In the vertebrate neural tube, cell cycle exit of neuronal progenitors is accompanied by the expression of transcription factors that define their generic and sub-type specific properties, but how the regulation of cell cycle withdrawal intersects with that of cell fate determination is poorly understood. Here we show by both loss- and gain-of-function experiments that the neuronal-subtype-specific homeodomain transcription factor Phox2b drives progenitor cells to become post-mitotic. In the absence of Phox2b, post-mitotic neuronal precursors are not generated in proper numbers. Conversely, forced expression of Phox2b in the embryonic chick spinal cord drives ventricular zone progenitors to become post-mitotic neurons and to relocate to the mantle layer. In the neurons thus generated, ectopic expression of Phox2b is sufficient to initiate a programme of motor neuronal differentiation characterised by expression of Islet1 and of the cholinergic transmitter phenotype, in line with our previous results showing that Phox2b is an essential determinant of cranial motor neurons. These results suggest that Phox2b coordinates quantitative and qualitative aspects of neurogenesis, thus ensuring that neurons of the correct phenotype are generated in proper numbers at the appropriate times and locations.
Motor neurons are a widely studied model of vertebrate neurogenesis. They can be subdivided in somatic, branchial and visceral motor neurons. Recent studies on the dorsoventral patterning of the rhombencephalon have implicated the homeobox genes Pax6 and Nkx2.2 in the early divergence of the transcriptional programme of hindbrain somatic and visceral motor neuronal differentiation. We provide genetic evidence that the paired-like homeodomain protein Phox2b is required for the formation of all branchial and visceral, but not somatic, motor neurons in the hindbrain. In mice lacking Phox2b, both the generic and subtype-specific programs of motoneuronal differentiation are disrupted at an early stage. Most motor neuron precursors die inside the neuroepithelium while those that emigrate to the mantle layer fail to switch on early postmitotic markers and to downregulate neuroepithelial markers. Thus, the loss of function of Phox2b in hindbrain motor neurons exemplifies a novel control point in the generation of CNS neurons.
Recent evidence suggests that specific families of homeodomain transcription factors control the generation and survival of distinct neuronal types. We had previously characterized the homeobox gene Phox2a, which is expressed in differentiating neurons of the central and peripheral autonomic nervous system as well as in motor nuclei of the hindbrain. Targeted deletion of the Phox2a gene affects part of the structures in which it is expressed: the locus coeruleus, visceral sensory and parasympathetic ganglia and, as we show here, the nuclei of the IIIrd and IVth cranial nerves. We now report on the characterization of Phox2b, a close relative of Phox2a, with an identical homeodomain. Phox2a and Phox2b are co-expressed at most sites, therefore suggesting a broader role for Phox2 genes in the specification of the autonomic nervous system and cranial motor nuclei than revealed by the Phox2a knock-out mice. A detailed analysis of the relative timing of Phox2a and Phox2b expression at various sites suggests positive cross-regulations, which are substantiated by the loss of Phox2b expression in cranial ganglia of Phox2a-deficient mice. In the major part of the rhombencephalon, Phox2b expression precedes that of Phox2a and starts in the proliferative neuroepithelium, in a pattern strikingly restricted on the dorsoventral axis and at rhombomeric borders. This suggests that Phox2b links early patterning events to the differentiation of defined neuronal populations in the hindbrain.