Molecular mechanisms underlying Mash1 function in oligodendrogenesis

Members of the basic helix-loop-helix (bHLH) proneural family of proteins, including Mash1, are crucial transcription factors (TFs) in neurogenesis. More recently, a role for Mash1 in the specification of oligodendrocyte precursor cells (OPCs) has been demonstrated. Here we investigate the role of Mash1 in lineage commitment of neural progenitors and more specifically the mechanisms underlying Mash1 activity in oligodendroglial cell fate specification. We use an in vitro cell culture system to perform Mash1 locational analysis. Mouse OPCs were cultured as oligospheres that expressed Mash1, a proportion of which also coexpressed the early OPC marker platelet-derived growth factor receptor \alpha (PDGFR\alpha) and oligodendrocyte promoting TFs including the bHLH TF Olig2 and the high mobility group (HMG) TF Sox9. We use a chromatin immunoprecipitation (ChIP)-on-chip strategy and found that Mash1 protein binds to proximal genomic regions of early OPC genes such as Olig1 and Sox8, late oligodendrocyte genes including myelin oligodendrocyte glycoprotein (Mog) and oligodendrocyte myelin glycoprotein (Omg), and other genes of interest including Brevican (Bcan), Notch1 and Sulfatase1 (Sulf1). Mash1 also bound distal genomic regions of Olig2 and Sox9 in oligosphere cultures. To formulate a TF combinatorial code for the activation of these putative enhancers, TF synergy were analysed with luciferase reporter assays. Furthermore, to isolate genomic regions with activity in the oligodendroglial lineage in vivo we used mouse transient transgenics. We hypothesise that Mash1 interacts with either neuronal- or oligodendroglial-specific cofactors, and that these interactions modulate Mash1 activity. To address this question we performed Sox9 and Olig2 ChIP and found that some Mash1 bound elements were also occupied by these TFs in oligosphere cultures. In conclusion, using an in vitro cellular system and ChIP-on-chip technology to interrogate proximal promoter regions bound by Mash1, we can begin to elucidate the molecular mechanisms of Mash1 function in oligodendroglial cell fate specification

SoxD Proteins Influence Multiple Stages of Oligodendrocyte Development and Modulate SoxE Protein Function

SummaryThe myelin-forming oligodendrocytes are an excellent model to study transcriptional regulation of specification events, lineage progression, and terminal differentiation in the central nervous system. Here, we show that the group D Sox transcription factors Sox5 and Sox6 jointly and cell-autonomously regulate several stages of oligodendrocyte development in the mouse spinal cord. They repress specification and terminal differentiation and influence migration patterns. As a consequence, oligodendrocyte precursors and terminally differentiating oligodendrocytes appear precociously in spinal cords deficient for both Sox proteins. Sox5 and Sox6 have opposite functions than the group E Sox proteins Sox9 and Sox10, which promote oligodendrocyte specification and terminal differentiation. Both genetic as well as molecular evidence suggests that Sox5 and Sox6 directly interfere with the function of group E Sox proteins. Our studies reveal a complex regulatory network between different groups of Sox proteins that is essential for proper progression of oligodendrocyte development

