Dichaete/Sox box protein 70D
In vertebrates, the delineation of the neural plate from a region of the primitive ectoderm is accompanied by the onset of specific gene expression, which in turn promotes the formation of the nervous system. SOX1, an HMG-box protein related to SRY, is one of the earliest transcription factors to be expressed in ectodermal cells committed to the neural fate: the onset of expression of SOX1 appears to coincide with the induction of neural ectoderm. In the mouse, expression of SOX1 is first detected at 7.5 days of development in the anterior half of the late-streak egg cylinder. Cross sections through the embryo at this stage reveal expression in columnar ectodermal cells, which appear to define the neural plate, while cells located more laterally are negative. SOX1 is maintained in all neuroepithelial cells along the entire anteroposterior axis as the neural plate bends and fuses to form the neural tube. The expression of SOX1 throughout the neural plate and early neural tube implies a similarity among these cells. After neural tube closure, neuroepithelial cells begin to differentiate into defined classes of neurons at specific dorsoventral (D/V) positions within the spinal cord. As development proceeds, SOX1 is downregulated in a stereotyped manner in cells along the D/V axis of the neural tube (Pevny, 1998).
In the spinal cord, expression is first downregulated in cells that occupy the ventral midline, then in the ventral motor horns and subsequently the dorsal regions. These regions appear to correlate with floor plate, motor neurons and sensory relay interneurons, respectively. SOX1 is expressed by early neural cells in the CNS and is downregulated in the developing neural tube coincident with neural differentiation. SOX1 expression marks proliferating cells within the embryonic neural tube. Using a series of antigenic markers that identify early neural cell types in combination with BrdU labeling, a temporal and spatial correlation has been demonstrated between the differentiation of cell types along the dorsoventral axis of the neural tube and the downregulation of SOX1 expression. Downregulation of SOX1 in the neural tube correlates with exit from mitosis. A role for SOX1 has been demonstrated in neural determination and differentiation using an inducible expression P19 cell system as an in vitro model of neurogenesis. Misexpression of SOX1 can substitute for the requirement of retinoic acid to impart neural fate to competent ectodermal P19 cells. In vitro SOX1 expression is initiated within 24 hours of the addition of retinoic acid to P19 aggregates coincident with the induction of neuropithelial markers such as NESTIN, Mash1 and Wnt1. SOX1, therefore, defines the dividing neural precursors of the embryonic central nervous system (Pevny, 1998).
A number of vertebrate transcription factors have previously been shown to impart neural fate on uncommitted ectodermal cells. Examples of these include XASH-3 and CASH-4 (the ASH-3 Xenopus and chicken homologs of the Drosophila AS-C genes); Neurogenin and NeuroD (vertebrate homologs of the Drosophila atonal genes), and MyT1, a Xenopus C2HC zinc finger protein. In animal cap assays, CASH-4, Neurogenin and NeuroD alone, as well as the combination of XASH-3 and MyT1, can induce neural differentiation in naive ectoderm in the absence of additional neural inducing molecules. This is analogous to the manner in which SOX1 promotes neural differentiation in P19 ectodermal cells in the absence of retinoic acid. However, the relatively late and restricted onset of expression of Neurogenin, NeuroD and MyT1, as well as the phenotype elicited by their misexpression in Xenopus embryos has led to the suggestion that these genes function in neuronal determination and differentiation rather than in neuroepithelial precursor determination. Only CASH-4, whose early expression in neural precursors is initiated by neural inducing signals and whose ectopic expression leads to the expansion of the neural plate, seems to function to promote the formation of neuroepithelial precursors. However, in vivo, the expression of CASH-4 is restricted to the posterior neural plate and therefore may play a role in specifying just posterior neural fate. The early expression of SOX1 throughout the anteroposterior axis of the neural plate and its ability to elicit a neural response in P19 cells implicates a general role for SOX1 in neuroepithelial cell fate determination (Pevny, 1998).
An important question is at what cellular stage does CNS
patterning arise in development. Is patterning already established in
stem cells, generating regional heterogeneity among these
cells, or in precursors downstream to the stem cell? Further,
once established, how is patterning maintained during the
multiple cell divisions occurring in embryogenesis and to what
extent is it reversible in response to progressive modifications
of the environment?
Sox2 is one of the earliest known transcription factors
expressed in the developing neural tube. Although it is
expressed throughout the early neuroepithelium, its later expression must depend on the activity of more
than one regionally restricted enhancer element. Thus, by
using transgenic assays and by homologous recombination-mediated
deletion, a region upstream of Sox2
(-5.7 to -3.3 kb) has been identified that can not only drive expression of a
beta-geo transgene to the developing dorsal telencephalon, but
is also required to do so in the context of the endogenous
gene. The critical enhancer can be further delimited to
an 800 bp fragment of DNA surrounding a nuclease
hypersensitive site within this region, as this is sufficient to
confer telencephalic expression to a 3.3 kb fragment
including the Sox2 promoter, which is otherwise inactive in
the CNS.
Expression of the 5.7 kb Sox2 beta-geo transgene localizes to
the neural plate and later to the telencephalic ventricular
zone. Transgene-expressing (and thus resistent to the antibiotic G418) ventricular
zone cells include cells displaying functional properties of
stem cells, i.e. self-renewal and multipotentiality. The majority of telencephalic stem cells
express the transgene, and this expression is largely
maintained over two months in culture (more than 40 cell
divisions) in the absence of G418 selective pressure. In
contrast, stem cells grown in parallel from the spinal cord
never express the transgene, and die in G418. Expression
of endogenous telencephalic genes has been similarly observed
in long-term cultures derived from the dorsal
telencephalon, but not in spinal cord-derived cultures.
