Banded hedgehog/Gli3 pathway is required for neural crest induction

TRISTÁN H. AGÜERO; SARA S. SÁNCHEZ; MANUEL J. AYBAR

Los Cocos, Córdoba, Argentina.

Workshop; Latest Concepts in Developmental Biology; 2006

Secreted morphogens of the Hedgehog (Hh) family in Xenopus laevis are sonic hedgehog, cephalic hedgehog and banded hedgehog, and direct a wide range of developmental processes. Members of the Hh family bind to the transmembrane receptor Patched (Ptc) resulting in the derepression of the protein Smoothened (Smo) that regulates the activity of the Gli family of transcription factors. In this work we analyzed in detail the expression of banded hedgehog (bhh) and all the components of the intracelular signaling cascade during early development of Xenopus laevis embryos. The comparative in situ hybridization analysis showed that bhh is expressed during neurulation stages at the lateral border of neural plate in territories that overlap with the expression of Gli2/Gli3 transcription factors and neural crest markers. In order to evaluate the participation of bhh pathway in neural crest development, we carried out a gain-of function approach by the microinjection of in vitro transcribed bhh mRNA in one blastomere of 8-16 cells embryos. Results showed that an experimental increase in bhh signaling leads to an increased expression of Gli transcription factors that correlates with an increase in the expression of neural crest markers (Slug, Snail, FoxD3). Additionally, the overexpression of Gli3 produced an expansion in the neural crest territory mimicking the effect produced by bhh gain of function. On the other hand, to evaluate the opposite effect we performed three approaches: 1) overexpression of a dominant negative construct of bhh (bhh∆N-C), 2) placing resin microspheres soaked with Cyclopamine, a specific inhibitor of Smo, 3) microinjection of morpholino antisense oligonucleotides (MO) against Bhh and Gli3. Results showed in all approaches the same effect, a reduction of the expression of neural crest markers suggesting that bhh/Gli3 signalling pathway is required for neural crest induction and migration. We also carried out a rescue experiment in which the inhibitor effects of loss-of function experiments were suppressed by the coinjection of full length bhh, Gli2 and Gli3. Our results show that bhh signaling and Gli3 transcription factors are key players in the early specification of Xenopus neural crest.