Transcriptional and epigenetic regulation of hindbrain development in the mouse

During embryonic development, proper vertebrate body patterning is achieved through a series of highly regulated transcriptional mechanisms that result in a precise spatial and temporal control of specific master genes. Hox transcription factors play a crucial role in the specification of posterior positional identity, acting as part of a downstream regulatory network responding to Retinoic Acid (RA) activity. RALDH2 enzyme is solely responsible for embryonic RA synthesis until E8.5, and its mutation affects dramatically the development of different structures and organs such as heart, somites, pharyngeal arches, limb and neural tube (Niederreither et al., 1999). Yet, little is known about the molecular mechanisms involved in its regulation. Previous literature showed that Pbx mutant mice phenocopy most of the defects exhibit by Raldh2–/– mutant animals (Capellini et al., 2006; Manley et al., 2004; Stankunas et al., 2008). Moreover, Pbx proteins are well characterized Hox cofactors (reviewed in Moens and Selleri, 2006). In the first part of this work we investigate the role of Hox and Pbx transcription factors in the maintenance of RALDH2 expression. Using genetic tools and biochemical assays such as in situ hybridization, reporter gene analysis of RA activity, chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and BAC recombineering we address this important question.
Furthermore, the generation of an early anterior boundary of RA activity, obtained through the complementary distribution of synthesizing and degrading enzymes, identifies a rostral embryonic domain devoid of Hox gene expression. Previously, it has been demonstrated that the maintenance of a Hox-negative domain is an essential condition required for the correct morphogenesis of vertebrate craniofacial structures (Couly et al., 1998; Creuzet et al., 2002). During early phases of neurogenesis, exogenous administration of RA or mutation of CYP degrading enzymes result in the anterior shift of Hox gene expression in the hindbrain and in the corresponding NCCs populating the pharyngeal regions (Hernandez et al., 2007; Mallo and Brändlin, 1997; Marshall et al., 1992; Mulder et al., 1998). These effects, associated with other RA-mediated molecular changes in the signalling epithelium of first pharyngeal arch, lead to impairment of craniofacial development (Mallo and Brändlin, 1997; Vieux-Rochas et al., 2007). Later on, anterior Hox genes become unresponsive to RA signalling and its exogenous administration no longer affects head and pharyngeal patterning. These evidences suggest a possible role of epigenetic silencing mechanisms in the maintenance of transcriptional repression of Hox gene in the face. Takihara and colleagues (Takihara et al., 1997) show that Phc1 disruption (the mouse homologue of the Drosophila polyhomeotic gene) leads to altered antero-posterior pattening and neural crest defects. Furthermore, Phc2 and Phc1 have been shown to act synergistically to establish a Polycomb-mediated repression of Hox genes (Isono et al, 2005). Although these works underscore the function PRC1 complex in the maintenance of transcriptional repression of Hox genes during antero-posterior specification, they do not account for the general function of Polycomb-mediated silencing in NCCs. Indeed, although a large majority of Polycomb targets are co-occupied by PRC2 and PCR1 complexes in ES cells, there is a substantial portion of target genes that show non-overlapping characteristics (Boyler et al., 2006; Ku et al., 2008). Moreover, a comprehensive analysis of craniofacial defects is missing, partly due to the early lethality of the analyzed mutant mice. In the second part of this work we address the genome-wide impact of cell-autonomous Ezh2 mutation during craniofacial development in the mouse and we discuss the implications of our results in the context of collinear expression of Hox genes and their chromatin architecture inside the nucleus. Using ChIP coupled with high throughput sequencing (ChIP-seq) and RNA-seq data, we identify the epigenomic and transcriptomic features of defined rostro-caudal cranial NCC populations. This study deciphers the role of PCR2 during head and pharyngeal morphogenesis.