Thus, neural stem cells of the midgestation embryo are
endowed with region-specific gene expression (at least with
respect to some networks of transcription factors, such
as those driving telencephalic expression of the Sox2
transgene), which can be inherited through multiple
divisions outside the embryonic environment (Zappone, 2000).
In a differential screen for downstream genes of the neural inducers, two
extremely early neural genes induced by Chordin and suppressed by BMP-4 have been identified: Zic-related-1
(Zic-r1), a zinc finger factor related to the Drosophila pair-rule gene odd-paired, and Sox-2, a
Sry-related HMG factor. Expression of the two genes is first detected widely in the
prospective neuroectoderm at the beginning of gastrulation, following the onset of Chordin
expression and preceding that of Neurogenin (Xngnr-1). Zic-r1 mRNA injection activates the
proneural gene Xngnr-1, and initiates neural and neuronal differentiation in isolated animal
caps and in vivo. In contrast, Sox-2 alone is not sufficient to cause neural differentiation, but
can work synergistically with FGF signaling to initiate neural induction. Thus, Zic-r1 acts in
the pathway bridging the neural inducer with the downstream proneural genes, while Sox-2
makes the ectoderm responsive to extracellular signals, demonstrating that the early phase
of neural induction involves simultaneous activation of multiple functions (Mizuseki, 1998).
Three chicken Sox (SRY-like box) genes have been identified that show an interactive pattern of
expression in the developing embryonic nervous system. cSox2 and cSox3 code for related
proteins and both are predominantly expressed in the immature neural epithelium of the entire CNS
of HH stage 10 to 34 embryos. cSox11 is related to cSox2 and cSox3 only by virtue of containing
an SRY-like HMG-box sequence but shows extensive homology with Sox-4 at its C-terminus.
cSox11 is expressed in the neural epithelium but is transiently upregulated in maturing neurons
after they leave the neural epithelium. These patterns of expression suggest that Sox genes play a
role in neural development and that the developmental program from immature to mature
neurons may involve switching of Sox gene expression. cSox11 also exhibits a lineage restricted
pattern of expression in the peripheral nervous system (Uwanogho, 1995).
Several stages in the lens determination process have been defined, though it is not known which gene
products control these events. At mid-gastrula stages in Xenopus, ectoderm is transiently competent to
respond to lens-inducing signals. Between late gastrula and neural tube stages, the presumptive lens
ectoderm acquires a lens-forming bias, becomes specified to form lens and begins differentiation. Several
genes have been identified, either by expression pattern, mutant phenotype or involvement in crystallin gene
regulation, that may play a role in lens bias and specification. Fate
mapping shows that the transcriptional regulators Otx-2, Pax-6 and Sox-3 are expressed in the presumptive
lens ectoderm prior to lens differentiation. Otx-2 appears first, followed by Pax-6, during the stages of lens
bias (late neural plate stages); expression of Sox-3 follows neural tube closure and lens specification. The expression of these genes is demonstrated in competent ectoderm transplanted to the lens-forming
region. Expression of these genes is maintained or activated preferentially in ectoderm in response to the
anterior head environment. Activation of these genes is examined in response to early and late
lens-inducing signals. Activation of Otx-2, Pax-6 and Sox-3 in competent ectoderm occurs in response to
the early inducing tissue, the anterior neural plate. Since Sox-3 is activated following neural tube closure, an examination was carried out of its dependence on the later inducing tissue, the optic vesicle, which contacts lens ectoderm at this
later stage. Sox-3 is not expressed in lens ectoderm, nor does a lens form, when the optic vesicle anlage is
removed at late neural plate stages. Expression of these genes demarcates patterning events preceding
differentiation and is tightly coupled to particular phases of lens induction (Zygar, 1998).
Activation of the first lens-specific gene of the chicken, (delta)1-crystallin, is dependent on a group of
lens nuclear factors, (delta)EF2, interacting with the (delta)1-crystallin minimal enhancer, DC5. One of
the (delta)EF2 factors was previously identified as SOX2. Two related SOX proteins,
SOX1 and SOX3, are shown to account for the remaining members of (delta)EF2. Activation of the DC5 enhancer is
dependent on the C-terminal domains of these proteins. Expression of Sox1-3 in the eye region during lens induction
was studied in comparison with Pax6 and (delta)1-crystallin. Pax6, known to be required for the
inductive response of the ectoderm, is broadly expressed in the lateral head ectoderm from before lens
induction. After tight association of the optic vesicle (around stage 10-11, 40 hours after egg incubation),
expression of Sox2 and Sox3 is activated in the vesicle-facing ectoderm at stage 12 (44 hours). These
cells, expressing together Pax6 and Sox2/3, subsequently give rise to the lens, beginning with
formation of the lens placode and expression of (delta)-crystallin at stage 13 (48 hours). Sox1 then starts
to be expressed in the lens-forming cells at stage 14. When the prospective retina area of the neural plate
is unilaterally ablated at stage 7, expression of Sox2/3 is lost in the side of lateral head ectoderm
lacking the optic cup, implying that an inductive signal from the optic cup activates Sox2/3 expression.
In the mouse embryonic lens, this subfamily of Sox genes is expressed in an analogous fashion,
although Sox3 transcripts have not been detected and Sox2 expression is down-regulated when Sox1
is activated. In ectodermal tissues of the chicken embryo, (delta)-crystallin expression occurs in a few
ectopic sites. These are always characterized by overlapping expression of Sox2/3 and Pax6. Thus, an
essential molecular event in lens induction is the 'turning on' of the transcriptional regulators SOX2/3 in
the Pax6-expressing ectoderm and these SOX proteins activate crystallin gene expression. Continued
activity, especially of SOX1, is then essential for further development of the lens (Kamachi, 1998).
