As a POU domain transcription factor, VVL is related to an evolutionary complex group of genes consisting of at least 5 classes. Pit-1 is a class one POU domain transcription factor. Pit-1, involved in the development of the anterior pituitary gland in mammals. (Billin, 1995 and Anderson, 1995). Class II POU domain transcription factors include mammalian Oct1, Oct2, Oct11 and Drosophila PDM-1 and PDM-2. Mammalian Brn1, Brn2, Brn4, SCIP/Oct6 and Xenopus XLPOU1 and XLPOU2 as well as Drosophila Ventral veins lacking (Drifter/Cf1a) and C. elegans ceh6 are Class III proteins. I-POU is in POU domain group IV, along with C. elegans unc86 and vertebrate Brn3 (Verrijzer, 1993). Oct-3/4 is a class V POU domain protein. There is no known Drosophila class 1 or class V homolog.

The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for 'POU' domain signatures.

A class III POU factor in C. elegans

C. elegans has three POU homeobox genes, unc-86, ceh-6 and ceh-18. ceh-6 is the ortholog of vertebrate Brn1, Brn2, SCIP/Oct6 and Brn4 and fly Cf1a/drifter/ventral veinless. Comparison of C. elegans and C. briggsae CEH-6 shows that it is highly conserved. C. elegans has only three POU homeobox genes, while Drosophila has five that fall into four families. Immunofluorescent detection of the CEH-6 protein reveals that it is expressed in particular head and ventral cord neurons, as well as in rectal epithelial cells, and in the excretory cell, which is required for osmoregulation. A deletion of the ceh-6 locus causes 80% embryonic lethality. During morphogenesis, embryos either extrude cells in the rectal region of the tail or rupture, indicative of a defect in the rectal epithelial cells that express ceh-6. Those embryos that hatch are sick and develop vacuoles, a phenotype similar to that caused by laser ablation of the excretory cell. A GFP reporter construct expressed in the excretory cell reveals inappropriate canal structures in the ceh-6 null mutant. Members of the POU-III family are expressed in tissues involved in osmoregulation and secretion in a number of species. It is proposed that one evolutionary conserved function of the POU-III transcription factor class could be the regulation of genes that mediate secretion/osmoregulation (Burglin, 2001).

ceh-6 is expressed in many different neuronal cell types. This is comparable with POU-III genes in Drosophila and vertebrates. In these organisms, the genes Brn1, Brn2, SCIP and Brn4 are also expressed in many different tissue types in the brain. Owing to the complexity of the vertebrate brain, it is difficult to make direct comparisons to the precisely defined neurons in C. elegans, but it seems clear that the vertebrate orthologs are expressed in some of the same different types of neurons, i.e. motoneurons, sensory neurons and interneurons. Furthermore, in vertebrates, expression is observed in the spinal cord. Both, ceh-6 and dfr/vvl are expressed in the ventral cord, while several of the vertebrate POU-III genes are expressed in the developing neural tube and the spinal cord. ceh-6 is transiently expressed in the ventral cord, and, for example, the vertebrate factor SCIP has also been shown to be transiently expressed in Schwann cells of the PNS. Although the phenotypes of ceh-6 in the nervous system were not examined, due to the early defects, it is nevertheless suspected that ceh-6 plays an important role in that system, like its cousin, unc-86 (Burglin, 2001).

The studies in Drosophila of dfr/vvl have mainly focused on the role in the developing tracheal system, since, similar to C. elegans, the mutants do not live long enough for an examination of their nervous system defects. dfr/vvl plays an important role during the development of the trachea, in particular for the differentiation and migration of these cells, and thus it might directly or indirectly regulate cell surface molecules. dfr/vvl is also expressed in the Filzkorper, the area where the tracheal system attaches to the cuticle in the rectal area of the fly. ceh-6 is required for the proper differentiation of the rectal cells, and it may directly or indirectly regulate cell surface molecules. Further, ceh-6 seems to regulate the outgrowth of the excretory canals. Thus, ceh-6 may regulate the same types of molecules in epithelial cells that are regulated by dfr/vvl. In vertebrates, POU-III genes have also been implicated in regulating cell surface molecules. For example, Brn2 directly regulates the expression of the cell surface adhesion molecule Po. In addition, Oct6 has been shown to be expressed in the epidermis, as well as other squamous epithelia. Thus, expression in subsets of epithelial cells appears to be another conserved feature of POU-III homeobox genes (Burglin, 2001).

