Summary Integration of anteroposterior and dorsoventral regulation of Phox 2 b transcription in cranial motoneuron progenitors by homeodomain proteins

How specific neuronal types are generated at defined locations within the developing neural tube is still poorly understood. During early development of the central nervous system (CNS), neural progenitors acquire regionally restricted positional addresses by responding to graded inductive signals intersecting along the AP and DV axes (Jessell, 2000; Lumsden and Krumlauf, 1996). A grid-like set of positional cues is established within an initially equivalent group of cells that, together with the activation of a neurogenesis program (Bertrand et al., 2002), is translated into specific neuronal fates. Moreover, distinct neuronal types are generated in specific temporal order, often from common pools of neural progenitors (Jessell, 2000; Pattyn et al., 2003a). In vertebrates, distinct sets of spatially restricted homeodomain (HD) transcription factors provide transcriptional readouts of AP and DV positional addresses in neural progenitors. Along the AP axis, distinct progenitor domains are generated by the nested expression patterns of the Hox HD-containing genes (Lumsden and Krumlauf, 1996). In the ventral neural tube, DV positional addresses are instead conferred by Nkx-, Dbx-, Paxand Irx-class HD proteins (Briscoe et al., 2000). Along both axes, autoand crossregulatory activities among HD factors are required to refine and/or maintain progenitor domains (Briscoe et al., 2000; Dasen et al., 2003; Maconochie et al., 1997; Popperl et al., 1995). The combined activity of HD genes is thought to activate other sets of transcription factors that, in turn, regulate the expression of unique neuronal phenotypes (Lee and Pfaff, 2001). Neuronal identity is therefore the result of a complex regulatory network of transcription factors acting sequentially. Despite the increasing knowledge about genetic cascades and epistatic relationships among HD factors, little is known about their direct downstream targets and how AP and DV molecular Little is known about the molecular mechanisms that integrate anteroposterior (AP) and dorsoventral (DV) positional information in neural progenitors that specify distinct neuronal types within the vertebrate neural tube. We have previously shown that in ventral rhombomere (r)4 of Hoxb1 and Hoxb2 mutant mouse embryos, Phox2b expression is not properly maintained in the visceral motoneuron progenitor domain (pMNv), resulting in a switch to serotonergic fate. Here, we show that Phox2b is a direct target of Hoxb1 and Hoxb2. We found a highly conserved Phox2b proximal enhancer that mediates rhombomere-restricted expression and contains separate Pbx-Hox (PH) and Prep/Meis (P/M) binding sites. We further show that both the PH and P/M sites are essential for Hox-Pbx-Prep ternary complex formation and regulation of the Phox2benhancer activity in ventral r4. Moreover, the DV factor Nkx2.2 enhances Hox-mediated transactivation via a derepression mechanism. Finally, we show that induction of ectopic Phox2b-expressing visceral motoneurons in the chick hindbrain requires the combined activities of Hox and Nkx2 homeodomain proteins. This study takes an important first step to understand how activators and repressors, induced along the AP and DV axes in response to signaling pathways, interact to regulate specific target gene promoters, leading to neuronal fate specification in the appropriate developmental context.