Pax6 is a key transcription factor in eye development, particularly in lens development, but its molecular action has not been clarified. Pax6 initiates lens development by forming a molecular complex with SOX2 (most closely related to Drosophila Sox Neuro) on the lens-specific enhancer elements, e.g., the delta-crystallin minimal enhancer DC5. DC5 shows a limited similarity to the binding consensus sequence of Pax6 and
is bound poorly by Pax6 alone. However, Pax6 binds cooperatively with SOX2 to the DC5 sequence, resulting in formation of a
high-mobility form of ternary complex in vitro, which correlates with the enhancer activation in vivo. Pax6 and SOX2-interdependent factor occupancy
of DC5 is observed in a chromatin environment in vivo, providing the molecular basis of synergistic activation by Pax6 and SOX2. Subtle alterations of the Pax6-binding-site
sequence of DC5 or of the inter-binding-sites distance diminish the cooperative binding and causes formation of a non-functional low-mobility form complex,
suggesting DNA sequence-guided and protein interaction-induced conformation change of the Pax6 protein. When ectopically expressed in embryo ectoderm, Pax6
and SOX2 in combination activate delta-crystallin gene and elicit lens placode development, indicating that the complex of Pax6 and SOX2 formed on
specific DNA sequences is the genetic switch for initiation of lens differentiation (Kamachi, 2001).
Group B Sox genes, Sox1, -2 and -3 are known to activate crystallin genes and to be involved in differentiation of lens and neural tissues. Screening of chicken genomic sequences for more Group B Sox genes has identified two additional genes, Sox14 and Sox21. Proteins encoded by Sox14 and Sox21 genes are similar to each other but distinct from those coded by Sox1-3 (subgroup B1) except for the HMG domain and Group B homology immediately C-proximal to the HMG domain. C-terminal domains of SOX21 and SOX14 proteins function as strong and weak repression domains, respectively, when linked to the GAL4 DNA binding domain. These SOX proteins strongly (SOX21) or moderately (SOX14) inhibit activation of delta1-crystallin DC5 enhancer by SOX1 or SOX2, establishing that Sox14 and Sox21 constitute a repressing subgroup (B2) of Group B Sox genes. This provides the first evidence for the occurrence of repressor SOX proteins. Activating (B1) and repressing (B2) subgroups of Group B Sox genes display interesting overlaps of expression domains in developing tissues (e.g. optic tectum, spinal cord, inner ear, alimentary tract, branchial arches). Within each subgroup, most expression domains of Sox1 and -3 are included in those of Sox2 (e.g. CNS, PNS, inner ear), while co-expression of Sox14 and Sox21 occurs in highly restricted sites of the CNS, with the likely temporal order of Sox21 preceding Sox14 (e.g. interneurons of the spinal cord). These expression patterns suggest that target genes of Group B SOX proteins are finely regulated by the counterbalance of activating and repressing SOX proteins (Uchikawa, 1999).
The Komeda miniature rat Ishikawa (KMI) is a naturally occurring mutant caused by an autosomal recessive mutation mri, that exhibits longitudinal growth retardation. The mri mutation has been identified as a deletion in the rat gene encoding cGMP-dependent protein kinase type II (cGKII). KMIs show an expanded growth plate and impaired bone healing with abnormal accumulation of postmitotic but nonhypertrophic chondrocytes. Ex vivo culture of KMI chondrocytes reproduce the differentiation impairment, which was restored by introducing the adenovirus-mediated cGKII gene. The expression of Sox9, an inhibitory regulator of hypertrophic differentiation, persists in the nuclei of postmitotic chondrocytes of the KMI growth plate. Transfection experiments in culture systems reveal that cGKII attenuates the Sox9 functions to induce the chondrogenic differentiation and to inhibit the hypertrophic differentiation of chondrocytes. This attenuation of Sox9 is due to the cGKII inhibition of nuclear entry of Sox9. The impaired differentiation of cultured KMI chondrocytes is restored by the silencing of Sox9 through RNA interference. Hence, the present study for the first time shed light on a novel role of cGKII as a molecular switch, coupling the cessation of proliferation and the start of hypertrophic differentiation of chondrocytes through attenuation of Sox9 function (Chikuda, 2004).
Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. This study demonstrates
induction of pluripotent stem cells from mouse embryonic or adult tail tip fibroblasts (TTFs) by introducing four factors, Oct3/4, Sox2, c-Myc,
and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These
cells, which have been designated iPS (induced pluripotent
stem) cells, exhibit the morphology and growth properties of ES cells and express ES
cell marker genes. Subcutaneous transplantation
of iPS cells into nude mice resulted in tumors containing a variety of tissues from all
three germ layers. Following injection into blastocysts,
iPS cells contributed to mouse embryonic development. These data demonstrate
that pluripotent stem cells can be directly generated
from fibroblast cultures by the addition of only a few defined factors (Takahashi, 2007).
Oct3/4, Sox2, and Nanog have been shown to function
as core transcription factors in maintaining pluripotency. Among the three,
it was found that Oct3/4 and Sox2 are essential for the generation
of iPS cells. c-Myc and Klf4 were also identified as essential factors.
These two tumor-related factors could not be replaced by
other oncogenes including E-Ras, Tcl1, β-catenin, and Stat3 (Takahashi, 2007).