A class III POU factor in other invertebrates

Three different POU-domain-containing genes have been found within the genome of the flatworm, Dugesia tigrina. Two of these genes appear to be close homologs of class-III POU genes previously identified in Dugesia japonica, while the third represents a new class-III POU gene. Nucleotide sequences encoding highly related POU domains within the two species of planaria exhibit a surprisingly poor degree of shared identity. To date, only class-III POU genes have been observed in flatworms. It is possible these genes may represent ancestral versions of the multiple classes of POU genes that now exist in vertebrates; if so, they may play important roles in the development of metazoan nervous tissue (Stuart, 1995).

Class III POU factor in fish

Zebrafish ZFPOU1 cDNA contains an open reading frame encoding a 425 amino acid peptide. The conserved POU domain is located near the carboxy terminus. The amino acid sequence is most similar to that of the mouse class III POU-domain gene, Brain-1. ZFPOU1 transcripts first appear at the early neurula stage of embryogenesis and transiently increase thereafter. However, a significant level of expression is not found in adult tissues, except in the brain. ZFPOU1 transcripts are localized in the neural tissues of embryos, but not in mesodermal, endodermal or ectodermal tissues. In adult zebrafish, the ZFPOU1 transcripts are detected in the restricted regions of the brain. Spatial and temporal expression patterns suggest that ZFPOU1 carries out distinct roles in the early neural development of zebrafish (Matsuzaki, 1992).

Zebrafish zp-50 class III POU domain gene is first activated in the prospective diencephalon after the end of the gastrula period. During somitogenesis, zp-50 is expressed in a very dynamic and complex fashion in all major subdivisions of the central nervous system. After one day of development, zp-50 transcripts are present in the fore- and midbrain in several distinct cell clusters. In the hindbrain, zp-50 expression is found in two types of domains. Correct zp-50 expression in the ventral fore- and midbrain requires genes known to be involved in dorsoventral patterning of the zebrafish CNS. Transcripts of the sonic hedgehog gene encoding an intercellular signaling molecule are detected in the forming diencephalon shortly prior to the appearance of zp-50 mRNA. Correct expression in this region of both shh, and zp-50, requires a functional cyclops locus: shh and zp-50 transcripts are likewise absent from the ventral rostral brain of mutant cyc-/- embryos. Injection of synthetic shh mRNA into fertilized eggs causes ectopic zp-50 expression at more dorsal positions of the embryonic brain. The close spatial and temporal coincidence of expression in the rostral brain, the similar response to the cyc- mutation, and the ectopic zp-50 expression in the injection experiments all suggest that zp-50 may directly respond to the reception of the Shh signal (Hauptmann, 1996).

The adult fish brain undergoes continuous neurogenesis and retains the capacity to regenerate. However, the cellular and molecular basis of this process is not well understood. A Brain-1-related, class III POU domain gene, tai-ji, has been characterized in the developing and adult zebrafish, as well as in a human cell line, hNT2. During development, as differentiation occurs, the expression of tai-ji is downregulated in the notochord, muscle, nervous system and dorsal fin. Similarly, tai-ji is expressed in the human neuronal precursor cell, hNT2, but is downregulated upon differentiation with retinoic acid. In the adult zebrafish nervous system, tai-ji persists in germinal zones, including cells in the germinal zone of the retina, the basal cells of the olfactory epithelium and cells of the subependymal zones in the optic tectum and telencephalon. Subsets of the tai-ji-expressing cells in these regions incorporate BrdU. Most of the tai-ji-expressing cells within these regions of the zebrafish brain are not differentiated and do not express a marker for post-mitotic neurons (acetylated tubulin) nor do they express a marker for glial cells [glial acidic fibrillary protein (GFAP)]. It is proposed that the majority of the tai-ji-expressing cells are neural stem or progenitor cell populations that may represent the cellular basis for continuous growth in the adult nervous system (Huang, 1998).

