventral pterylae. Comparison of the results of heterotopic transplantations of distal somatopleure in place of somatic mesoderm (Mauger 1972) or in place of proximal somatopleure (our data), leads to two conclusions. These are that the fate of the midventral apterium is not committed at day 2 of incubation and that the signals from the environment which specify the ventral and dorsal featherforming dermal progenitors are different. Effectively, Shh, but not Wnt -1 signalling can induce the formation of feather forming dermis from the embryonic somatopleure. Shh is not able, however, to trigger the formation of a feather forming dermis from the extra embryonic somatopleure. This brief report constitutes the first attempt, by comparing old and new preliminary results, to understand whether dermal progenitors at different sites are specified by different signalling pathways.

Skin field formation: morphogenetic events.

This chapter is mostly a review of the pioneering work of the Philippe Sengel school in Grenoble carried out in the late sixties and the seventies. The questions raised concerning the morphogenesis of feather tracts were approached by means of microsurgery on chick embryos. P. Sengel and his wife M. Kieny had the feeling that proteins synthesized by the neural tube were required for the formation of feather fields. It was my pleasure to carry on the story from the beginning. Although some clarifications concerning this morphogenesis have been contributed by my group and by a few other laboratories interested in this subject, the most important contributions to recent research have been the elucidation of the nature of the required messages, which will be explored further in other papers in this Issue.

The different steps of skin formation in vertebrates.

Skin morphogenesis occurs following a continuous series of cell-cell interactions which can be subdivided into three main stages: 1- the formation of a dense dermis and its overlying epidermis in the future appendage fields (macropattern); 2- the organization of these primary homogeneous fields into heterogeneous ones by the appearance of cutaneous appendage primordia (micropattern) and 3- cutaneous appendage organogenesis itself. In this review, we will first show, by synthesizing novel and previously published data from our laboratory, how heterogenetic and heterospecific dermal/epidermal recombinations have allowed us to distinguish between the respective roles of the dermis and the epidermis. We will then summarize what is known from the work of many different research groups about the molecular signaling which mediates these interactions in order to introduce the following articles of this Special Issue and to highlight what remains to done.

Drm/Gremlin, a BMP antagonist, defines the interbud region during feather development.

The pattern of feather buds in a tract is thought to result from the relative ratios between activator and inhibitor signals through a lateral inhibition process. We analyse the role of Drm/Gremlin, a BMPs antagonist expressed during feather pattern formation, in the dermal precursor, the dense dermis, the interbud dermis and in the posterior dermal condensation. We have altered the activity of Drm in embryonic chick skin using retroviral vectors expressing drm/ gremlin and bmps. We show that expression of endogenous drm is under the control of a feedback loop induced by the BMP pathway, and that overexpression of drm results in fusion between adjacent feather buds. We propose that endogenous BMP proteins induce drm expression in the interbud dermis. In turn, the Drm/Gremlin protein limits the inhibitory effect of BMPs, allowing the adjacent row of feathers to form. Thus, the balance between BMPs and its antagonist Drm would regulate the size and spacing of the buds.

Signaling dynamics of feather tract formation from the chick somatopleure.

In the chick, most feathers are restricted to specific areas of the skin, the feather tracts or pterylae, while other areas, such as the apteria, remain bare. In the embryo, the expansion and closure of the somatopleure leads to the juxtaposition of the ventral pteryla, midventral apterium and amnion. The embryonic proximal somatopleural mesoderm is determined to form a feather-forming dermis at 2 days of incubation (E2), while the embryonic distal and the extra-embryonic somatopleure remain open to determination. We found a progressive, lateral expression of Noggin in the embryonic area, and downregulation of Msx1, a BMP4 target gene, with Msx1 expression being ultimately restricted to the most distal embryonic and extra-embryonic somatopleural mesoderm. Msx1 downregulation thus correlates with the formation of the pterylae, and its maintenance to that of the apterium. Suspecting that the inhibition of BMP4 signaling might be linked to the determination of a feather-forming dermis, we grafted Noggin-expressing cells in the distal somatopleure at E2. This elicited the formation of a supplementary pteryla in the midventral apterium. Endogenous Noggin, which is secreted by the intermediate mesoderm at E2, then by the proximal somatopleure at E4, could be sufficient to suppress BMP4 signaling in the proximal somatopleural mesoderm and then in part of the distal somatopleure, thus in turn allowing the formation of the dense dermis of the future pterylae. The same result was obtained with the graft of Shh-producing cells, but Noggin and Shh are both required in order to change the future amnion into a feather-bearing skin. A possible synergistic role of endogenous Shh from the embryonic endoderm remains to be confirmed.

Transformation of amnion epithelium into skin and hair follicles.

There is increasing interest into the extent to which epithelial differentiation can be altered by mesenchymal influence, and the molecular basis for these changes. In this study, we investigated whether amnion epithelium could be transformed into skin and hair follicles by associating E12.5 to E14.5 mouse amnion from the ROSA 26 strain, with mouse embryonic hair-forming dermis from a wild-type strain. These associations were able to produce fully formed hair follicles with associated sebaceous glands, and skin epidermis. Using beta-galactosidase staining we were able to demonstrate that the follicular epithelium and skin epidermis, but not the associated dermal cells, originated from the amnion. As Noggin and Sonic hedgehog (Shh) were recently shown to be required for early chick ventral skin formation, and able to trigger skin and feather formation from chick amnion, we associated cells engineered to produce those two factors with mouse amnion. In a few cases, we obtained hair buds connected to a pluristratified epithelium; however, the transformation of the amnion was impeded by uncontrolled fibroblastic proliferation. In contrast to an earlier report, none of our control amnion specimens autonomously transformed into skin and hair follicles, indicating that specific influences are necessary to elicit follicle formation from the mouse amnion. The ability to turn amnion into skin and its appendages has practical potential for the tissue engineering of replacement skin, and related biotechnological approaches.