The role of Nkx proteins neuronal and glial specification

During development, different classes of neurons and glia are generated from proliferative progenitor cells lining the ventricles of the brain and the lumen of the spinal cord. A central issue in developmental neuroscience is to understand the mechanisms by which these cells are generated in space and over time. In the ventral spinal cord, the expression profile of homeodomain (HD) proteins defines five progenitor domains that each will give rise to a distinct type of neuron. Two closely related HD transcriptional repressors, Nkx6.1 and Nkx6.2 (Nkx6), are expressed by progenitors of the ventral spinal cord. We provide evidence that different levels of Nkx6 repressor activity in progenitor cells are a critical determinant of ventral neuronal fate, assigning both redundant and specific roles for these proteins in neuronal specification. A reduction in Nkx6 activity further permits V0 interneurons to be generated from progenitors that lack HD proteins normally required for their generation, providing direct evidence for a model where HD proteins direct specific cell fates by actively repressing the expression of transcription factors that direct alternative fates. In the ventral spinal cord, sMNs and oligodendrocyte precursors (OLPs) are sequentially generated from a domain defined by the expression of Olig2. We show that the generation of sMNs and OLPs in the ventral spinal cord is essentially missing in mice lacking Nkx6 function. In contrast, the same HD proteins instead act to suppress OLP specification in the ventral hindbrain. The divergent roles for Nkx6 proteins seem to reflect that OLPs in the spinal cord and hindbrain are produced by distinct ventral progenitor domains. While a ventral specification of OLPs is well established, it has remained unclear whether also more dorsal progenitor cells give rise to oligodendrocytes. We provide in vivo and in vitro evidence that oligodendrocytes are produced also by dorsal progenitors in the spinal cord and hindbrain and that the specification of these cells may result from the progressive evasion of dorsal BMP signalling over time. Together, our data suggest that oligodendrocytes are generated from multiple dorsoventral origins in the spinal cord and hindbrain, and indicate that the activation of Olig2 at different positions is controlled by distinct genetic programs. The observation that the loss of sMNs in the spinal cord of Nkx6 mutant mice correlates with an extinguished expression of the sMN determinant Olig2 has led to a model where Nkx6 proteins act strictly upstream of Olig2. However, in the hindbrain of Nkx6 mutant mice the initial expression of Olig2 is intact and despite this all sMNs are missing, indicating a parallel requirement for Nkx6 and Olig2 proteins in the generation of sMNs. Visceral motor neurons (vMNs) are generated immediately ventral to sMNs. The transcription factor Phox2b has been found to be an important determinant of these cells, but other factors involved have not been identified. We show that the HD protein Nkx2.2 is sufficient to mediate the expression of Phox2b. Furthermore, while the activities of Nkx6.1 and Nkx6.2 are dispensable for the initial generation of vMNs, they are required to prevent a parallel program of more dorsal interneuronal differentiation and to ensure a proper migration and axonal projection formation of vMNs. Thus, Nkx2 and Nkx6 proteins appear to have complementary roles in the establishment of vMN identity in the hindbrain. Taken together, our data suggest that both visceral and somatic motor neuron differentiation rely on the combined activity of cell intrinsic determinants, rather than on a singe key determinant of neuronal cell fate. Neuronal diversity is established by mechanisms that operate in space and over time. Advances have been made in regard to the mechanisms that restrict and direct neuronal generation in space, but less is known about the mechanisms that underlie how neural progenitors produce distinct types of neurons in a specific temporal order. We addressed this issue by studying a population of progenitor cells in the ventral hindbrain that gives rise to vMNs and serotonergic (S) neurons. Each hindbrain segment, or rhombomere (r), initially generates vMNs, but all the rhombomeres except for r4 switch to producing S neurons at a defined time point. We found that the temporal and spatial generation of vMNs and S neurons critically relies on the integrated activity of Nkxand Hox-class HD proteins. A primary function of these proteins is to coordinate the activation of Phox2b in space and time. Phox2b, in turn, functions as a binary switch in deciding whether progenitors differentiate into vMNs or serotonergic neurons. Taken together, these data indicate that determinants that control spatial patterning may be associated also with temporal patterning and require that expression patterns are dynamic and modulated over time. LIST OF PUBLICATIONS This thesis is based on the following papers, which will be referred to in the text by their roman numbers: I Vallstedt, A. , Muhr, J., Pattyn, A. , Pierani, A. , Mendelsohn, M. , Sander, M. , Jessell, T. M. , and Ericson, J. (2001). Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification. Neuron 31, 743-755. II. Pattyn, A*. , Vallstedt, A*. , Dias, J. M. , Sander, M. , and Ericson, J. (2003). Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain. Development 130, 4149-4159. III. Pattyn, A. , Vallstedt, A. , Dias, J. M. , Samad, O. A. , Krumlauf, R. , Rijli, F. M. , Brunet, J. F. , and Ericson, J. (2003). Coordinated temporal and spatial control of motor neuron and serotonergic neuron generation from a common pool of CNS progenitors. Genes Dev 17, 729-737. IV. Vallstedt, A*. , Klos, J*. , Ericson, J. (2004). Multiple Dorsoventral Origins of Oligdendrocyte Generation in the Spinal Cord and Hindbrain. Submitted. * These authors contributed equally to this work.