The role of Hoxa2 gene in oligodendrocyte development

Although numerous transcription factors (TFs) are expressed by oligodendrocytes (OGs), the role(s) of most of these TFs in oligodendrogenesis remains to be elucidated. One such TF is Hoxa2, which was recently shown to be expressed by O4-positive (+) pro-OGs. Hence, the main objectives of this thesis were to determine the expression profile and function(s) of Hoxa2 during OG development. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxa2 is expressed throughout oligodendrogenesis, diminishing only with the acquisition of a myelinating phenotype. Subsequently, immunohistochemical analysis suggested that Hoxa2 is expressed by migratory oligodendroglial cells in the embryonic spinal cord. However, double immunofluorescent analysis of Hoxa2 transgenic knockout mice showed that OG specification and early maturation proceed normally in the absence of Hoxa2 in the spinal cord. As Hoxa2 is one of 39 murine Hox genes, which exhibit extensive overlapping expression profiles in the spinal cord, we decided to examine the expression of an additional Hox TF, Hoxb4, during OG development. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxb4 is also expressed throughout OG development. Furthermore, comparison of the expression profiles of Hoxb4 and Olig2 suggested that Hoxb4 is expressed by oligodendroglial cells in the spinal cord. Hence, Hoxb4, as well as other Hox TFs could compensate for Hoxa2 in the spinal cord in its absence. As the anterior boundary of most Hox genes has been found to be in the hindbrain or spinal cord, we decided to look at the telencephalon which would be less likely to have compensatory mechanisms. Our results showed that similar to the spinal cord, Hoxa2 is expressed by oligodendroglial cells in the telencephalon. Subsequently, it was found that over-expressing Hoxa2 in CG4 cells, an oligodendroglial cell line derived from the perinatal rat cerebral cortex, impairs their differentiation. In an attempt to determine the mechanism by which it accomplishes this, we examined the expression of polysialylated neural cell adhesion molecule (PSA-NCAM), which has been implicated in this process. Contrary to our expectations, however, it was found that over-expressing Hoxa2 in CG4 cells results in significantly fewer PSA-NCAM+ cells. Hence, the results suggest that Hoxa2’s effect on OG differentiation is independent of its effect on PSA-NCAM expression. The expression of Hox genes is enhanced by retinoic acid (RA), which, in turn, both inhibits, as well as promotes OG differentiation. Although the reason for these opposing roles is uncertain, examination of the experimental protocols utilized by different research groups reveals disparities in age, CNS region, as well as RA concentration. As a result, RA’s effect on oligodendrogenesis could be stage- and/or concentration-dependent. In order to determine which of these factors could impact RA’s effect on OG differentiation we treated CG4 cells with two different concentrations of RA at two distinct time points. The results showed that both factors (concentration and time/stage) can impact RA’s effect on CG4 cell differentiation. In an attempt to determine the mechanism by which it accomplishes this, we examined the expression of PSA-NCAM. Contrary to our expectations, the results suggest that RA’s effect on CG4 differentiation is independent of its effect on PSA-NCAM expression. The results of this thesis suggest that Hoxa2 and RA could play multiple roles in OG development. Although these roles appear to be similar, further research will be needed to determine whether Hoxa2 acts a downstream effector in the RA signaling pathway in oligodendroglial cells

Long noncoding RNAs in neuronal-glial fate specification and oligodendrocyte lineage maturation

Background: Long non-protein-coding RNAs (ncRNAs) are emerging as important regulators of cellular differentiation and are widely expressed in the brain.Results: Here we show that many long ncRNAs exhibit dynamic expression patterns during neuronal and oligodendrocyte (OL) lineage specification, neuronal-glial fate transitions, and progressive stages of OL lineage elaboration including myelination. Consideration of the genomic context of these dynamically regulated ncRNAs showed they were part of complex transcriptional loci that encompass key neural developmental protein-coding genes, with which they exhibit concordant expression profiles as indicated by both microarray and in situ hybridization analyses. These included ncRNAs associated with differentiation-specific nuclear subdomains such as Gomafu and Neat1, and ncRNAs associated with developmental enhancers and genes encoding important transcription factors and homeotic proteins. We also observed changes in ncRNA expression profiles in response to treatment with trichostatin A, a histone deacetylase inhibitor that prevents the progression of OL progenitors into post-mitotic OLs by altering lineage-specific gene expression programs.Conclusion: This is the first report of long ncRNA expression in neuronal and glial cell differentiation and of the modulation of ncRNA expression by modification of chromatin architecture. These observations explicitly link ncRNA dynamics to neural stem cell fate decisions, specification and epigenetic reprogramming and may have important implications for understanding and treating neuropsychiatric diseases

Oligodendrocyte Population Dynamics and Plasticity Probed by Genetic Manipulation in Mice

During embryonic development, oliogdendrocyte precursor cells (OLPs) originate in the ventral region of the neural tube, and from this area proliferate and migrate throughout the entire CNS. Work from several labs has shown dorsal regions of the neural tube can also be an additional source of OLPs. Ventrally and dorsallyderived OLPs are thought to be specified differently, since morphogenic signals that are known to specify ventral OLPs are absent in the dorsal regions of the CNS. However, the question remains whether oligodendrocyte (OLs) with different developmental origins are functionally equivalent or not. If there are different specialized subtypes of OLPs and OLs, this might need to be taken into consideration when designing therapies for demyelinating diseases based on transplantation of pure OLPs from various sources. To address the question, I specifically ablated particular OL lineage cells with different developmental origins using a Cre/loxP system and Diphtheria toxin A chain (DTA). A transgenic mouse line was generated that carried DTA under the transcriptional control of Sox10, which is expressed in all OLPs regardless of their developmental origin. Upstream of the DTA open reading frame were sequences encoding the enhanced green fluorescent protein (EGFP) allowing expression of the toxin only after the floxed EGFP was excised. In order to selectively eliminate OLPs from ventral or dorsal origin, the Sox10-DTA mouse line was crossed with a line that expresses Cre recombinase selectively in dorsal or ventral regions of the developing telencephalon. I found that when either dorsal or ventral-derived OLPs were ablated with DTA, neighbouring OLP populations moved in to replace the missing cells. When ventrally-derived OLPs were ablated there was a slight delay in the accumulation of OLPs and in the onset of myelination. Even when all telencephalic-derived OLPs, both ventral and dorsal, were ablated, populations from more posterior brain regions made up for the loss. This work strongly suggests that OLPs, in the developing telencephalon, from dorsal and ventral origins are functionally equivalent