The c-Myc protein has many downstream targets that
enhance proliferation and transformation, many of which may have roles in the generation
of iPS cells. Of note, c-Myc associates with histone
acetyltransferase (HAT) complexes, including TRRAP,
which is a core subunit of the TIP60 and GCN5 HAT complexes, CREB binding protein
(CBP), and p300. Within the mammalian genome, there may be up to 25,000 c-Myc binding
sites, many more than the predicted number of Oct3/4 and Sox2 binding sites. c-Myc protein may induce global histone acetylation, thus allowing Oct3/4 and Sox2 to bind to their specific target loci. Klf4 has been shown to repress p53 directly, and p53 protein has been shown to suppress Nanog during ES cell differentiation. iPS cells showed levels of p53 protein lower than those in MEFs. Thus, Klf4 might contribute
to activation of Nanog and other ES cell-specific genes
through p53 repression. Alternatively, Klf4 might function
as an inhibitor of Myc-induced apoptosis through the repression
of p53 in this system. In contrast, Klf4 activates p21CIP1, thereby suppressing
cell proliferation. This antiproliferation function of Klf4 might be inhibited by c-Myc, which suppresses the expression of p21CIP1. The balance between c-Myc and Klf4 may be important for the generation of iPS cells (Takahashi, 2007).
One question that remains concerns the origin of the iPS cells. With the retroviral expression system, it is estimated that only a small portion of cells expressing the
four factors become iPS cells. The low frequency suggests that rare tissue stem/progenitor cells that coexisted in the fibroblast cultures might have given
rise to the iPS cells. Indeed, multipotent stem cells have
been isolated from skin. These studies showed that ~0.067% of
mouse skin cells are stem cells. One explanation for the
low frequency of iPS cell derivation is that the four factors
transform tissue stem cells. However, it was found that the
four factors induced iPS cells with comparably low efficiency
even from bone marrow stroma, which should be more enriched in mesenchymal stem cells and other multipotent cells. Furthermore, cells induced by the three factors were nullipotent. DNA microarray analyses suggested that iPS-MEF4 cells and iPS-MEF3 cells have the same origin. These results do not favor multipotent tissue stem cells as the origin of iPS cells (Takahashi, 2007).
There are several other possibilities for the low frequency
of iPS cell derivation. First, the levels of the four
factors required for generation of pluripotent cells may
have narrow ranges, and only a small portion of cells expressing
all four of the factors at the right levels can acquire
ES cell-like properties. Consistent with this idea, a mere 50% increase or decrease in Oct3/4 proteins induces differentiation of ES cells. iPS clones
overexpressed the four factors when RNA levels were analyzed,
but their protein levels were comparable to those in
ES cells, suggesting that the iPS clones possess a mechanism (or mechanisms) that
tightly regulates the protein levels of the four factors. It is
speculated that high amounts of the four factors are required
in the initial stage of iPS cell generation, but, once
they acquire ES cell-like status, too much of the factors
are detrimental for self-renewal. Only a small portion of
transduced cells show such appropriate transgene expression. Second, generation of pluripotent cells may require additional chromosomal alterations, which take place spontaneously during culture or are induced by some of the four factors. Although the iPS-TTFgfp4 clones had largely normal karyotypes, the existence of minor chromosomal alterations cannot be ruled out. Site-specific retroviral insertion may also play a role. Southern blot analyses showed that each iPS clone has ~20 retroviral
integrations. Some of these may have caused
silencing or fusion with endogenous genes. Further studies
will be required to determine the origin of iPS cells (Takahashi, 2007).
Another unsolved question is whether the four factors
identified play roles in reprogramming induced by fusion
with ES cells or nuclear transfer into oocytes. Since the four factors are expressed in ES cells at high levels, it is reasonable to speculate that they are involved in the
reprogramming machinery that exists in ES cells. These result
is also consistent with the finding that the reprogramming
activity resides in the nucleus, but not in the cytoplasm,
of ES cells. However, iPS cells were not identical to ES cells, as shown by the global
gene-expression patterns and DNA methylation status. It is possible that additional important factors have been missed. One such candidate is ECAT1, although its forced
expression in iPS cells did not consistently upregulate ES cell marker genes (Takahashi, 2007).
More obscure are the roles of the four factors, especially Klf4 and c-Myc, in the reprogramming observed in oocytes. Both Klf4 and c-Myc are dispensable for preimplantation mouse development. Furthermore, c-myc is not detected in oocytes. In contrast, L-myc is expressed maternally in oocytes. Klf17 and Klf7, but not Klf4, are found in expressed sequence-tag libraries derived from unfertilized mouse eggs. Klf4 and c-Myc might be compensated by these related proteins. It is highly likely that other factors are also required to induce complete reprogramming and totipotency in oocytes (Takahashi, 2007).
Sox2 is expressed in developing foregut endoderm, with highest levels in
the future esophagus and anterior stomach. By contrast, Nkx2.1 (Titf1) is
expressed ventrally, in the future trachea. In humans, heterozygosity for
SOX2 is associated with anopthalmia-esophageal-genital syndrome (OMIM
600992), a condition including esophageal atresia (EA) and tracheoesophageal
fistula (TEF), in which the trachea and esophagus fail to separate. Mouse
embryos heterozygous for the null allele, Sox2EGFP, appear
normal. However, further reductions in Sox2, using Sox2LP
and Sox2COND hypomorphic alleles, result in multiple
abnormalities. Approximately 60% of Sox2EGFP/COND embryos
have EA with distal TEF in which Sox2 is undetectable by immunohistochemistry
or western blot. The mutant esophagus morphologically resembles the trachea,
with ectopic expression of Nkx2.1, a columnar, ciliated epithelium, and very
few p63+ basal cells. By contrast, the abnormal foregut of
Nkx2.1-null embryos expresses elevated Sox2 and p63, suggesting reciprocal regulation of Sox2 and Nkx2.1 during early dorsal/ventral foregut patterning. Organ culture experiments further suggest that FGF signaling from the ventral mesenchyme regulates Sox2 expression in the endoderm. In the 40% Sox2EGFP/COND embryos in which Sox2 levels are ~18% of wild type there is no TEF. However, the esophagus is still abnormal, with luminal mucus-producing cells, fewer p63+ cells, and ectopic expression of genes normally expressed in glandular stomach and intestine. In
all hypomorphic embryos the forestomach has an abnormal phenotype, with
reduced keratinization, ectopic mucus cells and columnar epithelium. These
findings suggest that Sox2 plays a second role in establishing the boundary between the keratinized, squamous esophagus/forestomach and glandular hindstomach (Que, 2007).
