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

1The development of multicellular organisms and the maintenance of homeostasis in adulthood are achieved by complex control of basic cellular processes such as the maintenance of pluripotent stem cells, cell fate decision, differentiation, proliferation, and cell death. One of the key mechanisms involved in the control of developmental processes is based on the transcriptional regulation of gene expression. Through the activation and repression of the target genes, transcription factors (TFs) determine the fate of cells within tissues, organs and organisms, controlling the development. Most TFs act within complex regulatory networks, enabling combinatorial regulation of gene expression within the cells. Numerous families of genes encoding TFs involved in the control of embryonic development have been discovered, including the SOX gene family.

2Sry (Sex-determining Region Y), a founder member of the Sox gene family, was discovered in 1990 as a sex-determining gene necessary and sufficient to specify the male phenotype. During the course of cloning of Sry, the presence of related genes was discovered sharing the homology with the HMG box of Sry. These newly identified genes have been named by the acronym Sox/SOX (in mammals and human, respectively) standing for Sry-related HMG box genes. Further, it was shown that the SOX family is multigenic, with new members discovered both in vertebrates and invertebrates and being assigned by numbers based on the order of their discovery. After a detailed insight, the presence of 20 SOX genes in human genome was identified (Table 1) providing the basis for their final re-numeration and classification. Further research has shown that SOX genes encode the family of diverse and well conserved TFs.

3Based on the structure, expression profiles, as well as the similarity between the proteins they encode, human SOX genes are divided into 8 groups, A to H (Table 1), with group B being further split into subgroups B1 (SOX1, SOX2, and SOX3) and B2 (SOX14 and SOX21). SOX proteins within the same group show a high level of homology, both within and outside the HMG domain, while proteins from different groups show homology only within the HMG domain. Apart from the genes SRY and SOX3, other family members are located on autosomes and scattered throughout the genome (Table 1). Majority of SOX family members are single exon genes, with the exception of genes SOX5, SOX9, SOX10, SOX15, SOX17, and SOX18 that possess multiple exons.

4The SOX proteins display properties of both classical TFs and architectural components of chromatin (reviewed in ). SOX proteins carry an HMG domain of 79 amino acids that enables their specific binding to the sequence (A/T A/T CAA A/T) and additional domains involved in transcriptional regulation (reviewed in ). In contrast to the majority of DNA-binding proteins, SOX proteins interact with the minor groove and, upon binding, they introduce strong bends into DNA (reviewed in ). Consequently, SOX proteins act as architectural proteins by shaping the gene regulatory regions and by enabling establishment of physical contacts between TFs bound on the same target gene promoter or enhancer (reviewed in ). SOX TFs exert regulatory functions by activating or repressing gene transcription only through specific interactions with a partner factor(s) and by establishing contacts with the basic transcription machinery.

5The SOX TFs perform unique functions in different cell types and regulate different events in the same cell type. Several SOX proteins are demonstrated to have the ability to pair off with various types of TFs and their specificity is achieved via binding partners (reviewed in ). Consequently, transcriptional regulatory functions of SOX proteins usually require the cooperation with interacting partner factors that bind DNA in the vicinity of the SOX site and allow specific selection of target genes (reviewed in; ). SOX partner factor cooperation is dynamic and changes in partner factors enable SOX proteins to regulate different events in the same cell type and to drive the progression of developmental processes (reviewed in ).

6Since discovery, essential roles have been assigned to SOX TFs. Their critical functions have been revealed by both studying naturally occurring mutations in humans, as well as, by targeted mutations introduced in animal models. Numerous studies aimed at discovery of the roles of Sox/SOX genes are often being complicated by pleiotropy and by partial or extensive functional redundancy among co-expressed members of the same groups.

7It has been shown that many developmental processes depend on the presence of SOX proteins, ranging from blastocyst formation, gastrulation, germ layer formation to development of adult tissues and organs (reviewed in;; ). SOX TFs have been implicated in preimplantation development. Expression of Sox2 was detected from oocyte, through 2-cell to 8-cells embryo and morula to the blastocyst. Study of the effects of Sox2 knockdown in preimplantation embryo suggested that first essential function of Sox2 is to facilitate establishment of the trophectoderm lineage. Expression of Sox2 is detected in the inner cell mass of the murine blastocyst and subsequently in primitive ectoderm, extraembryonic ectoderm and the developing nervous system.

8The 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 (reviewed in; ). However, their roles are not limited to development since SOX TFs influence survival, regeneration, cell death and control homeostasis in adult tissues. As reported in numerous publications, most cells express at least one Sox/SOX gene and various members of the SOX gene family have roles in many tissues and stages of development (reviewed in;;; ). For instance, tissues that require Sox2 during development continue to express this factor in some adult stem and progenitor cells derived from that tissue. Thus, Sox2 marks stem and progenitor cell populations in adult tissues that depend on Sox2 expression during development.

9A unique set of TFs is required to establish embryonic stem cells (ESCs) and to maintain their pluripotent and proliferative state. The numerous evidences reveal the roles of SOX proteins in preservation of stem cell characteristics.

10As mentioned above, SOX proteins have the ability to pair off with various types of TFs and regulatory functions of SOX proteins usually require the cooperation with interacting partner factors. SOX2, together with OCT4 (octamer-binding transcription factor 4) and NANOG (named as abbreviation for the mythological Celtic land of the ever-young, “Tir nan Og”), establish the core transcriptional circuit that orchestrate self-renewal and maintenance of pluripotency of the stem cells (Figure 1).

11This pluripotency gene regulatory network relies on direct physical interaction between SOX2, NANOG, and OCT4. Through a cooperative interaction these factors drive pluripotent-specific expression of the numerous genes and play key roles in determining the fate of ESCs, regulating two distinct and opposing functions: self-renewal and differentiation (Figure 1). Besides directing the expression of target genes, SOX2, OCT4, and NANOG regulate their own expression via positive-feedback loops (Figure 1). In addition, this fully-connected triad has been implicated as recurring network motif among the transcriptional regulatory circuits that control the development and maintenance of cellular states. In addition to SOX2, SOX15 is expressed in mouse ESCs and associated with Oct3/4. It was found that SOX15 is able to replace the function of SOX2 in self-renewal of mouse ESCs.

