Development of the vertebrate nervous system is one of the most complex processes of embryogenesis. Due to its simple anatomy, the spinal cord has emerged as a model to study vertebrate neurogenesis (Jessel, 2000). In the ventral half of the spinal cord, five distinct neuronal subtypes are generated at characteristic dorsal–ventral positions; these are motor neurons (MN) and V3, V2, V1, and V0 interneurons, respectively (Briscoe and Ericson, 2001). The generation of these cell types is dependent on the secreted signalling molecule Sonic hedgehog (Shh; Chiang et al., 1996). Initially, Shh is produced by axial mesodermal midline cells of the notochord. Shh signaling from the notochord induces development of the floor plate—ventral midline cells of the neural tube—which also expresses Shh together with several other genes, including the winged helix transcription factor FoxA2 (Dodd et al., 1998). The different progenitor domains are defined by the combinatorial expression of subsets of transcription factors. These can be subdivided into two classes based on their mode of regulation by Shh signaling. Class I genes are repressed by Shh while class II genes require Shh signaling for their expression. Moreover, cross-repressive interactions between class I and class II proteins appear to refine and maintain the distinct progenitor domains (Briscoe et al., 2000). The class I group includes Pax7, Dbx1, Irx3, Pax6, and Dbx2, whereas Nkx2.2, Nkx6.1, and Olig2 belong to the class II proteins. A series of gain- and loss-of-function experiments have provided evidence of the cross-repressive interactions between Pax6 and Nkx2.2, and Nkx6.1 and Dbx2, respectively (Briscoe et al., 1999, 2000; Sander et al., 2000; Wijgerde et al., 2002; Vallstedt et al., 2001; Jacob and Briscoe, 2003). The expression of Nkx2.2 defines the ventral limit of MN generation: dorsal expansion of Nkx2.2 results in the production of V3 neurons at the expense of MNs (Ericson et al., 1997), whereas loss of Nkx2.2 results in the ventral expansion of MN generation. Conversely, the dorsal limit of MN generation appears to be determined by Irx3 as ventral misexpression of Irx3 represses Olig2 expression (Novitch et al., 2002), inducing V2 neurons at the expense of motor neurons (Briscoe et al., 2000). However, whether Irx3 or other Irx genes are necessary to determine the dorsal limit of MN generation remains unclear.
In Drosophila the Iroquois complex (IRO-C) consists of three homeobox genes, araucan (ara), caupolican (caup), and mirror (mirr), which are involved in development of the peripheral nervous system and the regionalization of the eye and wing imaginal discs (Gomez-Skarmeta and Modollel, 1996; McNeill et al., 1997; Grillenzoni et al., 1998). Ara and caup are suggested to function as positive regulators of the proneural genes achaete and scute, which encode for basic helix–loop–helix (bHLH) transcription factors (Gomez-Skarmeta and Modollel, 1996). Studies in Xenopus indicate that Iroquois (Irx) genes are expressed in a broad domain in the neural plate before the activation of the proneural gene XASH3 (Xenopus Achaete Scute Homologue; Gomez-Skarmeta et al., 1998). The mouse homologue Mash1 is expressed in the progenitor population of the dorsal neural tube in an overlapping domain with Irx expression.
The Fused toes (Ft) mouse mutation is characterized by a 1.6 Mb deletion on chromosome 8, which affects six genes, including the genes of the Iroquois B cluster, Irx3, Irx5, and Irx6 (Peters et al., 2002) and three other genes Fts, Fto, and Ftm. Embryos homozygous for the Ft mutation are retarded and die between embryonic day (E) 10.5 and E14.5, probably due to a malformation of the heart. Furthermore, they show severe abnormalities of craniofacial and forebrain structures, polydactyly of the forelimbs, and syndactyly of fore- and hindlimbs (van der Hoeven et al., 1994; Grotewold and Rüther, 2002). In addition, embryonic turning and heart looping are randomized (Heymer and Rüther, 1999), which suggests a defect in the midline and as a consequent disturbance in neural tube development (Izraeli et al., 1999; Murcia et al., 2000; Przemeck et al., 2003). Based on the described function of Irx genes in neural tube specification and the observed phenotypes in Ft/Ft embryos, we asked whether neural tube development was affected in Ft mutant mice and here we report this analysis.