The development of multicellular animals is initially controlled by maternal gene products deposited in the oocyte. During the maternal-to-zygotic transition, transcription of zygotic genes commences, and developmental control starts to be regulated by zygotic gene products. In Drosophila, the transcription factor Zelda specifically binds to promoters of the earliest zygotic genes and primes them for activation. It is unknown whether a similar regulation exists in other animals. This study found that zebrafish Pou5f1, a homolog of the mammalian pluripotency transcription factor Oct4, occupies SOX-POU binding sites before the onset of zygotic transcription and activates the earliest zygotic genes. These data position Pou5f1 and SOX-POU sites at the center of the zygotic gene activation network of vertebrates and provide a link between zygotic gene activation and pluripotency control (Leichsenring, 2013).

A class III POU factor in Xenopus

Two POU domain genes have been cloned from Xenopus laevis: XLPOU 1 and XLPOU 2. These POU domains have greater than 90% homology with that of the POU III class of proteins. XLPOU 1 gene expression begins at the neural plate stage, while XLPOU 2 gene expression is first detected at the neurula stage; both XLPOU 1 and XLPOU 2 transcripts continue to be expressed throughout development. In adults, XLPOU 1 expression is restricted to the skin and brain, while XLPOU 2 is observed in the kidney and brain. In dissected embryos, both XLPOU 1 and XLPOU 2 are expressed in the dorsoanterior portion of an early tailbud embryo. Consistent with this localization, uv treatment, a condition that "posteriorizes" embryos, greatly reduces the expression of XLPOU 1 and XLPOU 2. Whole-mount in situ hybridization demonstrates that at the neural fold stage, XLPOU 1 transcripts appear to be localized primarily in the anterior neural plate. In sections of embryos, in situ hybridization shows that XLPOU 1 transcripts are localized mostly in the anterior region of the nerve cord of neurula stage embryos. In tailbud stage embryos, the XLPOU 1 transcripts are found predominantly in the eye and brain, with weak expression along the length of the nerve cord. XLPOU 1 and XLPOU 2, because of their localized and early expression in embryos, may play an important role in the specification of neuronal phenotypes (Agarwal, 1991).

XIPOU 2, a member of the class III POU domain family, is expressed initially in Spemann's organizer, and later, in discrete regions of the developing nervous system in Xenopus laevis. XIPOU 2 may act downstream from initial neural induction events, since it is activated by the neural inducer, noggin. To determine if XIPOU 2 participates in the early events of neurogenesis, synthetic mRNA was microinjected into specific blastomeres of the 32-cell stage embryo. Misexpression of XIPOU 2 in the epidermis causes a direct switch in cell fate from an epidermal to a neuronal phenotype. In the absence of mesoderm induction, XIPOU 2 has the ability to induce a neuronal phenotype in uncommitted ectoderm. These data demonstrate the potential of XIPOU 2 to act as a master regulator of neurogenesis (Witta, 1995).

The POU domain gene, XlPOU 2, acts as a transcriptional activator during mid-gastrulation in Xenopus. Overexpression or misexpression of VP16-POU-GR, a fusion protein consisting of the strong activator domain of VP16 and the POU domain of XlPOU 2, results in ectopic expression of the neural-specific genes, nrp-1, en-2, and beta-tubulin. In contrast, overexpressing a dominant-inhibitory form of XlPOU 2 inhibits the chordin-induced neuralization of uncommitted ectoderm, and results in a loss of nrp-1 and en-2 expression in embryos. Furthermore, in uncommitted ectoderm, XlPOU 2 regulates the developmental neural program that includes a number of pre-pattern genes and at least one proneural gene, X-ngnr-1, thus playing a key role during neural determination. Thus XlPOU 2 is upstream from X-ngnr-1 (atonal-related), a neuronal determinant gene expressed in all primary neurons. Although XlPOU 2 induces X-ngnr-1 readily, X-ngnr-1 is not capable of inducing XlPOU 2 in an animal cap induction assay. XlPOU 2's activation of X-ngnr-1, then leads to the induction of beta-tubulin (Matsuo-Takasaki, 1999).

The activation of pre-pattern genes occurs at the beginning of gastrulation, and thus they are likely candidates to work upstream from XlPOU 2, which is not expressed in the neuroectoderm until mid-gastrulation. These data provide evidence for the potential cross-regulation that might occur between XlPOU 2 and the Zic genes during neural determination. The induction of XlPOU 2 by both Zic 3 and by Zic r1 was investigated after the overexpression of these genes in animal cap ectoderm. Both genes are capable of neuralizing animal cap ectoderm, as evidenced by the activation of nrp-1. They are also capable of weakly inducing XlPOU 2. Although XlPOU 2 cannot be responsible for the initial activation of the pre-pattern genes at the beginning of gastrulation, it is likely that XlPOU 2 may enhance pre-pattern gene expression later in gastrulation (Matsuo-Takasaki, 1999).