12Since SOX2 is a part of integrated and self-controlling network, the level of its expression is critical to sustain the stemness phenotype. Accordingly, SOX2 overexpression reduced the level of OCT4 and NANOG in human ESCs. In line with this data, we detected downregulation of OCT4 gene expression in pluripotent embryonal carcinoma stem cells NT2/D1 with constitutive SOX2 overexpression. We also demonstrate that transition from proliferation to retinoic acid (RA) induced neural differentiation of NT2/D1 cells coincides with complete OCT4 down-regulation. However, SOX2 overexpressing NT2/D1 cells retain ability to differentiate even in the presence of elevated SOX2 expression after 21 days of treatment with RA.

13In ESCs, SOX2 overexpression rapidly induces the expression of another SOXB member – SOX21 that further influences fate of these cells. Subsequently, SOX21 acting as a repressor, disrupts ESCs self-renewal and induces differentiation.

14In addition, SOX2 plays important role in reprogramming adult cells and generation of induced pluripotent stem cells (iPSCs). Reprogramming is achieved by overexpression of stem cell-associated genes in differentiated cells, such as adult fibroblasts. SOX2 is recognized as one of the “magical four” crucial TFs capable of cooperating to reprogram differentiated cells into an iPSCs. The fact that SOX2 is crucial factor for reversing the somatic cells back to their pluripotent state demonstrates its pivotal role in maintenance of cell pluripotency.

15Apart from Sox2, other members of the Sox gene family may also be involved in the reprogramming process. Sox1 yields iPSCs with a similar efficiency as Sox2, while Sox3, Sox15, and Sox18 genes are also capable to generate iPSCs, although with decreased efficiency.

16The SOX proteins also function as pioneer factors that occupy silenced target genes and keep them in a poised state for activation at subsequent stages of differentiation.

17demonstrated that binding of SOX proteins is developmental stage-specific and revealed sequential binding of SOX proteins to a common set of neural genes. Prebinding of SOX proteins to silent genes facilitates those genes to be activated at later stages of neural development. They showed that expression of many genes that are targeted by binding of SOX2 in ESCs and neural precursors is first initiated in neural precursors, while many neuronal genes that are prebound by SOX2 and SOX3 in neural precursors can only be activated by SOX11 in differentiating neurons (Figure 2). By these data, the authors reveal that sequentially acting SOX TFs coordinate neural gene expression from pluripotent cells to later stages of neuronal development.

18It has been demonstrated that glial-specific gene sets are extensively preselected in multipotent neural progenitor cells (NPCs) through prebinding by SOX3. Further, in the subsequent lineage-restricted glial precursor cells, it was shown that SOX3 performs a negative regulation of prebound astrocyte-specific genes and efficiently hinders SOX9 from activating their expression. Astrocyte-specific genes become additionally targeted and activated by SOX9, while oligodendrocyte-specific genes are prebound by SOX9 only and later on they are targeted and activated by SOX10 during oligodendrocyte maturation (Figure 2). Thus, the previous study demonstrated how sequentially expressed SOX proteins act on lineage-specific regulatory DNA elements to coordinate glial gene expression both in a temporal and in a sub-lineage-specific fashion.

19Together these interesting data demonstrated that sequentially acting SOX TFs orchestrate neural lineage development including neuronal-, astrocyte- and oligodendrocyte-specific gene expression.

20Neuronal differentiation is a complex process that relies on a timely and spatially controlled expression of transcriptional regulators. Numerous SOX TFs play widespread roles from initial phases of differentiation until generation of mature neurons. During neuronal differentiation SOXB and SOXC members act sequentially. SoxB1 genes are expressed in the neural precursor cells, while Sox21, Sox4, and Sox11 are mostly expressed in neural cells committed to neuronal differentiation (Figure 3, left panel).

21SOXB1 proteins are necessary for formation of neuroectoderm, maintenance of the neural progenitor state and suppression of neuronal differentiation (Figure 3, left panel). It has been revealed that forced expression of SoxB1 genes maintains neural cells in un undifferentiated state and inhibits neuronal differentiation, whereas their suppression induces upregulation of post-mitotic neuronal markers. Down-regulation of SoxB1 gene expression by Ngn2 (Neurogenin 2) is essential for neuronal differentiation.

22SOX1 is one of the earliest TFs expressed in cells committed to the neural fate. Its expression correlates with the formation of neural plate, while down-regulation of Sox1 expression in the developing neural tube correlates with the exit of cells from mitosis. Overexpression of Sox1 in NPCs is sufficient to promote neuronal lineage commitment while the loss of neurons in the ventral striatum was detected in the brains of Sox1 null mutant mice.

23Sox2 expression is detected in the early neuroectoderm and SOX2 transcription factor is necessary to maintain neural progenitor populations throughout the developing central nervous system (CNS). Constitutive Sox2 expression kept NPCs in a precursor state and inhibited neuronal differentiation, while expression of a dominant-interfering form of Sox2 led to exit from cell cycle, delamination of NPCs from the ventricular zone, loss of expression of progenitor markers and initiation of neuronal differentiation. We showed that constitutive SOX2 overexpression altered expression of neuronal markers and reduced number of mature MAP2 (Microtubule Associated Protein 2) positive neurons upon RA induced neural differentiation of NT2/D1 cells.

24Expression of Sox3 is detected throughout the developing CNS and activity of this gene is necessary for formation of the hypothalamo–pituitary axis. Ectopic Sox3 expression in zebrafish led cells of the ectoderm to acquire a neural fate, while reduction of neural ectoderm was seen in Sox3 knocked-down study.

25Sox21 expression is detected in NPCs while Sox14 is expressed in limited population of neurons in the developing brain and spinal cord. The balance of SoxB1 and SoxB2 expression determines whether NPCs remain as progenitors or become committed to differentiation (Figure 3, left panel). SOX21 promotes neuronal differentiation by counteracting the activity of SOXB1. Study conducted on Xenopus laevis shows that Sox21, like Sox2, functions in a dose-dependent manner and that its level of expression determines the decision between maintenance of neural progenitors and formation of neurons. Namely, proposed model which described how level of Sox21 expression regulates progression of NPCs during neurogenesis. When Sox21 expression is severely reduced, NPCs undergo cell death; with a minimal level of Sox21 expression NPCs differentiate to become neurons while higher expression of Sox21 inhibits neurogenesis, promotes SoxB1 expression and progenitor maintenance. On the other side, overexpression of Sox21 in the chick neural tube led to reduction of cell proliferation, downregulation of Sox3 expression and initiation of premature differentiation of NPCs. Furthermore, the maintenance of Sox21 expression in NPCs disabled their terminal differentiation. Interestingly, recent study shows that both SOX21 and SOX14 have their own unique gene targets and therefore these TFs do not compete for the same target genes. Thus, SOX21 is important for the maintenance, while SOX14 is necessary for terminal differentiation of the GABAergic neurons in the mouse brain.

