Study of vertebrate germ layer segregation with CRISPR/CAS9 genomic edition tool in Xenopus laevis

FAVAROLO, M. BELÉN; LÓPEZ, SILVIA L.

Capital Federal

Congreso; IV International Congress in Translational Medicine; 2018

Universidad de Buenos Aires

Notch is a single-pass membrane receptor that is cleaved to release an intracellular domain(NICD) after interacting with specific ligands (Delta, Jagged) presented by the neighboring cells.Once released, NICD translocates to the nucleus and activates transcription of target genes. Therole of Notch is context-dependent. As an inhibitory signal during lateral inhibition, Notchprevents equipotent cells to adopt the same fate. As an inductive signal, Notch induces theexpression of positively acting regulatory molecules in order to promote the developing of adifferent cellular type in the boundaries of non-equipotent cell populations by cell-cellinteraction, thus regulating the boundary between populations within morphogenetic fields.One known example is boundary generation between dorsal and ventral compartments in thewing imaginal disc in Drosophila, where Delta and Serrate (Jagged) are involved.Our lab demonstrated the major role of some members of the Notch pathway during thedevelopment of the Xenopus blastula and gastrula dorsal centers and their descendants, andduring the segregation of germ layers [López et al. Development 130, 2225?2238 (2003); Lópezet al. (2005) Development 132, 1035?1046; Revinski et al. (2010) Dev. Biol. 339, 477?492; Acostaet al. (2011) Development 138, 2567?79; Aguirre et al. (2013) PLoS One 8, e54777)]. Anomaliesduring the establishment and segregation of germ layers cause developmental failures Theirseverity varies from congenital malformations to embryonic lethality. In Revinski et al (2010), itwas possible to demonstrate by gain and loss-of-function that Delta1 (Dll1) and Notch1 areinvolved in endomesoderm and neural ectoderm delimitation. Notch1 also participates inendoderm and mesoderm delimitation, possibly involving another ligand.One of the developmental anomalies related with the Notch pathway in humans is the AlagilleSyndrome, which is caused by mutations in the gene encoding Jagged 1 (Jag1). However, Jag1knock-out mice do not reproduce the syndrome. We have found that Jag1 and Dll1 havecomplementary expression patterns in Xenopus during germ layer segregation. In situhybridizations at gastrula stage revealed Jag1 expression in the presumptive neuroectoderm andalso in the marginal zone, with a ?salt and pepper? cellular pattern, corresponding to the sub-blastoporal endoderm, adjacent to the mesoderm ring, where Dll1 is expressed. This patternsuggests that Jag1 might participate in unknown aspects of germ layer development. We areemploying the CRISPR/Cas9 gene editing tool for generating loss of function mutations in Jag1 inXenopus laevis. Preliminary results indicate that mutant embryos show a gastrulation defect,compatible with a role of Jag1 in germ layer segregation. Our aim is to study the role of Jag1during germ layer development and to establish a model of Alagille syndrome by employinggenomic edition with CRISPR/Cas9.