The esophagus, trachea and lung develop from the embryonic foregut, yet acquire and maintain distinct tissue phenotypes. Sox2 is necessary for foregut morphogenesis and esophagus development. Sox2 is also required for the normal development of the trachea and lung. In both the embryo and adult, Sox2 is exclusively expressed in the epithelium of the trachea and airways. An Nkx2.5-Cre transgene and a Sox2 floxed allele were used to conditionally delete Sox2 in the ventral epithelial domain of the early anterior foregut, which gives rise to the future trachea and lung buds. All conditional mutants die of respiratory distress at birth, probably due to abnormal differentiation of the laryngeal and tracheal cartilage as a result of defective epithelial-mesenchymal interaction. About 60% of the mutants have a short trachea, suggesting that the primary budding site of the lung shifts anteriorly. In the tracheal epithelium of all conditional mutants there are significantly more mucus-producing cells compared with wild type, and fewer basal stem cells, ciliated and Clara cells. Differentiation of the epithelium lining the conducting airways in the lung is abnormal, suggesting that Sox2 also plays a role in the differentiation of embryonic airway progenitors into specific lineages. Conditional deletion of Sox2 was then used to test its role in adult epithelium maintenance. It was found that epithelial cells, including basal stem cells, lacking Sox2 show a reduced capacity to proliferate in culture and to repair after injury in vivo. Taken together, these results define multiple roles for Sox2 in the developing and adult trachea (Que, 2009).
The mammalian hair represents an unparalleled model system to understand both developmental processes and stem cell biology. The hair follicle consists of several concentric epithelial sheaths with the outer root sheath (ORS) forming the outermost layer. Functionally, the ORS has been implicated in the migration of hair stem cells from the stem cell niche toward the hair bulb. However, factors required for the differentiation of this critical cell lineage remain to be identified. This study describes an unexpected role of the HMG-box-containing gene Sox9 in hair development. Sox9 expression can be first detected in the epithelial component of the hair placode but then becomes restricted to the outer root sheath (ORS) and the hair stem cell compartment (bulge). Using tissue-specific inactivation of Sox9, it was demonstrated that this gene serves a crucial role in hair differentiation and that skin deleted for Sox9 lacks external hair. Strikingly, the ORS acquires epidermal characteristics with ectopic expression of GATA3. Moreover, Sox9 knock hair show severe proliferative defects and the stem cell niche never forms. Finally, this study shows that Sox9 expression depends on sonic hedgehog (Shh) signaling and demonstrates overexpression in skin tumors in mouse and man. It is concluded that although Sox9 is dispensable for hair induction, it directs differentiation of the ORS and is required for the formation of the hair stem cell compartment. Genetic analysis places Sox9 in a molecular cascade downstream of sonic hedgehog and suggests that this gene is involved in basal cell carcinoma (Vidal, 2005).
The dermal papilla comprises the specialised mesenchymal cells at the base of the hair follicle. Communication between dermal papilla cells and the overlying epithelium is essential for differentiation of the hair follicle lineages. Sox2 is expressed in all dermal papillae at E16.5, but from E18.5 onwards expression is confined to a subset of dermal papillae. In postnatal skin, Sox2 is only expressed in the dermal papillae of guard/awl/auchene follicles, whereas CD133 is expressed both in guard/awl/auchene and in zigzag dermal papillae. Using transgenic mice that express GFP under the control of the Sox2 promoter, Sox2+ (GFP+) CD133+ cells were isolated and compared with Sox2- (GFP-) CD133+ dermal papilla cells. In addition to the 'core' dermal papilla gene signature, each subpopulation expressed distinct sets of genes. GFP+ CD133+ cells had upregulated Wnt, FGF and BMP pathways and expressed neural crest markers. In GFP- CD133+ cells, the hedgehog, IGF, Notch and integrin pathways were prominent. In skin reconstitution assays, hair follicles failed to form when dermis was depleted of both GFP+ CD133+ and GFP- CD133+ cells. In the absence of GFP+ CD133+ cells, awl/auchene hairs failed to form and only zigzag hairs were found. This study has thus demonstrated a previously unrecognised heterogeneity in dermal papilla cells and shown that Sox2-positive cells specify particular hair follicle types (Driskell, 2009).
In many species, the Sox2 transcription factor is a marker of the
nervous system from the beginning of its development, and Sox2 is expressed in embryonic neural stem cells. It is
also expressed in, and is essential for, totipotent inner cell mass stem cells
and other multipotent cell lineages, and its ablation causes early embryonic
lethality. To investigate the role of Sox2 in the nervous system, different mouse mutant alleles were generated: a null allele
(Sox2ß-geo `knock-in'), and a regulatory mutant
allele (Sox2DeltaENH), in which a neural cell-specific
enhancer is deleted. Sox2 is expressed in embryonic early neural
precursors of the ventricular zone and, in the adult, in ependyma (a
descendant of the ventricular zone). It is also expressed in the vast majority
of dividing precursors in the neurogenic regions, and in a small proportion of
differentiated neurons, particularly in the thalamus, striatum and septum.