26When NPCs start to differentiate into immature neurons, pro-neural proteins induce expression of SOXC TFs (Figure 3, left panel). SOXC TFs are necessary to ensure survival of NPCs and for establishment of their neuronal properties. Overexpression of Sox4 and Sox11 led to premature induction of neuronal markers while deficiency of both factors induced apoptosis in the developing nervous system. Furthermore, reduced level of Sox4 and Sox11 resulted in reduced number of mature neurons and decreased neurite length. Also, results obtained by indicate that high level of SOX11 expression, detected only in cortical neurons until birth, is necessary to suppress dendritic morphogenesis during radial migration.

27As already pointed out, SOXB and SOXC members are sequentially bound to a common set of neural genes during the process of neuronal differentiation (Figure 2). It has been found that 92% of the SOX3 binding sites will be targeted by SOX11 in newly formed neurons.

28SOXD members are also involved in the process of neuronal differentiation (Figure 3, left panel). They are expressed in proliferating progenitors in the ventricular and subventricular zones and in post-mitotic neurons. Sox5 promotes exit of neural progenitors from cell cycle and downregulation of its expression is necessary for the progression of neuronal differentiation. Furthermore, SOX5 post-mitotically controls the neuronal migration, molecular identity and subcortical axonal projections of subplate and deep-layer neurons. This TF is involved in the control of neurite outgrowth. SOX6 is important for positioning and maturation of cortical interneurons. Sox13 is expressed in a sub-population of post-mitotic differentiating neuronal cells and results obtained by suggest that this gene may have a role in the specification and/or differentiation of a specific subset of neurons in the developing CNS.

29Sox9, SoxE member, is expressed in neural stem cells (NSCs) and gain- and loss-of-function studies indicated that Sox9 was required for multipotentiality and maintenance of NSCs during development. On the other side, Sox9 overexpression led to reduction in the number of neuronal progenitors and neurons during the spinal cord development.

30The majority of existing knowledge regarding the functions of Sox genes in the process of neuronal differentiation is obtained by conducted experiments in mice and other animal models. However, comparative transcriptome analyses point to differences in gene expression between the human and the rodent brain. Thus, it would be interesting to investigate the roles of SOX genes in human neuronal differentiation using iPSCs and 3D brain organoids.

31Recent literature data has revealed that SOX gene variants are associated with neurodevelopmental disorders (NDDs), characterized by impairment of neuronal function during brain development. SOX gene variants associated with NDDs are listed in Supplementary Table 1.

32Contribution of SOX genes to NDDs is still not clear. Both deletions and duplications of SOX3 gene were detected in patients with intellectual disability. Also, SOX3 missense variant was detected in proband with mild intellectual disability. Furthermore, it was found that SOX4 heterozygous missense variants cause neurodevelopmental disease. On the other side, SOX5 haploinsufficiency and its loss of function variant have been found in probands with intellectual disability. Also, NDDs have been detected in individuals with heterozygous SOX6 variants. Significant down-regulation of SOX9 expression has been revealed in neural progenitors derived from Fragile X Syndrome ESCs while downregulation of SOX10 expression is detected in brains of patients with schizophrenia. Heterozygous missense variants within the HMG box of SOX11 gene are associated with intellectual disability while polymorphisms in distal 3′ untranslated region of this gene are associated with susceptibility for schizophrenia.

33All these data open a new avenue of research focused on discovering the roles of SOX TFs and their gene targets in NDDs, making them promising biomarkers and potential targets for future diagnostic and therapeutic strategies.

34In the mammalian brain, generation of neurons and astrocytes from NSCs throughout postnatal and adult life is mainly observed in the subventricular zone (SVZ) of lateral ventricle and in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus (reviewed in;; ). NSCs that reside in the SGZ generate dentate granular cells which play roles in learning, memory and pattern separation, whereas NSCs from the SVZ give rise to neuroblasts which migrate in the rostral migratory stream and differentiate to olfactory bulb neurons.

35Similar to neurogenesis in embryonic brain, the generation of new neurons from adult NSCs consists of the sequence of events including proliferation, differentiation and maturation, which are controlled by environment-derived signals and precise changes in the gene expression. However, despite many similarities between NSCs from embryonic and adult brain, emerging evidence suggests profound differences in these two cell populations, such as proliferation rates, neurogenic potential and gene expression profiles (reviewed in; ). In addition, there are important differences between NSCs from two major adult neurogenic niches, SVZ and SGZ in cellular and molecular properties which may arise as result of external signals received from different environments (reviewed in ).

36The roles of SOX TFs in the regulation of adult neurogenesis, particularly in the hippocampus, are extensively investigated, with the main focus on SOXB, SOXC, and SOXD proteins (Figure 4) (reviewed in;; ). In the SGZ (Figure 4A) and SVZ (Figure 4B) of the mouse adult brain, SOX2 is mostly expressed in both, quiescent NSCs and highly proliferating multipotent neuronal progenitor cells. Functional studies demonstrated that repression of Sox2 gene expression impaired neurogenesis in the adult mouse brain. SOX2 inhibits expression of pro-neurogenic TF NEUROD1 (Neuronal differentiation 1) via Wnt (Wingless-integration site)-signaling pathway, thus preventing neuronal differentiation and maintaining stem cells in a multipotent state.

37In contrast to SOX2, the expression of SOX1 has not been detected in NSCs, but it is revealed only in the highly proliferating neuronal progenitors of SGZ in the mouse adult hippocampus (Type 2a and Type 2b in the Figure 4A). In the mouse adult brain, SOX3 protein has been detected in cells within neurogenic niches, however, with different pattern of expression. In the SGZ, its robust expression was identified in slow dividing NSCs as well as in highly proliferating Type 2a and Type 2b neuronal progenitor cells (Figure 4A). In contrast, the expression of SOX3 was localized only in small population of SOX2 expressing cells in SVZ (Figure 4B). Despite robust expression of SOX1 and SOX3 in adult brain, their functional roles in the regulation of adult neurogenesis are yet to be determined.

38In the hippocampus of adult mouse, the expression of SOX21 has been detected in NSCs (Radial Type 1) and subset of neuronal progenitor cells (Type 2a) in SGZ (Figure 4A). Functional study indicated that Sox21 regulates the progression of adult neurogenesis in the hippocampus by direct repression of TF Hes5 (Hes Family bHLH Transcription Factor 5).