Expression of mammalian class III POU factors

Transcripts of a member of the POU-III class of the POU domain gene family, referred to as Brn-4, are initially widely expressed at all levels of the developing neural tube, but in contrast to these other POU-III genes, are subsequently restricted to only a few regions of the adult forebrain, including the supraoptic and paraventricular nuclei of the hypothalamus. Brn-4 was shown to bind to DNA sequences containing the octamer motif and to trans-activate promoters containing this DNA binding motif, based on the actions of a unique N-terminal information. This ontogenic pattern of Brn-4 expression in concert with that of Oct-2 and Pit-1, indicates that certain POU domain genes have the potential to exert their primary functions widely during early neural development, and in a very limited set of neurons in the mature brain (Mathis, 1992).

Direct conversion of fibroblasts to functional neurons by defined factors

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. It was hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, was found to suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine (Vierbuchen, 2010).

Class III POU factor transcriptional targets

The melanoma-specific octamer factor Brn-2/N-Oct3 is a POU domain protein previously known to be expressed in adult brain and in the developing nervous system. N-Oct3 mRNA has been detected in a range of human melanoma cell lines and is elevated approximately 10-fold, as compared to normal human melanocytes; mRNA for Brn-2 has also been detected in a mouse melanoblast cell line. Expression of Brn-2/N-Oct3, in melanoma cells in cotransfection assays activates the expression of the MHC class II DR alpha promoter but represses the activity of the melanocyte-specific tyrosinase promoter. Repression correlates with Brn-2/N-Oct3 binding in a mutually exclusive fashion with basic-helix-loop-helix-leucine-zipper (bHLH-LZ) transcription factor USF in vitro and with Brn-2 expression preventing activation of the tyrosinase promoter by the bHLH-LZ factor Microphthalmia in vivo (Eisen, 1995).

Transcription of a striatal dopamine gene transcription is regulated by Brn-4. Brn-4, a POU transcription factor, and member of the POU-III sublass of POU domain proteins that includes Drosophila Drifter, is expressed in the central nervous system. A functional Brn-4 responsive element in the intron of D1A dopamine receptor gene regulates D1A dopamine receptor gene expression. Brn-4 and DiA coexpressing neurons are found in the striatum in striosomes of the caudate-putamen with a small number in the matrix (Okazawa, 1996).

The mammalian proglucagon gene is expressed in a highly restricted tissue-specific manner in the alpha cells of the pancreatic islet, the hypothalamus, and the small and large intestines. Proglucagon is processed to glucagon and glucagon-like peptides GLP-1 and -2. Glucagon is expressed in alpha cells and regulates glucose homeostasis. GLP-1 is implicated in the control of insulin secretion, food intake, and satiety signaling, and GLP-2 is implicated in regulating small-bowel growth. Cell-specific expression of the proglucagon gene is mediated by proteins that interact with the proximal G1 promoter element, which contains several AT-rich domains with binding sites for homeodomain transcription factors. In an attempt to identify major homeodomain proteins involved in pancreatic alpha-cell-specific proglucagon expression, it was found that the POU domain transcription factor brain 4 is abundantly expressed in proglucagon-producing islet cell lines and rat pancreatic islets. In the latter, brain 4 and glucagon immunoreactivity colocalize in the outer mantle of the islets. Electrophoretic mobility shift assays with specific antisera identify brain 4 as a major constituent of nuclear proteins of glucagon-producing cells that bind to the G1 element of the proglucagon gene proximal promoter. Transcriptional transactivation experiments reveal that brain 4 is a major regulator of proglucagon gene expression by its interaction with the G1 element. The finding that a neuronal transcription factor is involved in glucagon gene transcription may explain the presence of proglucagon in certain areas of the brain as well as in pancreatic alpha cells. This finding supports the idea that the neuronal properties of endodermis-derived endocrine pancreatic cells may find their basis in regulation of gene expression by neuronal transcription factors (Hussain, 1997).

