In addition to the above genes that regulate BMN identities indirectly, a large number of genes have been identified that are expressed in the ventral neural tube and regulate motor neuron and interneuron identities more directly. Extensive functional analyses of these genes in chick and mouse have led to a three-step model of ventral cell type specification by Shh signaling (Briscoe and Ericson, 2001). In this model, different levels of the Shh gradient regulate the expression of genes encoding two classes of transcription factors: Class I factors (Nkx2.2, Nkx2.9, Nkx6.1, Nkx6.2) are induced by Shh, and Class II factors (Pax3, Pax6, Dbx1, Dbx 2, Irx3) are repressed. Cross-repressive interactions between Class I and Class II genes generate different domains of transcription factor expression in the ventral neural tube, leading to the induction of cell-type specific transcription factors such as Islet1, Mnr2, Hb9, Phox2a, and Phox2b in motor neuron progenitors (Ericson et al., 1992; Pattyn et al., 1997; Tanabe et al., 1998; Briscoe et al., 2000; for reviews, see Jacob et al., 2001; Shirasaki and Pfaff, 2002). Many of these genes are expressed in domains or progenitors that generate more dorsal cell types (Pax3, Dbx1, Dbx2, Irx3) or somatomotor neurons (Mnr2, Hb9, Pax6, Nkx6.1, Phox2a, Phox2b). Islet1 is expressed in motor neurons in all species (Ericson et al., 1992; Appel et al., 1995; Varela-Echavarria et al., 1996) and is essential for motor neuron differentiation (Pfaff et al., 1996). Nkx2.2, Nkx6.1, Nkx6.2, Phox2a, and Phox2b are expressed in branchiomotor and visceromotor neuron precursors, and interestingly, only knockout of Pax6, Nkx6.1, and Phox2b generate a BMN phenotype, which is discussed below. Such functional analyses will eventually decipher the transcription factor code that completely specifies the identities of hindbrain motor neuron progenitors (Briscoe and Ericson, 2001; Takahashi and Osumi, 2002; Pattyn et al., 2003), preceding the expression of SMN-specific (Varela-Echavarria et al., 1996), VMN-specific (Pattyn et al., 2003), and BMN-specific transcription factors.
Pax6 and Nkx2.2
Pax6 is expressed in the progenitors of the nVI and nXII somatomotor neurons (Ericson et al., 1997; Osumi et al., 1997), and Pax6 function is required for the proper specification of nVI and nXII progenitor domains (Takahashi and Osumi, 2002). In mouse Pax6 mutants, these somatomotor neurons are missing but there is a dorsal expansion of the Nkx2.2 domain (containing BMN progenitors) and a corresponding increase in the number of nVII and nX BMNs (Ericson et al., 1997; Osumi et al., 1997). Interestingly, in mouse Nkx2.2 mutants, the Pax6 expression domain does not expand ventrally and there is no effect on the formation of various BMN subtypes (Briscoe et al., 1999). Clearly, Nkx2.2 expression is sufficient to generate BMN progenitors in ectopic locations (as in Pax6 mutants), but its function is not necessary within its normal ventral domain, likely due to redundancy with Nkx2.9 (Briscoe et al., 1999).
Nkx6.1 and Nkx6.2
Mouse Nkx6.1 is expressed in all branchiomotor and visceromotor neurons, and Nkx6.2 is expressed in the nV and nVII BMNs (Muller et al., 2003; Pattyn et al., 2003). It must be noted that the VMNs described by Pattyn et al. (2003) include the VMNs and BMNs as defined in this review. In Nkx6.1 mutants, all BMNs appear to differentiate normally, but the migration of nV and nVII motor neurons is greatly reduced (Muller et al., 2003; Pattyn et al., 2003; see below). In addition, the nV, nIX, and nX motor axons exhibit varying degrees of pathfinding defects in Nkx6.1 single and Nkx6.1:Nkx6.2 double mutants (Muller et al., 2003; Pattyn et al., 2003). Nkx6.2 knockout mice are viable and exhibit no motor neuron defects, possibly due to complete compensation by Nkx6.1 (Pattyn et al., 2003; M. Sander, personal communication). The inability of Nkx6.2 to rescue motor neuron defects in Nkx6.1 mutants may reflect temporal differences in onset of expression of the two genes.
Phox2a and Phox2b
Both homeodomain genes are expressed in somatic, visceral, and branchial motor neuron progenitors in the mouse hindbrain (Pattyn et al., 1997). However, Phox2a expression precedes Phox2b expression in the nIII and nIV somatomotor neurons, while Phox2b expression precedes Phox2a expression in all BMN progenitors. Hence, only the nIII and nIV neurons are missing in Phox2a knockout mice (Pattyn et al., 1997), while all BMN progenitors fail to differentiate into motor neurons in Phox2b knockout mice (Pattyn et al., 2000). In zebrafish phox2a mutants, nIII and nIV motor neurons are lost, as in mutant mice (Guo et al., 1999). However, the roles of zebrafish phox2a and phox2b genes in BMN development have not been examined.