39In the adult mammalian brain, the expression of SOX4 and SOX11 proteins is detected prominently in the both main neurogenic niches, SGZ (Figure 4A) and SVZ (Figure 4B). Similar to the expression pattern in embryonic brain, onset of SOX4 and SOX11 expression in cells coincides with the down-regulation of SOX2 and up-regulation of DCX (Doublecortin) expression, and remains throughout the period when newborn neurons migrate to their final destination (Figure 4). Finally, SOX4 and SOX11 expression was detected in extremely low number of mature neurons. Overexpression of these two TFs promoted expression of neuronal-specific genes in NPCs, whereas their repression disturbed neurogenesis, but not gliogenesis.

40Among SOXE TFs, SOX9 expression has been detected in the NSCs and different subset of neuronal progenitor cells in the SVZ in the adult mouse brain (Type B1 and Type C neuronal progenitors in the Figure 4B). Functional studies provided evidence that this TF is necessary for maintaining the multipotency of NSCs in SVZ. Furthermore, knockdown of miR-124, which targets Sox9, increase SOX9 expression and decrease neurogenesis.

41Adult neurogenesis has been implicated as a major contributor of brain homeostasis, restoring neurological functions under physiological or pathological conditions. Differentiation and maturation of new neurons from NSCs in adult brain are dynamically regulated by numerous intrinsic and extrinsic factors, such as neurotrophic factors, transcriptional programs, inflammatory cytokine, cell cycle regulators, neurotransmitters and hormones. On the other hand, the most studied negative regulators of NSC fate during adult neurogenesis include aging, stress, inflammation and alcohol abuse. In addition, wide spectrum of neurological conditions is a consequence of neuron loss after injury. Although increase in neurogenesis is detected in response to injury, the capacity for restoring of neurological function in damaged areas is limited (review in ).

42Neurodegenerative diseases are heterogeneous group of deleterious conditions with multifactorial etiologies caused by progressive damage of neurons and glial cells and, consequently, the loss of cognitive and physical functions. Recent findings provided multiple evidence for deregulated adult neurogenesis in several neurodegenerative diseases that display symptoms related to hippocampal and olfactory dysfunction, including Parkinson’s, Alzheimer’s, and Huntington diseases (reviewed in;;; ).

43Despite numerous data implicating the key role of Sox/SOX genes in regulation of embryonic and adult neurogenesis, their function under pathological conditions are largely unknown. Alzheimer’s disease, the most common adult onset-dementia, is characterized by deteriorating hippocampus, memory impairment, and other cognitive and olfactory deficits. Recently, have demonstrated reduction in SOX2 positive NSCs in the hippocampus of Alzheimer’s disease patients which correlated with the severity of the disease or the patient’s cognitive capacity.

44In our previous work, we analyzed the expression of selected members of SOXB group (SOX1, SOX2, and SOX21) in the hippocampus of 2 months old 5xFAD mice, which represent a transgenic model of Alzheimer’s disease. Immunohistochemical analysis revealed a significant decrease in the number of cells expressing SOX1, SOX2, and SOX21 TFs within the SGZ of 5xFAD mice in comparison to their non-transgenic counterparts. Our comparative study also revealed, for the first time, significant difference in the number of SOX1 positive cells between genders in both, transgenic and non-transgenic animals. Considering previous findings that epigenetic-related mechanisms are involved in brain development, we speculate that sex dependent level of SOX1 expression could be result of epigenetic regulation. However, further studies are needed to clarify these findings.

45During embryonic development of the CNS, multipotent neural precursor cells undergo a characteristic temporal pattern of differentiation wherein neurons are generated ahead of the production of glial cells. This developmental transition consists of two distinct molecular processes: the termination of neurogenesis and the initiation of gliogenesis. This developmental interval, often named “gliogenic switch,” is fundamental to the entire developing CNS and is conserved throughout all the vertebrate species. The genesis of neural cells by chronological order in vertebrates provides a good biological sense. In fact, the generated neurons create the functional neuronal circuits and, when the scaffold is formed, then the numbers and positions of glia are fitted in the preformed platform. The integrated glial cells further provide mechanical, metabolic and trophic support to neurons. The sequential production of neurons and glia is best characterized in the ventral region of the mouse and chick embryonic spinal cord. SOX9 TF has been reported as a crucial molecular component in triggering the switch from neurogenic to gliogenic program in the developing mouse spinal cord. In particular, the absence of Sox9 in NSCs caused defects in the specification of oligodendrocytes and astrocytes, the two main types of glial cells in CNS, and induced a transient increase in the number of motoneurons. However, findings in the developing cerebellum indicate that the primary functional role of Sox9 in modulating the neuron-versus-glia switch is to suppress neurogenesis, rather than to actively trigger the initiation of gliogenesis. The discrepancy in results obtained by studying different developmental CNS regions suggests that the importance of SOX9 transcriptional factor in orchestrating NSCs fate decision toward gliogenesis may be tissue or organ dependent. During gliogenic switch SOX9 actively promotes glial lineage progression by controlling a set of genes that contribute to early gliogenesis. As previously mentioned, an important feature of SOX proteins is that they generally display their gene regulatory functions by forming complexes with partner TFs. Thus, the NFIA (Nuclear factor-1 A) directly regulated by SOX9, has been identified as the crucial transcriptional partner of SOX9 necessary for the onset of gliogenesis. Subsequently, SOX9 and NFIA form a complex and co-activate multiple genetics programs that regulate the activities of astroglial precursors. It is important to note that this data has been collected from the embryonic chick and mouse spinal cord, whether the same mechanism is active in the other regions of developing CNS is still unknown. Recent findings have indicated that the synergistic activation of astrocyte genes by SOX9 and NFIA is repressed by SOX3 binding in glial precursor cells of mouse spinal cord. Indeed, while both astrocyte- and oligodendrocyte-specific genes are prebound by SOX9 in glial progenitor cells, a specificity of astrocyte genes is that this prebinding occurs in combination with SOX3. Sox9 continues to be expressed in maturing astrocytes but its expression decreases during oligodendrocyte-lineage progression (Figure 3, right panel). At the later stages of development, maturing oligodendrocytes become fully dependent on Sox10, as evident from the severe disruption of both terminal oligodendrocyte differentiation and myelination in the CNS of Sox10-deficient mice.

46Although Sox8 is also expressed in oligodendrocyte precursors, it performs only supportive role during oligodendrocyte development. Despite functional redundancy, SOX8 is only able to partially rescue the compromised oligodendrocyte differentiation in Sox10-deficient mice. However, differentiated oligodendrocytes rely to a much greater extent on SOX8 than oligodendroglial precursors, since recent findings have indicated that SOX8 and SOX10 are jointly required for maintaining the myelinated state.

