Hes6 inhibits astrocyte differentiation and promotes neurogenesis through different mechanisms.

The mechanisms regulating the generation of cell diversity in the mammalian cerebral cortex are beginning to be elucidated. In that regard, Hairy/Enhancer of split (Hes) 1 and 5 are basic helix-loop-helix (bHLH) factors that inhibit the differentiation of pluripotent cortical progenitors into neurons. In contrast, a related Hes family member termed Hes6 promotes neurogenesis. It is shown here that knockdown of endogenous Hes6 causes supernumerary cortical progenitors to differentiate into cells that exhibit an astrocytic morphology and express the astrocyte marker protein GFAP. Conversely, exogenous Hes6 expression in cortical progenitors inhibits astrocyte differentiation. The negative effect of Hes6 on astrocyte differentiation is independent of its ability to promote neuronal differentiation. We also show that neither its proneuronal nor its anti-gliogenic functions appear to depend on Hes6 ability to bind to DNA via the basic arm of its bHLH domain. Both of these activities require Hes6 to be localized to nuclei, but only its anti-gliogenic function depends on two short peptides, LNHLL and WRPW, that are conserved in all Hes6 proteins. These findings suggest that Hes6 is an important regulator of the neurogenic phase of cortical development by promoting the neuronal fate while suppressing astrocyte differentiation. They suggest further that separate molecular mechanisms underlie the proneuronal and anti-gliogenic activities of Hes6 in cortical progenitor cells.

Hes6 promotes cortical neurogenesis and inhibits Hes1 transcription repression activity by multiple mechanisms.

Hes1 is a mammalian basic helix-loop-helix transcriptional repressor that inhibits neuronal differentiation together with corepressors of the Groucho (Gro)/Transducin-like Enhancer of split (TLE) family. The interaction of Hes1 with Gro/TLE is mediated by a WRPW tetrapeptide present in all Hairy/Enhancer of split (Hes) family members. In contrast to Hes1, the related protein Hes6 promotes neuronal differentiation. Little is known about the molecular mechanisms that underlie the neurogenic activity of Hes6. It is shown here that Hes6 antagonizes Hes1 function by two mechanisms. Hes6 inhibits the interaction of Hes1 with its transcriptional corepressor Gro/TLE. Moreover, it promotes proteolytic degradation of Hes1. This effect is maximal when both Hes1 and Hes6 contain the WRPW motif and is reduced when Hes6 is mutated to eliminate a conserved site (Ser183) that can be phosphorylated by protein kinase CK2. Consistent with these findings, Hes6 inhibits Hes1-mediated transcriptional repression in cortical neural progenitor cells and promotes the differentiation of cortical neurons, a process that is normally inhibited by Hes1. Mutation of Ser183 impairs the neurogenic ability of Hes6. Taken together, these findings clarify the molecular events underlying the neurogenic function of Hes6 and suggest that this factor can antagonize Hes1 activity by multiple mechanisms.

Role for Runx1 in the proliferation and neuronal differentiation of selected progenitor cells in the mammalian nervous system.

Neurogenesis requires factors that regulate the decision of dividing progenitors to leave the cell cycle and activate the neuronal differentiation program. It is shown here that the murine runt-related gene Runx1 is expressed in proliferating cells on the basal side of the olfactory epithelium. These include both Mash1+ olfactory receptor neuron (ORN) progenitors and NeuroD+ ORN precursors. Disruption of Runx1 function in vivo does not cause a change in Mash1 expression but leads to a decrease in the number of NeuroD+ neuronal precursors and an increase in differentiated ORNs. These effects result in premature and ectopic ORN differentiation. It is shown further that exogenous Runx1 expression in cultured olfactory neural progenitors causes an expansion of the mitotic cell population. In agreement with these findings, exogenous Runx1 expression also promotes cortical neural progenitor cell proliferation without inhibiting neuronal differentiation. These effects are phenocopied by a chimeric protein containing ETO, the eight twenty one transcriptional repressor, fused to the Runx1 DNA-binding domain, which suggests the involvement of transcription repression mechanisms. Consistent with this possibility, Runx1 represses transcription driven by the promoter of the cell cycle inhibitor p21Cip 1 in cortical progenitors. Together, these findings suggest a previously unrecognized role for Runx1 in coordinating the proliferation and neuronal differentiation of selected populations of neural progenitors.

AML1/Runx1 is important for the development of hindbrain cholinergic branchiovisceral motor neurons and selected cranial sensory neurons.

The mechanisms that regulate the acquisition of distinctive neuronal traits in the developing nervous system are poorly defined. It is shown here that the mammalian runt-related gene Runx1 is expressed in selected populations of postmitotic neurons of the embryonic central and peripheral nervous systems. These include cholinergic branchial and visceral motor neurons in the hindbrain, restricted populations of somatic motor neurons of the median and lateral motor columns in the spinal cord, as well as nociceptive and mechanoreceptor neurons in trigeminal and vestibulocochlear ganglia. In mouse embryos lacking Runx1 activity, hindbrain branchiovisceral motor neuron precursors of the cholinergic lineage are correctly specified but then fail to progress to a more differentiated state and undergo increased cell death, resulting in a neuronal loss in the mantle layer. In contrast, the development of cholinergic somatic motor neurons is unaffected. Runx1 inactivation also leads to a loss of selected sensory neurons in trigeminal and vestibulocochlear ganglia. These findings uncover previously unrecognized roles for Runx1 in the regulation of mammalian neuronal subtype development.