Compound Sox2ß-geo/DeltaENH heterozygotes show
important cerebral malformations, with parenchymal loss and ventricle
enlargement, and L-dopa-rescuable circling behaviour and epilepsy. Striking abnormalities were observed in neurons; degeneration and cytoplasmic protein
aggregates, a feature common to diverse human neurodegenerative diseases, are
observed in thalamus, striatum and septum. Furthermore, ependymal cells show
ciliary loss and pathological lipid inclusions. Finally, precursor cell
proliferation and the generation of new neurons in adult neurogenic regions
are greatly decreased, and GFAP/nestin-positive hippocampal cells, which
include the earliest neurogenic precursors, are strikingly diminished. These
findings highlight a crucial and unexpected role for Sox2 in the
maintenance of neurons in selected brain areas, and suggest a contribution of
neural cell proliferative defects to the pathological phenotype (Ferri, 2004).
The otic placode, a specialized region of ectoderm, gives rise to components of the inner ear and shares many characteristics with the neural crest, including expression of the key transcription factor Sox10. This study shows that in avian embryos, a highly conserved cranial neural crest enhancer, Sox10E2, also controls the onset of Sox10 expression in the otic placode. Interestingly, this study showed that different combinations of paralogous transcription factors (Sox8, Pea3 and cMyb versus Sox9, Ets1 and cMyb) are required to mediate Sox10E2 activity in the ear and neural crest, respectively. Mutating their binding motifs within Sox10E2 greatly reduces enhancer activity in the ear. Moreover, simultaneous knockdown of Sox8, Pea3 and cMyb eliminates not only the enhancer-driven reporter expression, but also the onset of endogenous Sox10 expression in the ear. Rescue experiments confirm that the specific combination of Myb together with Sox8 and Pea3 is responsible for the onset of Sox10 expression in the otic placode, as opposed to Myb plus Sox9 and Ets1 for neural crest Sox10 expression. Whereas SUMOylation of Sox8 is not required for the initial onset of Sox10 expression, it is necessary for later otic vesicle formation. This new role of Sox8, Pea3 and cMyb in controlling Sox10 expression via a common otic/neural crest enhancer suggests an evolutionarily conserved function for the combination of paralogous transcription factors in these tissues of distinct embryological origin (Betancur, 2011).
The highly related transcription factors Sox4 and Sox11 are expressed in the developing sympathetic nervous system. In the mouse, Sox11 appears first, whereas Sox4 is prevalent later. Using mouse mutagenesis and overexpression strategies in chicken, the role of both SoxC proteins was studed in this tissue. Neither Sox4 nor Sox11 predominantly functioned by promoting pan-neuronal or noradrenergic differentiation of sympathetic neurons as might have been expected from studies in neuronal precursors of the central nervous system. The transcriptional network that regulates the differentiation of sympathetic neurons remained intact and expression of noradrenergic markers showed only minor alterations. Instead, Sox11 was required in early sympathetic ganglia for proliferation of tyrosine hydroxylase-expressing cells, whereas Sox4 ensured the survival of these cells at later stages. In the absence of both Sox4 and Sox11, sympathetic ganglia remained hypoplastic throughout embryogenesis because of consecutive proliferation and survival defects. As a consequence, sympathetic ganglia were rudimentary in the adult and sympathetic innervation of target tissues was impaired leading to severe dysautonomia (Potzner, 2010).
The optic vesicle is a multipotential primordium of the retina, which becomes subdivided into the neural retina and retinal pigmented epithelium domains. Although the roles of several paracrine factors in patterning the optic vesicle have been studied extensively, little is known about cell-autonomous mechanisms that regulate coordinated cell morphogenesis and cytodifferentiation of the retinal pigmented epithelium. This study demonstrates that members of the SoxB1 gene family, Sox1, Sox2 and Sox3, are all downregulated in the presumptive retinal pigmented epithelium. Constitutive maintenance of SoxB1 expression in the presumptive retinal pigmented epithelium both in vivo and in vitro resulted in the absence of cuboidal morphology and pigmentation, and in concomitant induction of neural differentiation markers. It was also demonstrated that exogenous Fgf4 inhibits downregulation of all SoxB1 family members in the presumptive retinal pigment epithelium. These results suggest that retinal pigment epithelium morphogenesis and cytodifferentiation requires SoxB1 downregulation, which depends on the absence of exposure to an FGF-like signal (Ishii, 2009).
Neural precursors in the developing olfactory epithelium (OE) give rise to three major neuronal classes - olfactory receptor (ORNs), vomeronasal (VRNs) and gonadotropin releasing hormone (GnRH) neurons. Nevertheless, the molecular and proliferative identities of these precursors are largely unknown. Two precursor classes were characterized in the olfactory epithelium (OE) shortly after it becomes a distinct tissue at midgestation in the mouse: slowly dividing self-renewing precursors that express Meis1/2 at high levels, and rapidly dividing neurogenic precursors that express high levels of Sox2 and Ascl1. Precursors expressing high levels of Meis genes primarily reside in the lateral OE, whereas precursors expressing high levels of Sox2 and Ascl1 primarily reside in the medial OE. Fgf8 maintains these expression signatures and proliferative identities. Using electroporation in the wild-type embryonic OE in vitro as well as Fgf8, Sox2 and Ascl1 mutant mice in vivo, it was found that Sox2 dose and Meis1 -- independent of Pbx co-factors -- regulate Ascl1 expression and the transition from lateral to medial precursor state. Thus, proliferative characteristics and a dose-dependent transcriptional network were characterized that define distinct OE precursors: medial precursors that are most probably transit amplifying neurogenic progenitors for ORNs, VRNs and GnRH neurons, and lateral precursors that include multi-potent self-renewing OE neural stem cells (Tucker, 2010).