The transcription factor Pou3f1 promotes neural fate commitment via activation of neural lineage genes and inhibition of external signaling pathways

The neural fate commitment of pluripotent stem cells requires the repression of extrinsic inhibitory signals and the activation of intrinsic positivetranscription factors. However, how these two events are integrated to ensure appropriate neural conversion remains unclear. This study showed that Pou3f1 is essential for the neural differentiation of mouse embryonic stem cells (ESCs), specifically during the transition from epiblast stem cells (EpiSCs) to neural progenitor cells (NPCs). Chimeric analysis showed that Pou3f1 knockdown leads to a markedly decreased incorporation of ESCs in the neuroectoderm. By contrast, Pou3f1-overexpressing ESC derivatives preferentially contribute to the neuroectoderm. Genome-wide ChIP-seq and RNA-seq analyses indicated that Pou3f1 is an upstream activator of neural lineage genes, and also is a repressor of BMP and Wnt signaling. These results established that Pou3f1 promotes the neural fate commitment of pluripotent stem cells through a dual role, activating internal neural induction programs and antagonizing extrinsic neural inhibitory signals (Zhu, 2014).

Pou3f1 was previously reported to be a transcription factor that participates in Schwann cell development and myelination. The Pou3f1 gene expression profiles in mouse embryos in vivo and of ESC differentiation in vitro imply that Pou3f1 may also participate in early neural development. Indeed, the shRNA-mediated knockdown of Pou3f1 in ESCs results in the reduced expression of the neural markers Sox1, Pax6, and Tuj1 in serum-free medium. However, the compensation of the POU III member Brn2 may be one of the reasons for the mild effects observed during ESC neural differentiation after Pou3f1 depletion. Brn2 compensation and the different ESC lines and culture system used potentially explain why the Pou3f1 knockdown effects are not reported in a previous. On the other hand, the results are consistent with their results indicating that the forced expression of Pou3f1 promotes the expression of neural markers. Clearly, Pou3f1 is necessary and sufficient for ESC neural differentiation. Pou3f1-overexpressing or Pou3f1-knockdown ESCs generate EpiSC-like colonies that are similar to the control ESCs. However, the neural differentiation of Pou3f1-overexpressing or Pou3f1-knockdown EpiSCs is markedly different from the control EpiSCs, suggesting that Pou3f1 functions specifically during the neural transition from the epiblast to neural progenitor cells. Furthermore, in our blastocyst injection study, the contribution of Pou3f1-knockdown ESCs to the neuroectoderm was severely impaired, indicating that Pou3f1 most likely functions cell-autonomously during the neural fate commitment of pluripotent stem cells in vivo. The findings revealed that Pou3f1 is an essential transcription factor required for the intrinsic neural differentiation of pluripotent stem cells (Zhu, 2014).

Cell fate determination is regulated in a step-wise fashion via the activation or inhibition of lineage specification factors. Several transcription factors, including Pax6, Sox2, Zfp521, Zic1, and Zic2, promote neural gene expression and play roles in the derivation of the anterior neural plate. Zfp521 and Zic1/2 are important for neural fate consolidation rather than initiation. To date, the intrinsic modulators essential for the early neural initiation event have not been identified. In this study, the combination of RNA-seq and ChIP-seq enabled investigation of the underlying molecular mechanisms governing Pou3f1-mediated neural fate commitment in ESCs at the genome-wide level and to determine whether Pou3f1 is involved in the initiation of neural differentiation. The results indicate that Pax6, Sox2, Zfp521, and dozens of other known neural fate-promoting genes are enhanced by Pou3f1 overexpression during ESC differentiation. Furthermore, ChIP-seq data reveal that Pou3f1 is enriched at the regulatory regions of Pax6, Sox2, Zfp521, Zic1, and Zic2 genomic loci, indicating that Pou3f1 directly activates these neural fate-promoting genes. Surprisingly, Pou3f1 did not bind the Sox2N1 enhancer, which controls Sox2 posterior neural plate expression; Pou3f1 preferentially binds to the Sox2N2 enhancer, which drives Sox2 anterior neural plate expression. This result is consistent with the in vivo Pou3f1 and Sox2 overlapping expression patterns during neural fate commitment. The results are also consistent with the notion that the anterior-most portion of the epiblast constitutes the primitive neural identity following neural induction. Moreover, the observations confirm the hypothesis proposed in a recent study that Pou3f1 functions upstream of Zfp521 during ESC neural differentiation. Taken together, these findings demonstrate that Pou3f1 is most likely an intrinsic neural initiation factor that participates in the transition of pluripotent stem cells to NPCs by directly activating a group of key neural fate-promoting genes (Zhu, 2014).

