Nkx 6 are central regulators in dorsoventral patterning of the Drosophila brain

INTRODUCTION The development of the central nervous system in vertebrates and invertebrates involves the transformation of a two-dimensional sheet of neuroectodermal cells into a complex three-dimensional structure comprising a variety of different neural cell types. The specification of neural cell types is a multi-step process, which at early stages of embryogenesis critically depends on conveying positional information in the neuroectoderm (NE) to neural stem cells. The underlying molecular genetic mechanisms have been extensively studied in the embryonic ventral nerve cord (VNC) of the trunk (reviewed by Dessaud et al., 2008; Skeath and Thor, 2003). In Drosophila, the VNC is generated by segmental arrays of neural stem cells, called neuroblasts (NBs), which delaminate from the truncal NE. Each NB acquires a unique identity that is finally reflected in the production of a specific cell lineage (Bossing et al., 1996; Schmidt et al., 1997). NB identity is specified by the combinatorial code of positional cues in the NE provided by the products of early patterning genes, which act in the anteroposterior (AP) and dorsoventral (DV) axes (reviewed by Skeath and Thor, 2003). The graded activities of the nuclear factor Dorsal, BMP and Epidermal growth factor receptor (Egfr) signaling pathways determine the DV boundaries of the NE, and further regulate the expression of a set of evolutionary conserved DV patterning genes (Hong et al., 2008; Mizutani et al., 2006; Skeath, 1998; von Ohlen and Doe, 2000). The expression of these ‘DV genes’ subdivides the trunk NE into three longitudinal columns along the DV axis: ventral nervous system defective (vnd/Nkx2) in the ventral, intermediate neuroblasts defective (ind/Gsx) in the intermediate, and muscle segment homeobox (msh/Msx; Drop – FlyBase) in the dorsal NE column (Chu et al., 1998; Isshiki et al., 1997; McDonald et al., 1998; Weiss et al., 1998). In Drosophila, these homeobox genes interact in a hierarchical cascade of transcriptional repression, according to which vnd represses ind (and msh) in the ventral column, and ind represses msh in the intermediate column. The DV genes encode key regulators of NB identity and each NE column thereby gives rise to a population of distinctly specified NBs. However, whereas vnd, ind and Egfr have also been shown to be central for the formation of NBs in their respective NE column, this role appears dispensable for msh (reviewed by Cornell and von Ohlen, 2000; Skeath, 1999). Much less is known about the genetic mechanisms of DV regionalization in the developing brain. The procephalic NE and the descending population of ~100 brain NBs in each hemisphere can be subdivided (from anterior to posterior) into the presumptive proto(PC), deuto(DC) and tritocerebrum (TC) (Urbach et al., 2003). We previously showed that DV genes are expressed in a segment-specific manner in the procephalic NE and brain NBs (Urbach and Technau, 2003a; Urbach and Technau, 2003b). It was further shown that the way in which vnd controls expression of ind and msh differs between the brain and the VNC, and that vnd is necessary for proper development of the ventral procephalic NE and of the brain NBs that descend from these NE domains. These experiments suggested that the role of vnd/Nkx2 in brain development exhibits striking parallels between vertebrates and Drosophila (Urbach et al., 2006). In this work, we uncover a novel regulatory network of homeodomain transcription factors in Drosophila that is specifically necessary to pattern the TC and DC in the DV axis, whereas the underlying genetic interactions in the PC diverge. We provide evidence that empty spiracles (ems) and Nk6 homeobox (Nkx6; HGTX – FlyBase) encode key regulators in the DV genetic network. Intriguingly, ems, a cephalic gap gene, is crucial for the brainEms and Nkx6 are central regulators in dorsoventral patterning of the Drosophila brain