SOX Transcription Factors as Important Regulators of Neuronal and Glial Differentiation During Nervous System Development and Adult Neurogenesis

The SOX proteins belong to the superfamily of transcription factors (TFs) that display properties of both classical TFs and architectural components of chromatin. Since the cloning of the Sox/SOX genes, remarkable progress has been made in illuminating their roles as key players in the regulation of multiple developmental and physiological processes. SOX TFs govern diverse cellular processes during development, such as maintaining the pluripotency of stem cells, cell proliferation, cell fate decisions/germ layer formation as well as terminal cell differentiation into tissues and organs. However, their roles are not limited to development since SOX proteins influence survival, regeneration, cell death and control homeostasis in adult tissues. This review summarized current knowledge of the roles of SOX proteins in control of central nervous system development. Some SOX TFs suspend neural progenitors in proliferative, stem-like state and prevent their differentiation. SOX proteins function as pioneer factors that occupy silenced target genes and keep them in a poised state for activation at subsequent stages of differentiation. At appropriate stage of development, SOX members that maintain stemness are down-regulated in cells that are competent to differentiate, while other SOX members take over their functions and govern the process of differentiation. Distinct SOX members determine down-stream processes of neuronal and glial differentiation. Thus, sequentially acting SOX TFs orchestrate neural lineage development defining neuronal and glial phenotypes. In line with their crucial roles in the nervous system development, deregulation of specific SOX proteins activities is associated with neurodevelopmental disorders (NDDs). The overview of the current knowledge about the link between SOX gene variants and NDDs is presented. We outline the roles of SOX TFs in adult neurogenesis and brain homeostasis and discuss whether impaired adult neurogenesis, detected in neurodegenerative diseases, could be associated with deregulation of SOX proteins activities. We present the current data regarding the interaction between SOX proteins and signaling pathways and microRNAs that play roles in nervous system development. Finally, future research directions that will improve the knowledge about distinct and various roles of SOX TFs in health and diseases are presented and discussed

The role of SOX9 in neural progenitor identity

Recent evidence has shown that SOX9 is required for the proliferation and multipotentiality of neural progenitors in the developing CNS. Notably, these findings suggest that in contrast to previous studies, SOX9 is important for differentiation along the neuronal lineage, both in the adult and embryonic CNS. Here, a phenotypic analysis of the CNS-specific Sox9-null forebrain, including detailed analysis of cortical lamination, shows that neurons of the appropriate layer-identity are born and migrate to their destined layers. All other parameters in this analysis were normal, with the exception of the formation of glia from the ventral and dorsal telencephalons, and midline glial structures, which were absent in the mutant. Since Sox9 is expressed long before the onset of gliogenesis in these brain regions, the possibility that Sox9 may ‘prime’ the progenitors of the ventricular zone to respond to a gliogenic signal arose. To investigate this, populations of Sox9-deficient and wild-type dorsal telencephalon cells were enriched for progenitors and subjected to transcriptional profiling. Bioinformatic analysis revealed that ‘vascular endothelial growth factor’ receptors, which are important for gliogenesis, were down-regulated, in addition to two transcription factors. Previously, Sox9-deficient neural progenitors have been shown to generate neurospheres poorly, and so the dataset of potential targets was used to identify candidates that might mediate this reduced neurosphere-forming ability. Thirteen down-regulated targets were confirmed by qPCR, six of which were expressed in the same distribution as Sox9 in the embryonic telencephalon; three were also expressed in neurosphere cultures. Of these, one encoded a K+ channel (Kir4.1), and the other a modulator of the GABAA channel (DBI). In order to show that reduced expression of one of these might contribute to the Sox9-deficient neurosphere phenotype, pharmacological modulators were used and showed that blockade of Kir4.1 or enhancement of GABAA channels mimicked the effect of Sox9 loss, leaving open the possibility that Kir4.1 or DBI expression might mediate this effect