Multiple steps in the induction of the neural crest

C. TRÍBULO; A. GLAVIC; M. AYBAR; S. VILLANUEVA; G. ACUÑA; E. RODRIGUEZ; J. DECALISTO; R. CARPIO; C. ARAYA; R. MAYOR

Timberline Lodge, Mt. Hood, Oregon, USA.

Workshop; The Neural Crest: new perspectives on lineage and morphogenesis; 2003

The neural crest cell is a migratory embryonic cell population that forms from the neural folds at the border between the neural plate and the future epidermis. The neural fold surrounds the entire neural plate but only the lateral and posterior portion of the fold give rise to neural crest cells. In this work we have focused on neural crest induction in Xenopus laevis embryos. The neural crest is induced by an interaction between neural plate and epidermis. This interaction leads to the specification of the neural plate border by a gradient of BMP activity. The anterior neural plate border is transformed into neural crest cell by the posteriorizing signals, Wnts, FGF and retinoic acid. Once the neural crest is specified by these signals, a genetic cascade of transcription factors is activated. We have studied the role that Snail, Slug, Msx and Notch have on neural crest development. In order to understand the molecular mechanism of neural crest specification we have analyzed the role that the downstream BMP target, msx1, plays on neural crest development. We shown that msx1 expression is able to induce all other early neural crest markers tested. Furthermore, the expression of a dominant negative of msx leads to the inhibition of all the neural crest markers analyzed. These results indicate that msx play a critical role in neural crest specification. We have also shown that Snail is one of the earliest genes acting in the neural crest genetic cascade. In order to study the hierarchical relationship between msx1 and Snail/Slug we performed several rescue experiments using dominant negatives for these genes. The rescuing activity by Snail and Slug on neural crest development of the msx1 dominant negative, together with the inability of Slug or Snail to rescue the dominant negative msx1 strongly argues that msx1 is upstream of Snail and Slug in the genetic cascade that specifies the neural crest in the ectoderm. As msx1 is an expected direct downstream gene of BMP, we experimentally modified the level of BMP activity by use of a dominant negative BMP4 or BMP receptor in Xenopus and zebrafish embryos and also through BMP pathway component mutants in the zebrafihs and msx expression were analyzed. In addition, by reaching different levels of BMP activity in animal cap ectoderm, we show that a specific concentration of BMP induces msx1 expression, which is similar to the level required to induce neural crest. In addition we have analyzed the molecular mechanisms through which Notch signalling and the homeoprotein Xiro1 regulate the generation of the neural crest. Activation of Xiro1 or Notch signalling led to an enlargement of the neural crest markers. We show that Xiro1 and Notch signalling upregulates Hairy2A and inhibitis Bmp4 transcription during neural crest specification. In addition we shown that Delta1 expression at the borders of the neural crest region, is positively controlled by Xiro1 and repressed by Snail. These results, in conjunction with rescue experiments, allow us to propose a model where Xiro1 lies upstream of the cascade regulating Delta1 transcription, and the interaction of Delta1 with Notch induces Hairy2A expression at the border of neural plate. Here, Hairy2A acts as a repressor of Bmp4 transcription ensuring that the optimal levels of BMP are maintained to specify msx1 expression and the genesis of neural crest cells. We propose a model where Delta1, Hairy2A and Xiro1 control the level of msx in the neural fold region, and that this expression is essential for the early specification of the neural crest cells.