2713 RESEARCH ARTICLE INTRODUCTION Motoneurons and different interneuronal subtypes are specified in the ventral spinal cord in response to different concentrations of the morphogen sonic hedgehog (Shh). Close to the source of Shh (notochord, floor plate) V3 interneurons form, while, at a further distance, motoneurons and V2, V1 and V0 interneurons differentiate. The decreasing concentrations of Shh at a distance from the sources are interpreted by transcription factors, the expression of which is either repressed (class 1 transcription factors) or induced (class 2 transcription factors) by Shh (Briscoe and Ericson, 2001; Ingham and McMahon, 2001; Jessell, 2000). The cells adjacent to the medial floor plate express the homeobox transcription factor genes nkx2.2 and nkx2.9, which are induced by Shh (Cheesman et al., 2004; Guner and Karlstrom, 2007; Schäfer et al., 2005; Schäfer et al., 2007; Xu et al., 2006). In this region of the zebrafish spinal cord (the lateral floor plate), nkx2.9 and two related nkx2.2 genes, nkx2.2a and nkx2.2b, are expressed (Barth and Wilson, 1995; Schäfer et al., 2005; Schäfer et al., 2007; Strahle et al., 2004; Xu et al., 2006). Knockout of Nkx2.2 in the mouse abolishes the formation of V3 interneurons (Briscoe et al., 1999), whereas inactivation of the related transcription factor Nkx2.9 does not result in a phenotype in the mouse spinal cord (Pabst et al., 2003). The zebrafish lateral floor plate is the origin of two distinct neuronal types: V3 interneurons and GABAergic Kolmer-Agduhr (KA) cells (Fig. 1A,B) (Bernhardt et al., 1992; Schäfer et al., 2007). The KA cells of the lateral floor plate and the more dorsally located KA cells form a special class of neurons that stay in contact with the ventricular lumen (Martin et al., 1998) (see also Fig. 1A,B). Whereas the ventral KA cells develop from the lateral floor plate, the more dorsal KA cells are derived from olig2- expressing motoneuron precursors (Park et al., 2004). At least some of the KA cells are part of the neuronal network that controls spontaneous swimming movement (Wyart et al., 2009). Although all these cell types depend on Shh signaling (Pinheiro et al., 2004; Schäfer et al., 2007), it is less clear how the Shh signals are interpreted to trigger the differentiation of KA interneurons. The zinc-finger transcription factors Gli1 (detour) and Gli2 (you-too) are the immediate mediators of the Shh signal in the spinal cord (Karlstrom et al., 1999; Karlstrom et al., 2003). The primary targets of the Gli factors appear to be nkx2.2a, nkx2.2b and nkx2.9 (Cheesman et al., 2004; Guner and Karlstrom, 2007; Xu et al., 2006). As in mammals, zebrafish V3 interneurons express the leucine zipper/PAS domain transcription factor Sim1. In addition, a number of other transcription factors, such as the C4 zinc-finger transcription factors Gata2 and Gata3 and the basic helix-loop-helix transcription factor Tal2, are expressed in the lateral floor plate, but also in other cells at more dorsal aspects of the spinal cord (Batista et al., 2008; Pinheiro et al., 2004; Schäfer et al., 2007). The precise functional relationships of these factors with respect to the different neuronal subtypes are not understood. Morpholino knockdown of nkx2.2a and nkx2.2b does not abolish tal2 expression in the zebrafish spinal cord (Schäfer et al., 2007). In the lateral floor plate, cells that co-express either tal2 and nkx2.2b or foxa2 and nkx2.2b can be distinguished (Schäfer et al., 2007). Although not affecting the tal2-positive cells, double knockdown of nkx2.2a and nkx2.2b abolishes the foxa2/nkx2.2b co-expressing cells. These latter cells seem to differ also in their dependence on hedgehog (Hh) signaling from tal2-positive cells. Moderate reduction of Shh signaling abolishes the foxa2-expressing cells, whereas complete removal of Shh signaling is necessary to prevent the differentiation of tal2- positive cells. The foxa2/nkx2.2b co-expressing and tal2/nkx2.2b co-expressing cells were suggested to represent proliferating and post-mitotic cells, respectively (Schäfer et al., 2007). Development 137, 2713-2722 (2010) doi:10.1242/dev.048470 © 2010. Published by The Company of Biologists Ltd Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Postfach 3640, 76021 Karlsruhe, Germany. *Author for correspondence (uwe.straehle@kit.edu) Accepted 28 May 2010 SUMMARY In the zebrafish spinal cord, two classes of neurons develop from the lateral floor plate: Kolmer-Agduhr (KA) and V3 interneurons. We show here that the differentiation of the correct number of KA cells depends on the activity of the homeobox transcription factor Nkx2.9. This factor acts in concert with Nkx2.2a and Nkx2.2b. These factors are also required for the expression of the zinc-finger transcription factor Gata2 in the lateral floor plate. In turn, Gata2 is necessary for expression of the basic helix-loop-helix transcription factor Tal2 that acts upstream of the GABA-synthesizing enzyme glutamic acid decarboxylase 67 gene (gad67) in KA cells. Expression of the transcription factor Sim1, which marks the V3 interneurons in the lateral floor plate, depends also on the three Nkx2 factors. sim1 expression does not require, however, gata2 and tal2. KA cells of the lateral floor plate and the KA cells located more dorsally in the spinal cord share expression of transcription factors. The functional connections between the different regulatory genes, however, differ in the two GABAergic cell types: although gata2 and tal2 are expressed in KA cells, they are dispensable for gad67 expression in these cells. Instead, olig2 and gata3 are required for the differentiation of gad67-expressing KA cells. This suggests that the layout of regulatory networks is crucially dependent on the lineage that differs between KA and KA cells. KEY WORDS: Kolmer-Agduhr interneuron, V3 interneuron, Zebrafish Regulatory interactions specifying Kolmer-Agduhr interneurons Lixin Yang, Sepand Rastegar and Uwe Strähle* DEVELOPMENT