In addition to intrinsic factors, several extrinsic signals involved in early neural fate commitment have been intensively studied, including BMPs and Wnts. However, how BMP/Wnt inhibitory activities are alleviated to secure neural fate commitment has not been fully elucidated. BMP and Wnt signals function partially through their downstream genes. Unlike Zfp521, which did not affect BMP signaling, the expression of a few genes related to BMP and Wnt pathways was regulated by Pou3f1 knockdown or by overexpressing in EBs at day 4. However, this regulation was not evident in ESCs or in EBs at day 2. This finding suggests that Pou3f1 interferes with the BMP/Wnt signaling pathways during the process of neural conversion from epiblast to NPCs. Moreover, Pou3f1 is recruited to the genomic loci of many downstream targets of BMP and Wnt signals, such as Id1, Id2, Myc, and Axin2. It was also found that Pou3f1 represses the transcriptional activation of a BMP responsive element (BRE) by BMP4 and of a TCF optimal promoter (TOP) by Wnt3a. The data further suggest that the binding of pSmad1 to the BRE locus is potentially compromised in the presence of Pou3f1, which results in the repression of BMP signaling pathway activity. However, other possibilities, such as the recruitment of repressing cofactors by Pou3f1, could not be excluded by the present study. Notably, Pou3f1 overexpression enables neural differentiation even in the presence of BMP4 or Wnt3a. It is proposed that the Pou3f1-dependent repression of the BMP and Wnt signaling pathways and the activation of intrinsic neural lineage genes together are involved in the neural fate-promoting activity of Pou3f1 (Zhu, 2014).

In summary, this study establishes Pou3f1 as a critical dual-regulator of intrinsic transcription factors and extrinsic signals to promote neural fate commitment. This study provides a better understanding of the internal mechanism of neural initiation. Nonetheless, many questions concerning this process remain unanswered, such as whether the dual regulatory mechanism of Pou3f1 is also utilized to initiate the mouse neural program in vivo, whether this two-way modulating processes occurs simultaneously or in a sequential, temporal manner, and how the controversial activation/inhibition activities of the Pou3f1 transcription factor is achieved. All these unanswered questions lay the foundation for exciting future work concerning the interplay between the extrinsic and intrinsic cues during early embryonic neural fate commitment (Zhu, 2014).

Mammalian class III POU factors and myelination

Despite the importance of myelinating Schwann cells in health and disease, little is known about the genetic mechanisms underlying their development. The POU domain transcription factor pou3f1 (Tst-1, SCIP, Oct-6) is required for the normal differentiation of myelinating Schwann cells, but its precise role requires identification of the genes that it regulates. Six genes have been isolated whose expression is reduced in the absence of pou3f1. Only one of these genes, the fatty acid transport protein P2, was known previously to be expressed in Schwann cells. The LIM domain proteins cysteine-rich protein-1 (CRP1) and CRP2 are expressed in sciatic nerve and induced by forskolin in cultured Schwann cells, but only CRP2 requires pou3f1 for normal expression. pou3f1 appears to require the claw paw gene product for activation of at least some of its downstream effector genes. Expression of the novel Schwann cell genes after nerve injury suggests that they are myelin related. One of the genes, tramdorin1, encodes a novel amino acid transport protein that is localized to paranodes and incisures. These results suggest that pou3f1 functions to activate gene expression in the differentiation of myelinating Schwann cells (Bermingham, 2002).

Members of the POU and SOX transcription factor families exemplify the partnerships established between various transcriptional regulators during early embryonic development. Although functional cooperativity between key regulator proteins is pivotal for milestone decisions in mammalian development, little is known about the underlying molecular mechanisms. In this study, focus was placed on two transcription factors, Oct4 and Sox2, since their combination on DNA is considered to direct the establishment of the first three lineages in the mammalian embryo. Using experimental high-resolution structure determination, followed by model building and experimental validation, it was found that Oct4 and Sox2 were able to dimerize onto DNA in distinct conformational arrangements. The DNA enhancer region of their target genes is responsible for the correct spatial alignment of glue-like interaction domains on their surface. Interestingly, these surfaces frequently have redundant functions and are instrumental in recruiting various interacting protein partners (Reményi, 2003).