47SOX10 directly controls the expression of genes encoding the major myelin proteins. This TF is an essential general determinant of myelination in both CNS and the peripheral nervous system. Although both oligodendrocytes in CNS and Schwann cells in peripheral nervous system represent myelinating glia, they achieve myelination in distinct ways. However, while the interacting partners of SOX10 in Schwann cells are well described, less is known about its transcription partners in oligodendrocytes. Expression of Sox5 and Sox6 overlaps strongly with SOXE protein activity during oligodendrocytes specification and terminal differentiation (Figure 3, right panel). Nevertheless, while SOX9 and SOX10 promote oligodendrocyte lineage progression, SOX5 and SOX6 have the opposite effects as evident from both premature specification and precocious terminal differentiation of oligodendrocyte precursors in Sox5/Sox6-deficient mice. Direct physical interaction between the SOXD and SOXE TFs during oligodendrocyte differentiation has not been reported. Actually, the SOXD proteins counteract the SOXE proteins by competition for the same binding sites and the recruitment of co-repressors to target gene promoters.

48Interestingly, growing evidence indicates that Sox genes also play additional roles in adult tissue homeostasis and regeneration. However, in comparison to the data about the roles of Sox genes during developmental, this research field is less explored. Unlike neurons and oligodendrocytes, which become post-mitotic and take on a distinct morphology upon terminal differentiation, astrocytes are not permanently post-mitotic. In response to injury, these cells transform from quiescent into reactive state and dedifferentiate to a progenitor cell-like state, which serves as a compensatory response that modulates tissue damage and recovery. The re-expression of Sox genes in reactivated astrocytes of adult mouse brain has been reported, but the better understanding of their roles in this process is still needed. Peripheral nerve regeneration is a good example for the role of Sox2 gene in tissue repair. Upon injury, mature adult Schwann cells re-express Sox2 and re-acquire progenitor cell-like characteristics. Sox2 re-expression seems to play a direct role in Schwann cell clustering, a key event during nerve regeneration that enables Schwann cells to form multicellular cords to guide axon re-growth across the site of injury.

49The link between neural functions of SOXE genes and human nervous system pathologies has been reported throughout different studies. Thus, the increased number of SOX9- and SOX10-positive early glial progenitors in brains of multiple sclerosis patients has been reported. Additionally, SOX8 has been identified as genetic risk loci for Multiple Sclerosis in humans. Also, dysfunction of oligodendrocytes in patients with schizophrenia was correlated with increased DNA methylation of SOX10 gene. Taking together, these data imply that SOX genes could be considered as potential therapeutic targets. Modulation of SOX genes expression may change the functional properties of glial cells for more efficient remyelination of neurons or the repopulation of damaged areas upon CNS trauma. Full understanding of SOX genes function in nervous system development as well as homeostasis maintenance and regeneration holds the promise for development of novel therapeutic strategies.

50During last decade numerous results established SOX TFs as key players in various signaling pathways. Their interplay with Wnt/β-catenin and RA signaling pathways is of particular interest since SOXB1 neural-specific interpretation of these signaling cascades are involved in the maintenance of stemness and neural differentiation.

51β-catenin is central signaling molecule in canonical Wnt pathway. In the absence of the Wnt ligand, cytosolic β-catenin level is low due to activation of destruction complex. Wnt stimulation results in the inhibition of cytosolic β-catenin degradation and its shuttle to nucleus. In the nucleus β-catenin interacts with the TCF/LEF (T cell factor/Lymphoid enhancer factor) family of DNA-binding TFs on Wnt Response Elements and enhances expression of Wnt target genes.

52Both canonical Wnt signaling and SOXB1 proteins promote self-renewal of NPCs. In addition, SOXB1/β-catenin interplay fine tunes the complex mechanism involved in pluripotency/differentiation switch. Activation of canonical Wnt signaling enhances self-renewal of mouse and human ESCs and embryonal carcinoma cells.

53In neural progenitors Wnt/β-catenin signaling activates expression of the pro-neural gene NeuroD1 by counteracting SOX2-mediated repression on DNA element containing overlapping SOX2 and TCF/LEF-binding sites (SOX/LEF) in its promoter. We showed that lithium induced activation of Wnt/β-catenin signaling increased expression of all SOXB1 proteins in NT2/D1 cells. We also demonstrated that increase in SOX2 and SOX3 protein expression is β-catenin dependent, while overexpression of SOX1 is governed by β-catenin-independent manner (Figure 5B).

54SOX1, SOX2, and SOX3 also affect Wnt/β-catenin signaling. SOX1 binds to β-catenin via their C-terminal regions and inhibits β-catenin/TCF transcription activity in mouse and human NPCs in the onset of neural differentiation. SOX2 overexpression reduced β-catenin protein level and down-regulated Wnt/β-catenin signaling in NT2/D1 cells, suggesting negative feedback loop between β-catenin and SOX2 (Figure 5B). However, overexpression of SOX1 and SOX3 genes has no effect on endogenous β-catenin level in NT2/D1 cells (Figure 5B).

55SOX3 down-regulates Wnt signaling by interactions with β-catenin or by direct binding to the regulatory regions of Wnt target genes. SOX21 restricts Wnt activity by interacting with β-catenin and subsequent interfering with the binding of TCF4/β-catenin complex to the WNT8B (Wingless-type MMTV integration site family, member 8B) enhancer.

56The SOX proteins are considered as nuclear regulators of β-catenin/TCF activity responsible for fine tuning of transcriptional responses to Wnt signaling. They regulate β-catenin/TCF activity by recruitment of various mechanisms in cell context-dependent manner (review in ). SOX proteins can physically interact with both β-catenin and TCF/LEF. SOX and TCF bind to similar DNA sequences in the DNA minor groove and induce DNA bending which enables assemble of SOX/TCF complexes regardless of distance of their binding sites. Post-translational modifications of SOX, β-catenin and TCF also affect their interactions. SOX proteins bind to the promoters of Wnt target genes and recruit transcriptional co-activators or co-repressors thus controlling β-catenin dependent transcriptional activity. SOX proteins activate the expression of Wnt signaling pathway repressors. In addition, SOX proteins control endogenous β-catenin protein level by promoting either, proteosome-mediated β-catenin degradation or its stabilization (Figure 5A).

