MASH1 ACTIVATES EXPRESSION OF THE PAIRED HOMEODOMAIN TRANSCRIPTION FACTOR PHOX2A, AND COUPLES PAN-NEURONAL AND SUBTYPE-SPECIFIC COMPONENTS OF AUTONOMIC NEURONAL IDENTITY

We have investigated the genetic circuitry underlying the determinatio n of neuronal identity, using mammalian peripheral autonomic neurons a s a model system, Previously, we showed that treatment of neural crest stem cells (NCSCs) with bone morphogenetic protein-2 (BMP-2) leads to an induction of MASH1 expression and consequent autonomic neuronal di fferentiation, We now show that BMP2 also induces expression of the pa ired homeodomain transcription factor Phox2a, and the GDNF/NTN signall ing receptor tyrosine kinase c-RET Constitutive expression of MASH1 in NCSCs from a retroviral vector, in the absence of exogenous BMP2, ind uces expression of both Phox2a and c-RET in a large fraction of infect ed colonies, and also promotes morphological neuronal differentiation and expression of pan-neuronal markers, In vivo, expression of Phox2a in autonomic ganglia is strongly reduced in Mash1 -/- embryos, These l oss-and gain-of-function data suggest that MASH1 positively regulates expression of Phox2a, either directly or indirectly, Constitutive expr ession of Phox2a, by contrast to MASH1, fails to induce expression of neuronal markers or a neuronal morphology, but does induce expression of c-RET. These data suggest that MASH1 couples expression of pan-neur onal and subtype-specific components of autonomic neuronal identity, a nd support the general idea that identity is established by combining subprograms involving cascades of transcription factors, which specify distinct components of neuronal phenotype.