The transcription factors Nkx2.2 and Nkx2.9 play a novel role in floor plate development and commissural axon guidance

Andreas Holz 2 Heike Kollmus 2 4 Jesper Ryge 1 Vera Niederkofler 0 Jose Dias 3 Johan Ericson 3 EstherT.Stoeckli 0 Ole Kiehn 1 Hans-Henning Arnold () 2 0 Institute of Molecular Life Sciences, University of Zurich , 8057 Zurich , Switzerland 1 Mammalian Locomotor Laboratory, Department of Neuroscience, Karolinska Institute , 17177 Stockholm , Sweden 2 Cell and Molecular Biology, University of Braunschweig , Spielmannstrae 7, 38106 Braunschweig , Germany 3 Institute of Cell and Molecular Biology, Karolinska Institute , 17177 Stockholm , Sweden 4 Present address: Department of Infection Genetics, Helmholtz Centre for Infection Research , Inhoffenstrae 7, 38124 Braunschweig , Germany SUMMARY The transcription factors Nkx2.2 and Nkx2.9 have been proposed to execute partially overlapping functions in neuronal patterning of the ventral spinal cord in response to graded sonic hedgehog signaling. The present report shows that in mice lacking both Nkx2 proteins, the presumptive progenitor cells in the p3 domain of the neural tube convert to motor neurons (MN) and never acquire the fate of V3 interneurons. This result supports the concept that Nkx2 transcription factors are required to establish V3 progenitor cells by repressing the early MN lineage-specific program, including genes like Olig2. Nkx2.2 and Nkx2.9 proteins also perform an additional, hitherto unknown, function in the development of non-neuronal floor plate cells. Here, we demonstrate that loss of both Nkx2 genes results in an anatomically smaller and functionally impaired floor plate causing severe defects in axonal pathfinding of commissural neurons. Defective floor plates were also seen in Nkx2.2+/-;Nkx2.9-/- compound mutants and even in single Nkx2.9-/- mutants, suggesting that floor plate development is sensitive to dose and/or timing of Nkx2 expression. Interestingly, adult Nkx2.2+/-;Nkx2.9-/- compound-mutant mice exhibit abnormal locomotion, including a permanent or intermittent hopping gait. Drug-induced locomotor-like activity in spinal cords of mutant neonates is also affected, demonstrating increased variability of left-right and flexor-extensor coordination. Our data argue that the Nkx2.2 and Nkx2.9 transcription factors contribute crucially to the formation of neuronal networks that function as central pattern generators for locomotor activity in the spinal cord. As both factors affect floor plate development, control of commissural axon trajectories might be the underlying mechanism. INTRODUCTION The spinal cord contains anatomically distinct classes of neurons that contribute to sensory and motor tasks. Neurons that exert and regulate motor control reside in the ventral spinal cord, and their development in the mammalian embryo is largely controlled by genetic programs (for reviews, see Briscoe and Novitch, 2008; Jessell, 2000; Shirasaki and Pfaff, 2002) that specify at least five classes of neuronal progenitor cells. These progenitors give rise to motor neurons (MNs) and four cardinal classes of ventral interneurons, referred to as V0 to V3 neurons (Ericson et al., 1997a; Pierani et al., 1999). The different progenitor cell types are generated in response to the graded activity of the signaling protein sonic hedgehog (Shh), which is first produced by the notochord and subsequently by the overlying floor plate, the most ventral structure of the neural tube (Chiang et al., 1996; Ericson et al., 1996; Marti et al., 1995; Roelink et al., 1995). The concentration gradient of Shh along the dorsoventral (DV) axis of the neural tube leads to regional activation or repression of homeodomain (HD) transcription factors, generating a transcriptional code for distinct progenitor cell populations (Briscoe and Ericson, 2001; Briscoe et al., 2000; Briscoe et al., 1999; Ericson et al., 1997a; Ericson et al., 1997b; Novitch et al., 2001). The most ventral neuronal population in the spinal cord, referred to as Sim1-expressing (Sim1+) V3 interneurons, is derived from progenitor cells expressing Nkx2.2 and Nkx2.9 HD transcription factors. This cell population is located immediately dorsal to the floor plate and encounters high concentrations of Shh signal. Both Nkx2 genes are structurally, and presumably functionally, related and exhibit very similar spatiotemporal expression patterns in the central nervous system (CNS) in a Shh-dependent manner (Briscoe et al., 1999; Pabst et al., 1998; Pabst et al., 2000). In Nkx2.2-deficient mice, the domain (p3) of early V3 progenitor cells forms and is indistinguishable from wild type between the ventral floor plate and dorsal Pax6expressing cells. However, p3 progenitors in the mutant mouse undergo a ventral-to-dorsal transformation and generate MNs in place of Sim1+ V3 interneurons (Briscoe et al., 1999; Ericson et al., 1997a). This indicates that Nkx2.2 is essential for the correct differentiation of V3 neurons but not for establishing the p3 progenitor domain. Nkx2.9, which is initially co-expressed with Nkx2.2, might substitute for the missing Nkx2.2 function. Slightly later, when expression of Nkx2.9 stops in the neural tube at embryonic day (E) 10.5 (Briscoe et al., 1999), the essential requirement of Nkx2.2 to establish V3 neuronal fate becomes apparent. Alternatively, Nkx2.2 might play no role in setting up p3 progenitor cells and entirely different factors could be responsible. Targeted disruption of the Nkx2.9 gene in mouse causes no obvious neuronal phenotype in spinal cord, consistent with the idea that Nkx2.2, which is expressed for a longer time period, provides redundant functions and thereby rescues V3 neurons (Pabst et al., 2003). Thus, the individual phenotypes of single Nkx2.2 and Nkx2.9 mutant mice suggest that the two genes have overlapping roles in neuronal patterning of the ventral spinal cord. In order to clarify the extent of shared functions between the two transcription factors, we examined double-mutant mice lacking both Nkx2.2 and Nkx2.9 proteins with particular focus on their individual and combined roles in early cell lineage decisions in the spinal cord. Although floor plates were reported to be anatomically normal in Nkx2.2 and Nkx2.9 single-mutant mice (Briscoe et al., 1999; Pabst et al., 2003), early expression of both regulators in the floor plate area suggests that they might have a function in development and/or maintenance of this important organizing and signaling center. Nkx2.2 expression in the ventral spinal cord of zebrafish and chicken embryos has been shown to extend beyond the p3 domain into the lateral floor plate (Chapman et al., 2002; Strahle et al., 2004). In the mouse, expression of Nkx2.2 in early floor plate has also been described (Jeong and McMahon, 2005) and preliminary evidence showed that the Nkx2.9 knock-in allele directs lacZ expression in the most ventral part of the neural tube (Pabst et al., 2003). The floor plate is not composed of a single population of uniform cells but consists of different cell groups, most n (...truncated)