57Wnt and SOX interplay and mutual control led to a hypothesis pointing out that interactions between lineage-specific SOX TFs and β-catenin/TCF govern specificity of Wnt/β-catenin dependent transcription. This idea is supported by the study conducted by. They studied the transition between stem cells and rapidly dividing progenitors in mouse cortex and proposed model of SOXB1-dependent bi-phasic repressive mechanism. High level of SOXB1 in stem cells represses pro-proliferative genes, primarily Ccnd1 (Cyclin D1), by binding to low-affinity SOX binding sites in Ccnd1 promoter, by interactions with TCF/LEF proteins and by recruitment of GRO/TLE (Groucho/Transducin-like Enhancers) co-repressors. Upon differentiation, pro-neural proteins reduce SOXB1 level, thus only the high affinity SOX binding sites stay occupied, while loss of binding to low-affinity sites de-represses Ccnd1 and promotes proliferation of progenitor cells. Proposed model provides the explanation how presence of low- and high-affinity SOX binding sites enables graded SOXB1 target gene regulation and how differences in the expression levels of SOXB1 proteins can be interpreted by determining the response of target genes.

58It has been shown that complex network between Wnt/β-catenin pathway, SOX2 and proneural genes regulates the progression from progenitors to neurons and glia cells. Lack of this coordination leads to aberrant neuronal proliferation and differentiation and contributes to the pathology of psychiatric disorders. In the search for specific targets of lithium resistance in the patients with bipolar disorder, conducted comparative transcriptome analysis of the hippocampal dentate gyrus-like neurons derived from iPSCs of lithium-responsive and lithium-non-responsive patients. First, they have demonstrated that neurons generated from both cohorts exhibited neuronal hyperexcitability compared to control neurons that could be reversed by lithium treatment of the lithium-responsive neurons only. The study showed that neurons from lithium-non-responsive patients acquire distinct phenotypic characteristics, electrophysiological properties and the response to lithium during differentiation due to the severely affected function of canonic Wnt/β-catenin signaling with a significant decrease in expression of LEF1 (Lymphoid enhancer-binding factor 1). Interestingly, SOX2 was also up-regulated in lithium-non-responsive neurons compared to control neurons.

59Retinoic acid exerts its pleiotropic effects through binding to retinoic acid receptors (RARs), members of the nuclear receptor superfamily (reviewed by ). RARs act in heterodimeric combinations with retinoid X receptors (RXRs). It was suggested that RXRs act as scaffolding proteins and facilitate DNA binding of the RAR-RXR complex. In the nucleus, RAR/RXR dimers can interact with cis-acting RA response elements (RAREs), atypical RARE and composite response units within the promoters of different RA-target genes. Ligand binding induces conformational changes that lead to release of co-repressors, binding of co-activators and subsequent initiation of transcription.

60Our group conducted comprehensive analyses of the SOXB1 protein expression during RA induced neural differentiation of NT2/D1 cells. Obtained results showed dynamic changes in the expression profiles of SOX1, SOX2 and SOX3 proteins during 4-weeks course of RA induction (Figure 6).

61Retinoic acid induced transient up-regulation of SOX1 at the day 4 of RA induction and oscillating expression followed by decrease at 3 and 4 weeks of treatment (Figure 6). After initial downregulation in the first 48h of induction, SOX2 was up-regulated in all time points of induction (Figure 6). However, in mature neurons (NT2-N) expression of SOX2 is abolished. Similar results were obtained in the studies of the effects of RA on Sox2 expression in mouse P19 and F9 embryonal carcinoma cell lines.

62SOX3 expression was transiently up-regulated during 48h of RA treatment and then gradually decreased up to 4 weeks of RA treatment (Figure 6). Comprehensive examination of the promoter of human SOX3 gene revealed the presence of two RA response elements, DR-3-like RXR RE and atypical RA/RXR RE. In addition, we have identified numerous TFs involved in the modulation of RA induced activation of human SOX3 promoter.

63Beside RA involvement in transcriptional regulation of SOXB proteins, SOXB1 neural-specific interpretation of signaling morphogens add an additional level of complexity to the RA/SOXB1 interplay in developing CNS. Genome-wide characterization of cis-regulatory modules (CRMs) in neural-specific target genes showed that interpretation of pleiotropic signals is the result of integration of SOXB1 and signaling morphogens on CRMs. CRMs of RA target genes contains RARE and SOX binding sites both required for synergistic activation of CRMs. One of genes enriched for functions in neural development whose CRM was analyzed in the study is Dbx1 (Developing brain homeobox 1). In another study conducted by Dbx1 was identified as a direct and exclusive SOX3 target gene in NPCs both in vitro and in vivo. The fact that RA regulates SOX3 expression through multiple RARE confirms that SOXB1/RA signaling interplay is complex and fine-tuned at multiple levels.

64Many evolutionary conserved microRNAs (miRNAs) present key factors in fine regulation of self-renewal and proliferation of NSCs and NPCs. By interaction with complementary sequence motifs in 3′ untranslated region of target genes, miRNAs regulate the gene expression during different stages of neurogenesis, thus affecting the development of nervous system. Also, acting in synergy with TFs, miRNAs form regulatory networks that can influence cell fate decision. Therefore, it is not surprising that miRNAs are often called “master regulators” or “fine-tuners” of gene expression orchestrating important processes during neural development. SOX TFs and miRNAs represent one of the most important regulatory networks that control whether NSCs will self-renew or differentiate into neurons, astrocytes or oligodendrocytes (Figure 7). Particularly, SOX1, SOX2, SOX4, SOX5, SOX6, SOX9, and SOX10 are shown to interact with different miRNAs and orchestrate differentiation into neurons and oligodendrocytes (Figure 7).

65It is suggested that miR-200 family members target Sox2, thus regulating the transition from NSCs to postmitotic and differentiated cells. It turns out to be one of the most important regulatory networks during neural differentiation, whereas Sox2 is regulated by miR-200c, forming a negative-feedback loop that further decreases expression of Sox2 during neural differentiation. Another axis shown to be important for neuronal differentiation is miR-135a-5p/Sox6/CD44, where miR-135a-5p acts through Sox6, affecting not only differentiation of neurons, but also development of dendrites. Moreover, it was shown that overexpression of Sox6 could reverse miR-135a-5p-mediated neuronal differentiation and dendrite development of P19 cells. Sox9 is target of miR-124 during adult neurogenesis in the mouse SVZ where inhibition of Sox9 expression leads to differentiation into neurons. Further, miR-145 is important for differentiation of neurons through regulation of Sox2–Lin28/let-7 signaling pathway that represents important mechanism for proliferation of NPCs. Additionally, miR-145 directly regulates Sox2 and suppresses its expression in NPCs leading to induction of neurogenesis.