Neural progenitors of the vertebrate CNS are defined by generic cellular characteristics, including their pseudoepithelial morphology and their ability to divide and differentiate. SOXB1 transcription factors, including the three closely related genes Sox1, Sox2, and Sox3, universally mark neural progenitor and stem cells throughout the vertebrate CNS. Constitutive expression of SOX2 inhibits neuronal differentiation and results in the maintenance of progenitor characteristics. Conversely, inhibition of SOX2 signaling results in the delamination of neural progenitor cells from the ventricular zone and exit from cell cycle, which is associated with a loss of progenitor markers and the onset of early neuronal differentiation markers. The phenotype elicited by inhibition of SOX2 signaling can be rescued by coexpression of SOX1, providing evidence for redundant SOXB1 function in CNS progenitors. Taken together, these data indicate that SOXB1 signaling is both necessary and sufficient to maintain panneural properties of neural progenitor cells (Graham, 2003).
Collectively, several lines of evidence suggest that the members of the SOXB1 subfamily are functionally redundant. (1) Microinjection of dominant-negative forms of Sox2 mRNA in Xenopus that inhibit neural differentiation of animal caps can be rescued by injection of Sox3 but not divergent Sox genes such as Sox9 and SoxD. (2) Midline glial defects in Drosophila Dichaete mutants can be rescued by directed expression of SOX1 and SOX2 proteins. (3) The elimination of both members of the Drosophila SOXB subfamily, SoxNeuro and Dichaete, simultaneously results in much more severe phenotypes in the neuroectoderm than the single mutants. Thus, functional redundancy appears to be confined to SOXB1 subfamily and does not extend to more divergent SOX family members. The data provides further evidence that the members of the SOXB1 subfamily are functionally redundant: the phenotypic consequences of the inhibition of SOX2 signaling in chick neural progenitors can be rescued by the coexpression of SOXB1 subfamily member SOX1, and the forced expression of SOX1 in CNS cells phenocopies forced expression of SOX2 (Graham, 2003).
There is now increasing evidence that SOX factors may play a global role in maintaining progenitor/stem cell fates in a variety of tissues including the nervous system. Members of the SOX gene family are expressed in a variety of embryonic and adult tissues where their expression, and in some cases function, is associated with the specification and/or maintenance of progenitor identity. For example, SRY is transiently expressed in the progenitor of Sertoli cells of the XY genital ridge and is responsible for triggering development of the male phenotype. SOX9 is expressed in immature chondrocytes and plays a role in their proliferation and differentiation. Intriguingly, a recent report describing the function of SOX10 in the PNS reveals many functional parallels between the role of SOX10 in the PNS stem/progenitor cells and those described here for SOXB1 factors in CNS progenitor cells. SOX10 is expressed in multipotent neural crest stem cells and is downregulated during their neuronal differentiation. Forced expression of SOX10 is able to override both antigliogenic activity of BMP2 and antineurogenic (antiproliferative) activity of TGFbeta and thus maintain multipotential differentiation capacity of NCSCs. Furthermore, by directly inhibiting terminal neuronal differentiation, SOX10 appears to provide a permissive environment for glial differentiation. It will be interesting to determine if any of the molecular pathways by which SOX10 maintains neural crest stem cell fate in the PNS are also used in the CNS (Graham, 2003 and references therein).
Purified rat oligodendrocyte precursor cells (OPCs) can be induced by extracellular signals to convert to multipotent neural stem-like cells (NSLCs), which can then generate both neurons and glial cells. Because the conversion of precursor cells to stem-like cells is of both intellectual and practical interest, it is important to understand its molecular basis. The conversion of OPCs to NSLCs depends on the reactivation of the sox2 gene, which in turn depends on the recruitment of the tumor suppressor protein Brca1 and the chromatin-remodeling protein Brahma (Brm) to an enhancer in the sox2 promoter. Moreover, the conversion is associated with the modification of Lys 4 and Lys 9 of histone H3 at the same enhancer. These findings suggest that the conversion of OPCs to NSLCs depends on progressive chromatin remodeling, mediated in part by Brca1 and Brm (Kondo, 2004).
Pluripotent embryonic stem (ES) cells can generate all cell types, but how cell lineages are initially specified and maintained during development remains largely unknown. Different classes of Sox transcription factors are expressed during neurogenesis and have been assigned important roles from early lineage specification to neuronal differentiation. This study characterize the genome-wide binding for Sox2, Sox3, and Sox11, which have vital functions in ES cells, neural precursor cells (NPCs), and maturing neurons, respectively. The data demonstrate that Sox factor binding depends on developmental stage-specific constraints and reveal a remarkable sequential binding of Sox proteins to a common set of neural genes. Interestingly, in ES cells, Sox2 preselects for neural lineage-specific genes destined to be bound and activated by Sox3 in NPCs. In NPCs, Sox3 binds genes that are later bound and activated by Sox11 in differentiating neurons. Genes prebound by Sox proteins are associated with a bivalent chromatin signature, which is resolved into a permissive monovalent state upon binding of activating Sox factors. These data indicate that a single key transcription factor family acts sequentially to coordinate neural gene expression from the early lineage specification in pluripotent cells to later stages of neuronal development (Bergsland, 2011).
