Corticogenesis is a complex process requiring proliferation of progenitor cells in the ventricular and subventricular zones, neuronal migration, and synaptogenesis. Early in neural development, radial glia divide to produce excitatory neurons of the cortex. Later on in development, radial glia produce intermediate progenitor cells (IPCs) that detach from the surface of the ventricular zone (VZ) to relocate to the subventricular zone (SVZ). IPCs have limited mitotic potential relative to radial glia, and also produce cells that migrate into the cortex. The timing of activation of molecular pathways involved in proliferation and neural differentiation is critical during corticogenesis, and relies on various transcriptional regulators.
The Sox family of transcriptional regulators is known to alter gene expression and determine cell fate, and they partner with other proteins to exert their effects. The SoxC subfamily of transcriptional regulators (Sox4, Sox11, and Sox12) is thought to be functionally identical and expressed in all neuron subtypes. Chen et al. (2015) assessed the expression levels, protein localization, and putative function of both Sox11 and Sox4 in the developing mouse cortex. Sox4 and Sox11 had similar expression levels across cortical development from E10.5-P10 measured by qRT-PCR, peaking between E12.5-E16.5, a time corresponding to early neuronal differentiation.
While expression levels were similar between Sox11 and Sox4 across corticogenesis, immunohistochemical analysis showed distinct protein localizations. Sox11 localized to differentiated neurons, as expected, and also co-localized with Neurogenin1 (Ngn1), a transient marker of early differentiated neurons, at E12.5. Sox4 was localized to both differentiated neurons as well as IPCs in the SVZ. Given the difference in protein expression patterns seen via IHC, Chen et al. examined the role of Sox4 and Sox11 in neuronal differentiation separately. In vitro analysis of differentiated neuron cultures transfected to either overexpress (GOF) or underexpress (LOF) Sox11 was performed. GOF cultures showed increased neuronal polarization and increased neurite length, whereas LOF cultures showed less mature neurons and decreased neurite length. These results support a role of Sox11 in early neuronal differentiation.
So what might Sox11 be doing functionally in these early differentiating neurons? In order to answer this question, Chen et al. used mice with Sox11 coding sequence flanked by loxP sites, and crossed them with a Cre line expressed from the Emx1 promoter, knocking out Sox11 in all cells derived from cortical germinal zones. At early stages of corticogenesis, Sox11 conditional mutants showed reduced immunohistochemical staining for the immature neuronal marker Tuj1. At later stages, mutants showed reduced levels of the deep cortical layer markers Tbr1 and Ctip2. In vivo electroporation to create LOF Sox11 mutants further illustrated that Sox11 promotes the creation of early born neurons by sacrificing apical progenitor cells, as LOF mutants showed an increased proportion of Sox9+ progenitor cells compared to controls.
Sox11 co-localized with Ngn1, a known transcription factor critical for neuronal maturation, and co-immunoprecipitation experiments were performed using E14.5 cortex to assess the ability of Sox11 to bind Ngn1. Sox11 selectively bound Ngn1, suggesting it may be a binding partner with Sox11. Chen et al. also demonstrated that Sox11 could bind regulatory sequences in NeuroD1, an early neuronal differentiation marker in the forebrain regulated by Ngn1, via ChIP analysis. Follow up luciferase assays using an expression vector containing the NeuroD1 promoter linked to luciferase showed that Sox11, when transfected with Ngn1, had a synergistic effect on the activation of the NeuroD1 promoter. These results suggest that Ngn1 and Sox11 act as binding partners to regulate the expression of NeuroD1.
Chen et al. next assessed the function of Sox4 in IPCs in the SVZ. IHC showed that Sox4 co-localized with Ngn2, a transcription factor previously shown to be present in IPCs, as well as Tbr2, another marker of IPCs. In vivo manipulation of Sox4 levels was performed to ascertain the function of Sox4 in IPCs. Cells were transfected with GFP, along with either a Sox4 GOF or Sox4 LOF vector. Cortex was then stained for Tbr2. GOF cortex had significantly more Tbr2+ cells, and LOF cortex had significantly less Tbr2+ cells compared to control cells. At E12.5, conditional Sox4 mutants had a reduction in the number of Tbr2+ IPCs, indicating Sox4 involvement in IPC specification.
Given the co-localization of Sox4 with both Ngn2 and Tbr2, co-immunoprecipitation experiments using E14.5 cortex were performed, showing that Sox4 binds to both Ngn2 and Tbr2. Tbr2 is a known target of Ngn2, and ChIP analysis revealed that Sox4 is able to bind a promoter region on Tbr2. Transactivation experiments showed that Sox4, but not Ngn2, can increase Tbr2 expression, and that cotransfection of Sox4 and Ngn2 produces similar Tbr2 levels to Sox4 alone. These results suggest that Sox4 and Ngn2 may act via distinct molecular pathways to influence Tbr2 levels in IPCs. Luciferase assays showed that Tbr2 cannot increase Tbr2 levels alone, but when cotransfected with Sox4, Tbr2 levels were elevated over Sox4 alone. These results taken together suggest that Ngn2 and Tbr2 are involved in pathways used by Sox4 to induce IPC specification.
Taken together, these results suggest that Sox4 and Sox11 have distinct roles during corticogenesis, and suggest a model by which temporal regulation of corticogenesis may occur (see figure below). To hear more about Dr. Maria Donoghue’s research, please attend her talk on Tuesday, January 17 at 4pm in the CNCB Marilyn G. Farquhar Seminar Room. To learn more about her lab and for a list of recent publications, please visit her lab website: http://www.donoghuelab.org/.
Molly Kwiatkowski is a third year MD/PhD candidate, currently working in the Consortium for Translational Research in Neuropsychopharmacology.