66Surprisingly, to the best of our knowledge, there is only one study suggesting that interaction between miRNA and SOX TFs plays role in differentiation of astrocytes. miR-124, with pivotal role in differentiation of neurons and astrocytes, can induce differentiation of NSCs to astrocytes through regulation of Sox2 and Sox9 expression in NSCs of amyotrophic lateral sclerosis transgenic mice.

67Interestingly, it was shown that miR-184, which is one of the key miRNAs throughout all stages of oligodendrocytes differentiation, directly targets Sox1, leading to differentiation of NPCs to oligodendrocytes. During differentiation of NPCs the loss of Sox2 also results in terminal differentiation of oligodendrocytes through negative regulation by miR-145. miR-219, miR-138 and miR-338 directly inhibit the expression of Sox6 gene which results in reduced proliferation of oligodendrocyte progenitors and induction of oligodendrocyte differentiation and myelination. Further, SOX6 TF is directly regulated by miR-219 and additionally this TF represses the expression of Sox10 in oligodendrocyte progenitor cells which results in differentiation of mature oligodendrocytes. miR-204 overexpression also leads to differentiation of oligodendrocyte progenitors to mature oligodendrocytes through control of Sox4 gene, while the expression of this miRNA is regulated by SOX9. SOX10 also regulates miR-338 that is important for differentiation of oligodendrocytes through inhibition of Hes5 and Hes6 genes and it is suggest that Sox10 exerts its role in maturation of oligodendrocytes specifically through regulation of miR-338. Another study shows that SOX10 TF directly targets miR-338 and miR-335, and then these two miRNAs further repress Sox9 in oligodendroglial cells. This fine tuning of Sox9 and Sox10 expression levels leads to terminal differentiation of oligodendrocytes.

68It is evident that SOX TFs and miRNAs establish the functional interactions important for cell fate decision of NPCs during neural development. This can be achieved either through post-transcriptional regulation of Sox genes by miRNAs, through SOX-dependent control of miRNAs expression or by combination of both. Even though SOX3, SOX14, and SOX21 play important roles in neural differentiation, there is a lack of data regarding their regulation by miRNAs. Although SOX TFs are involved in regulation of gliogenesis, more studies are required to clarify posttranscriptional regulation of Sox/SOX gene expression by miRNAs during this process. Additionally, we previously reported that specific SOX genes and miRNAs can be potentially used as biomarkers for monitoring radiation response during early phase of neural differentiation. This result suggests that SOX and miRNAs have additional important roles during neural differentiation. Overall, future studies are needed in order to gain better insight into the complex interactions between miRNAs and SOX TFs during neural development.

69SOX proteins belong to the family of TFs that exerts multiple important roles during nervous system development, starting from preimplantation embryo to the adulthood. Many Sox genes are expressed in the developing and adult nervous system in the overlapping manners, covering various cell types, beginning with the neural stem cell (NSC) stage until terminal maturation of neurons and macroglia (reviewed in ). During embryonic development SOX family members are involved, both in maintaining multipotency of neural progenitors, as well as in the promotion of neuronal differentiation (Table 2). They are also implicated in the control of glial differentiation by promoting differentiation of astrocytes and oligodendrocytes. SOX proteins may have dual roles in the regulation of target gene expression, acting as either activators or repressors, depending on cellular and genomic context. For instance, SOX3 is acting as activator of genes in neural progenitors, while suppressing neuronal differentiation by repression of neuronal and glial specific genes in the same cells (Figure 2). It is interesting to point out that SOXB and SOXC family members are sequentially bound to the common set of neural genes during the process of neuronal differentiation, highlighting the context-dependent nature of their actions. It has been reported that majority of the SOX3 binding sites will be targeted by SOX11 in newly formed neurons. The presence of low- and high-affinity SOX binding sites enables graded control of target genes, while the levels of SOX proteins is interpreted by the corresponding response of the target gene expression. Thus, sequentially acting SOX TFs orchestrate neuronal-, astrocyte- and oligodendrocyte-specific gene expression defining neuronal and glial phenotypes.

70The capability of SOX TFs to orchestrate the process of neural differentiation strongly relies on epigenetic regulation. We already showed that SOXB1 genes were controlled by different epigenetic mechanisms during neural differentiation. Additional study of the differences in histone signatures will provide further insight into the epigenetic regulation of pluripotency for proper differentiation of neurons or glial cells.

71The roles of SOX proteins are not limited to development since these factors influence survival, regeneration, cell death and control of homeostasis in adult tissues. Adult neurogenesis has been recognized as a major contributor of brain homeostasis, restoring neurological functions under physiological or pathological conditions. We provide the overview of the current data implicating at least seven SOX proteins (members of SOXB, SOXC, and SOXE groups) in control of adult neurogenesis (Figure 4).

72The majority of current knowledge regarding the roles of Sox/SOX genes in neural development is based on research mainly conducted in mice and other animal models, and to the lesser extent, on the in vitro cell based models of human neural differentiation. Research based on animal models provides important information about the roles of Sox genes in neural development. However, significant differences in brain development between species have been revealed and evident divergence among species is discovered regarding gene expression at the earliest stages of brain development.

73The emerging data associates SOX gene variants with NDDs characterized by impairment of neuronal function during brain development (Supplementary Table 1). Interestingly, down-regulation of SOX9 expression has been detected in neural progenitors derived from Fragile X Syndrome human ESCs. However, contribution of SOX proteins to NDDs is still not fully explored and further research is needed to clarify their roles in the underlying pathologies.

74The rising field of research is devoted to study the roles of SOX TFs in neurodegenerative diseases. Neurodegenerative diseases are characterized by progressive damage of neurons and glial cells and, consequently, loss of cognitive and physical functions. Recent years provided multiple evidences of impaired adult neurogenesis in several neurodegenerative diseases (reviewed in ). Reduction in SOX2 positive NSCs detected in the hippocampus of Alzheimer’s disease patients is correlated with the severity of the disease or the patient’s cognitive capacity.

