INTRODUCTION It is still a major challenge to understand how vast numbers of different neuronal types are generated and assigned their fates in the vertebrate nervous system. Among the transcriptional regula- tors implicated in vertebrate neural development, homeodomain proteins expressed in differentiating neurons are good candidates to control final neuronal phenotypes. Targeted gene inactivation experiments have demonstrated that homeodomain proteins are required for the generation of various classes of neurons (Erkman et al., 1996; Gan et al., 1996; Xiang et al., 1996; McEvilly et al., 1996; Pfaff et al., 1996; Morin et al., 1997). An emerging concept from these and other studies is that structurally similar homeo- domain proteins could insure the determination of subsets of related neuronal phenotypes, suggesting that duplication of tran- scription factor genes is causally linked to the appearance of new subclasses of neurons during evolution. For example, the three known POU-domain proteins of the Brn-3 family are expressed in overlapping but distinct patterns (Xiang et al., 1995, 1996; Turner et al., 1994). Each is uniquely expressed in some classes of neurons and is necessary for their generation as shown by the knock-out phenotypes (Erkman et al., 1996; Gan et al., 1996; McEvilly et al., 1996; Xiang et al., 1996). They are co-expressed in other neurons, most of which are apparently spared in the knock-out mice. In these cells, they could be functionally redundant with each other or, alternatively, co-operate to diversify phenotypes on a combinatorial mode. The latter model has been proposed for the closely homologous LIM-homeodomain proteins expressed in motoneurons and commissural neurons of the spinal cord (Tsuchida et al., 1994; Tanabe and Jessell, 1996). Phox2a is a homeodomain protein specific to the nervous system (Valarché et al., 1993; Tiveron et al., 1996). Its expression pattern reveals two striking correlates. The first is with a neuro- transmitter phenotype: Phox2a is expressed in all neurons that transiently or permanently express dopamine-β-hydroxylase (DBH), the last enzyme in the pathway of noradrenaline synthesis, suggesting that Phox2a is a determinant of the nora- drenergic phenotype. The second correlate is with neuronal circuitry: Phox2a expression is largely restricted to circuits involved in medullary control of autonomic functions (Tiveron et al., 1996). Inactivation of the Phox2a gene leads to agenesis of the locus coeruleus and of parasympathetic ganglia, to altered morphology of the superior cervical ganglion and to massive atrophy of cranial sensory ganglia (Morin et al., 1997). In the cranial ganglia, it was possible to show the dependence of DBH expression on Phox2a, providing the first in vivo evidence that Phox2a may indeed regulate the noradrenergic phenotype (Morin et al., 1997). The gene coding for the GDNF (glial cell line- derived neurotrophic factor) receptor subunit Ret is also regulated, directly or indirectly, by Phox2a in these ganglia, sug- 4065 Development 124, 4065-4075 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 DEV1198 Recent evidence suggests that specific families of homeo- domain transcription factors control the generation and survival of distinct neuronal types. We had previously char- acterized 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 homeo- domain. 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 neu- roepithelium, 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. Key words: Phox2a, Phox2b, neurogenesis, homeodomain, mouse, nervous system SUMMARY Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis Alexandre Pattyn, Xavier Morin, Harold Cremer, Christo Goridis and Jean-François Brunet* Laboratoire de Génétique et Physiologie du Développement, Institut de Biologie du Développement de Marseille, CNRS/INSERM/Université de la Méditerranée, Campus de Luminy, Case 907, F-13288 Marseille Cedex 9, France *Address for correspondence: (e-mail: brunet@ibdm.univ-mrs.fr)