During development of the CNS, neurons and glia are generated from self-renewing neural progenitor cells (NPCs) that are directed to leave the cell cycle, down-regulate progenitor identities, and activate neuronal or glial gene expression in a spatially and temporally defined manner. The mechanisms regulating gene expression in NPCs and their differentiated progeny have been extensively characterized, but it is largely unknown how neural lineage-specific gene expression programs are initially specified and activated during the course of differentiation (Bergsland, 2011).
An important property of pluripotent stem cells is their capacity to induce gene programs characteristic of all cell lineages. Previous studies in embryonic stem (ES) cells have demonstrated that many genes destined to become activated at later stages of development are already bound by ES cell regulatory transcription factors, including Sox2, Oct4, Nanog, and FoxD3. Moreover, genes poised for activation are often associated with bivalent histone domains consisting of repressive histone modifications combined with modifications associated with transcriptional activation (H3K27me3 and H3K4me3). Bivalent histone marks are subsequently resolved as genes become activated or terminally repressed during development. Together, these findings indicate that many silent genes in ES cells are prebound by transcription factors and epigenetically prepared for activation, but they do not demonstrate how lineage-specific gene expression programs are initially selected and later activated. Insights into these questions come from studies of the liver-specific enhancer Alb1. In ES cells, the Alb1 enhancer is prebound by FoxD3, which ensures the assembly of permissive chromatin. Interestingly, upon endodermal differentiation, FoxD3 binding is replaced by FoxA1, which helps to induce Alb1 expression in a liver-specific manner. Studies of the B-cell-specific lambda5-VpreB1 locus constitute an additional example of how a transcription factor prepares the enhancer for later activation by an alternative member of the same transcription factor family. This locus contains an intergenic enhancer to which Sox2 binds and adds an epigenetic active mark in ES cells. In pro-B cells, Sox2 binding is replaced by Sox4, which leads to the activation of lambda5 expression. Although these studies indicate the importance of pioneering functions of transcription factors, experiments are focused on specific enhancers in individual genes, and it remains unclear whether the sequential regulatory strategy is a more general requirement for activation of larger sets of gene batteries in differentiating cell lineages (Bergsland, 2011).
Apart from the above-mentioned gene regulatory functions in ES cells and roles in early B-cell development, transcription factors of the Sox gene family have important sequential roles in regulating the maintenance and differentiation of progenitor cells from early pluripotent stages to late steps of neurogenesis. Sox2 is necessary for the establishment and maintenance of ES cells. All three SoxB1 proteins (Sox1, Sox2, and Sox3) are expressed in most neural precursors in both the developing and adult CNS, and studies conducted in chick and mouse embryos demonstrate that they act redundantly to maintain neural cells in a progenitor state and counteract neuronal differentiation. The SoxC proteins (Sox4, Sox11, and Sox12) are expressed complementary to Sox1-3 in the developing CNS and can mostly be detected in post-mitotic differentiating neurons. Misexpression experiments in chicks demonstrate that SoxC proteins have the opposite function compared with Sox1-3 and can induce the expression of neuronal proteins, whereas deletion of the SoxC proteins in the embryonic mouse spinal cord leads to a significant decrease in differentiated neurons and an associated increased cell death (Bergsland, 2011).
Despite the importance of Sox factors during the course of neural development, there is very limited information concerning the control of appropriate gene expression programs that are activated in CNS progenitors and their differentiated progeny. This is partly due to the limited number of identified Sox target genes. This study analyzed Sox transcription factors during neural lineage development by generating and comparing genome-wide binding data for Sox2, Sox3, and Sox11 from early lineage specification stages with the onset of neuronal gene expression. The data indicate that sequentially acting Sox transcription factors control neural lineage-specific gene expression by predisposing gene programs to become activated in NPCs and during neuronal and glial differentiation (Bergsland, 2011).
The HMG-Box transcription factor SOX2 is expressed in neural progenitor populations throughout the developing and adult central nervous system and is necessary to maintain their progenitor identity. However, it is unclear whether SOX2 levels are uniformly expressed across all neural progenitor populations. In the developing dorsal telencephalon, two distinct populations of neural progenitors, radial glia and intermediate progenitor cells, are responsible for generating a majority of excitatory neurons found in the adult neocortex. This study demonstrates, using both cellular and molecular analyses, that SOX2 is differentially expressed between radial glial and intermediate progenitor populations. Moreover, utilizing a SOX2(EGFP) mouse line, this differential expression can be used to prospectively isolate distinct, viable populations of radial glia and intermediate cells for in vitro analysis. Given the limited repertoire of cell-surface markers currently available for neural progenitor cells, this provides an invaluable tool for prospectively identifying and isolating distinct classes of neural progenitor cells from the central nervous system (Button, 2011).
The vertebrate forebrain is patterned during gastrulation into telencephalic, retinal, hypothalamic and diencephalic primordia. Specification of each of these domains requires the concerted activity of combinations of transcription factors (TFs). Paradoxically, some of these factors are widely expressed in the forebrain, which raises the question of how they can mediate regional differences. To address this issue, focus was placed on the homeobox TF Six3.2. With genomic and functional approaches it was demonstrated that, in medaka fish, Six3.2 regulates, in a concentration-dependent manner, telencephalic and retinal specification under the direct control of Sox2. Six3.2 and Sox2 have antagonistic functions in hypothalamic development. These activities are, in part, executed by Foxg1 and Rx3, which seem to be differentially and directly regulated by Six3.2 and Sox2. Together, these data delineate the mechanisms by which Six3.2 diversifies its activity in the forebrain and highlight a novel function for Sox2 as one of the main regulators of anterior forebrain development. They also demonstrate that graded levels of the same TF, probably operating in partially independent transcriptional networks, pattern the vertebrate forebrain along the anterior-posterior axis (Beccari, 2012).
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