75Most of the knowledge regarding human neurodegenerative diseases has been acquired from post-mortem patient samples since human brain tissue is inaccessible and highly difficult to obtain. Although many animal models mimicking diseases have been available for the research, they have provided only limited success in identification of the molecular mechanisms underlying human brain diseases. In the recent years, generation of patient-specific iPSCs provides remarkable opportunity to recapitulate both normal and pathologic human tissue formation in vitro, enabling genuine disease investigation (reviewed in ). Furthermore, iPSCs have potential to replace affected neurons in neurodegenerative disorders. Various human brain diseases across the spectrum of neurodevelopmental, neurodegenerative and neuropsychiatric are being studying by iPSC –based disease modeling (reviewed in ). Studying the differentiation of patient-specific iPSCs into neurons or glial cells provides valuable insight into the molecular mechanisms underlying brain diseases in patient-specific genetic background. In the last decades remarkable efforts have been made in developing protocols for fast and efficient differentiation of iPSCs in specific neuronal sub-types. Recently, described a fast, robust and reproducible protocol for differentiation of human iPSCs into functionally maturing forebrain neurons in vitro, which will facilitate studies of neurodevelopmental and neurodegenerative disorders. Obtained neurons have ability of in vivo integration which makes the protocol compatible with cell therapy-based strategies. Apart from enabling research of disease phenotype in vitro, iPSCs are providing the tool for gene defect/s repair ex vivo. Moreover, iPSCs from healthy donors can be modified by introducing disease–specific mutation by genome editing allowing the study of the effect of specific gene defect in “healthy” background.

76In an aging society, regenerative therapies based on iPSCs could provide significant potential therapeutic benefits, in particular for the patients suffering from neurodegenerative diseases including Parkinson’s and Alzheimer’s diseases. Study of the impacts of donor age on iPSC- derived cell functionality indicate that aging may reduce reprogramming efficiency having no significant effects on iPSCs maintenance or differentiation capacity. These data suggest that donor age does not limit applications of iPSCs based methodology for modeling genetic diseases and for development of therapies for age-related diseases, especially in combination with recently developed gene-editing tools such as CRISPR/Cas9 technology. The same authors indicate that burden of age-associated somatic mutations that iPSCs inherit from donor cells cannot be reduced, increasing the risk of abnormalities in iPSCs. A low number of healthy and elderly donors serving as a source of control cells present a great challenge in research and applications in the field of iPSCs. Recently a collection of iPSCs derived from old male and female healthy subjects has been reported that can be used as controls for other disease lines derived from geriatric patients and for studying the roles of SOX TF in neurodegenerative disorders and aging.

77Traditionally, brain diseases have been generally assigned to malfunction or loss of neurons. However, in the last decade, it has been shown that astrocytes play essential roles in the regulation of various brain functions. Astrocytes process and control synaptic information, modulate synaptic formation and elimination at all stages of development and in adulthood. Patient-specific iPSC-based models as human platforms for research accelerated the study of molecular mechanisms underlying neurogenesis, synapse formation, maintenance and plasticity. However, whether astrocytes contribute to the pathology of underlying brain disorders and potential contribution of SOX TFs to the pathologies is yet to be discovered. Accordingly, astrocytes became a promising target for drug discovery and the development of novel therapies.

78Furthermore, wide spectrum of neurological and neurodegenerative conditions are consequence of neuron loss after ischemic injury. Although increase in neurogenesis is detected in response to injury, the capacity for restoring neurological function in damaged areas is limited (review in ). Accordingly, iPSCs can be used for developing effective therapies aimed to increase neurogenesis by modulating SOX gene expression toward enhancement of regenerative potential for repair of damaged or aged neural cells.

79Most recently advances in biotechnology, including stem cell propagation and novel biomaterials enable development of 3D models for studying human brain development. Brain organoids represent 3D-aggregates generated from human pluripotent cells (ESCs and iPSCs) resembling the embryonic human brain regarding the cell types, cells’ architectures and maturation. The 3D models, ranging from region-specific organoids to more complex whole-brain organoids, are mimicking cell interactions and interconnectivity between multiple brain regions providing novel tools for studying more complex phenotypes involving different neuronal networks, tissue architecture, and organ morphogenesis. Advances in 3D modeling including extracellular matrix composition, optimized media transitions and agitation of the tissues led to the formation of cerebral organoids with various brain region identities. These advances revealed the remarkable fidelity with which organogenesis can occur in vitro leading to accurate modeling of events occurring during the first half of gestation in humans (reviewed in ). While cerebral organoids are capable to spontaneously acquired forebrain, midbrain and hindbrain identities, it is feasible to generate particular brain regions of interest by applying novel modified protocols for guiding and directing regional identity. Brain organoids recapitulate many features of the fetal human brain, including cytoarchitecture, cell diversity and maturation and comprise a variety of cell types comparable, to some extent, to the complex composition of the cells present in the brain (reviewed in ). Importantly, spontaneous neuronal activity has been detected in brain organoids suggesting the existence of functional communication among neuronal cells. Brain organoids have been used for modeling neurological diseases and NDDs, providing remarkable advantage in studying diseases in vitro, in a 3D environment resembling the affected tissue (reviewed in ). The position of organoids at the interface of in vitro and in vivo neurobiology makes them a unique model system that will provide further progress in understanding brain development. Combined with single cell transcriptomics technology, the brain organoids would enable to decipher cellular heterogeneity and transcriptional landscape at single cell resolution. These novel tools will open innovative approaches for studying the roles of SOX TFs in brain development at the single cell level in physiological and pathological conditions.

80In the past decade, the “omics” technologies, such as genomics, transcriptomics, miRNomics, and proteomics have become integrated parts of the research in biology and medicine, enabling progress in collecting, processing and integrating huge amounts of health-related information. While genomic analyses provide the insight into variation at DNA level, RNAseq data reveal transcriptome diversity in patients compared to healthy controls. For instance, integrative transcriptomic analysis may lead to the identification of key deregulated candidate genes and pathways shared between various developmental disorders. Such innovative approach may help in identifying novel roles of SOX proteins in pathology of NDDs.

81Advance in proteomic technologies enables mapping of specific gene interactome providing the insights into the network of interacting partners. Thus, the study of Nanog interactome identified SOX2 as interacting factor. Mapping the interactome of specific SOX protein will provide deeper insight into interacting factors and disruption of interactions in diverse pathological conditions. Appropriate bioinformatics analysis will reveal networks of TFs and signaling pathways differentially regulated between different cell states. Such analysis will identified position of SOX proteins within signaling cascades active in particular cell context and pinpoint their functionally relevant links in complex regulatory networks.

82Although many important roles during neural development have been assigned to SOX TFs, we strongly believe that many novel functions are yet to be discovered.

83All authors wrote the manuscript and contributed to literature collection. MiS designed the concept of the manuscript and supervised and edited the manuscript. MiS, DD, and DSN contributed to the preparation of the tables. MaS, AL, MM, and DSN designed the figures. All authors contributed to the article and approved the submitted version.

84The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.