Regulation of neurogenesis in the vertebrate CNS

Abstract

During development, neurons are generated from self-renewing progenitor cells in the ventricular zone of the neural tube. Proneural bHLH proteins are involved in the regulation of neurogenesis and can direct the exit of neural progenitors from the cell cycle and promote the expression of downstream differentiation markers. The expression of proneural bHLH proteins is, in turn, regulated by the activation of the Notch signaling pathway. Thus, whether neural stem cells differentiate or remain as progenitors is dependent on the interaction between Notch receptors and their ligands and the expression of proneural proteins. However, proneural proteins are expressed in proliferating cells that are not yet committed to differentiate, indicating the presence of other factors that actively counteract neurogenesis and keep cells undifferentiated.

The HMG-box containing transcription factors, Sox1, Sox2 and Sox3 (Sox1-3) are expressed by most progenitor cells in the developing CNS. Sox1-3 are generally downregulated when neural cells start to differentiate, indicating a regulatory role of Sox1-3 during neuronal differentiation. In paper I, we found that Sox1-3 proteins have the ability to maintain progenitor cells in an undifferentiated state and suppress neuronal differentiation. Our analysis showed that the activity of Sox1-3 does not influence the expression of proneural bHLH genes, but instead blocks the ability of proneural proteins to induce downstream events of neuronal differentiation. Furthermore, the capacity of proneural proteins to induce differentiation seems to be dependent on their ability to repress the expression of Sox1-3 genes. We also showed that active repression of Sox1-3 target genes results in a premature differentiation of progenitor cells. Thus, in paper I we showed that Sox1-3 can maintain progenitors undifferentiated and that the proneural protein-mediated repression of Sox1-3 proteins represent an irreversible commitment step of neuronal differentiation.

Although proneural proteins are known to promote neuronal differentiation, it is not yet known how these factors that function as transcriptional activators can induce neural cells to suppress progenitor characteristics and commit to neuronal differentiation. The expression pattern of Sox21, a member of the Sox gene family, within the ventricular zone indicated that this protein might have a more general role in the regulation of neurogenesis and, therefore we investigated the role of Sox21 during the progression of neurogenesis in the chick spinal cord. In paper II, we showed that Sox21 has the ability to promote differentiation of neural cell. Interestingly, Sox21 has the opposite effect compared to Sox1-3, even though these proteins belong to the same group of the Sox gene family. We showed that the different activities of Sox21 and Sox1-3 appear to reside in the C-terminal domain of these proteins and that Sox21 mediates its function by counteracting the activity of Sox1-3. Thus, the balance between the repressive activity of Sox21 and the activity of Sox1-3 appears to determine if neural cells remain as progenitors or commit to differentiation. Finally, we demonstrate that the ability of Sox21 to promote neural cells to differentiation is independent of the cell intrinsic levels of proneural protein activity. However, the ability of proneural bHLH proteins to drive neurogenesis seems to be dependent on their ability to upregulate the levels of Sox21 expression. Together, these findings establish a key role for Sox21 in the progression of neurogenesis and further indicate that an important function of proneural proteins during neurogenesis is their capacity to upregulate the expression of Sox21.

Even though 50-70 million years has passed since the human and rodent genomes diverged, it is still possible to align 40% of the human and mouse genomes at the nucleotide level. A fraction of the aligned sequences are Highly Conserved Non-coding Regions (HCNRs) and Ultra Conserved Regions (UCRs) that exhibit an extremely high level of conservation. In paper III, we examined the functional role of these HCNRs by starting with a set of twelve genes encoding Homeodomain (HD) proteins that function as transcriptional repressors and are involved in dorsal-ventral patterning of the spinal cord. We could demonstrate that a majority of HCNRs associated with these HD genes are enriched for binding sites of Sox (S), POU (P) and HD transcription factors. By using a predictive computational model we could show that a significant portion of the HCNRs in vertebrate genomes contain binding sites of Sox, POU and HD transcription factors (SPHD+). Furthermore, these SPHD+ HCNRs can be linked to hundreds of genes that are expressed in the developing CNS. They are transcriptionally active in neural progenitor cells and the activity of these HCNRs are dependent on Sox and POU proteins.

In summary, our data revealed an unifying feature for a large portion of vertebrate HCNRs and imply that SPHD+ HCNRs are involved in a transcriptional core program, which is involved in the neural expression of a large set of genes during development. It also suggests a common transcriptional logic for these HCNR-linked genes in which Sox/POU proteins function as generic drivers of CNS expression, while the positional control of gene expression is regulated by HD-mediated transcriptional repression.