The interaction of Oct1 and Oct4 with Sox2 was investigated on two different DNA enhancers to test whether a previously discovered regulation mechanism of DNA-mediated swapping of the arrangement of homodimers may also be applicable for unrelated transcription factor assemblies. The crystal structure of the ternary Oct1/Sox2/FGF4 enhancer element complex was solved and then homology modeling tools were used to construct an Oct4/Sox2/FGF4 as well as an Oct4/Sox2/UTF1 structural model. These models reveal that the FGF4 and the Undifferentiated Transcription Factor 1 (UTF1) enhancers mediate the assembly of distinct POU/HMG complexes, leading to different quaternary arrangements by swapping protein-protein interaction surfaces of Sox2. Moreover, it has been demonstrated that Sox2 uses one of its two protein interacting surfaces to assemble a ternary complex with another unrelated transcription factor on a late-embryonic-stage-specific enhancer (Pax6/Sox2 on the DC5 element). These findings outline a simple mechanism for promiscuous yet highly specific assembly of transcription factors, in which the sequence of DNA enhancers governs a combinatorial use of redundant protein-protein interaction surfaces (Reményi, 2003).

The genetic hierarchy that controls myelination of peripheral nerves by Schwann cells includes the POU domain Oct-6/Scip/Tst-1and the zinc-finger Krox-20/Egr2 transcription factors. These pivotal transcription factors act to control the onset of myelination during development and tissue regeneration in adults following damage. This report demonstrates the involvement of a third transcription factor, the POU domain factor Brn-2. Schwann cells express Brn-2 in a developmental profile similar to that of Oct-6; Brn-2 gene activation does not depend on Oct-6. Overexpression of Brn-2 in Oct-6-deficient Schwann cells, under control of the Oct-6 Schwann cell enhancer (SCE), results in partial rescue of the developmental delay phenotype, whereas compound disruption of both Brn-2 and Oct-6 results in a much more severe phenotype. Together these data strongly indicate that Brn-2 function largely overlaps with that of Oct-6 in driving the transition from promyelinating to myelinating Schwann cells (Jaegle, 2003).

Class III POU factors and the development of the hypothalamus

Neurons comprising the endocrine hypothalamus are disposed in several nuclei that develop in tandem with their ultimate target the pituitary gland, and arise from a primordium in which three related class III POU domain factors, Brn-2, Brn-4, and Brn-1, are initially coexpressed. Subsequently, these factors exhibit stratified patterns of ontogenic expression, correlating with the appearance of distinct neuropeptides that define three major endocrine hypothalamic cell types. Strikingly, deletion of the Brn-2 genomic locus results in loss of endocrine hypothalamic nuclei and the posterior pituitary gland. Lack of Brn-2 does not affect initial hypothalamic developmental events, but instead results in a failure of differentiation to mature neurosecretory neurons of the paraventricular and supraoptic nuclei, characterized by an inability to activate genes encoding regulatory neuropeptides or to make correct axonal projections, with subsequent loss of these neurons. Thus, both neuronal and endocrine components of the hypothalamic-pituitary axis are critically dependent on the action of specific POU domain factors at a penultimate step in the sequential events that underlie the appearance of mature cellular phenotypes (Schonemann, 1995).

The neuroendocrine system consists of two sets of hypothalamic neurons: the magnocellular and the parvocellular neurons. The magnocellular neurosecretory system projects to the posterior pituitary where it releases vasopressin (AVP) and oxytocin (OT) directly into the general circulation. Vasopressin participates in the control of blood volume, osmolality, and pressure, whereas OT promotes parturition and lactation. The magnocellular neurons are located in two nuclei of the anterior hypothalamus, the paraventricular (PVN) and the supraoptic (SON) nuclei. Within the PVN and the SON, AVP and OT are produced by mutually exclusive sets of neurons. The sum of AVP- and OT-producing cells corresponds to the total number of magnocellular neurons, indicating that AVP and OT define the two cell types of this neurosecretory system. The PVN, which contains both magnocellular and parvocellular neurons, and the SON, which is mainly composed of magnocellular neurons, originate from a small patch of neuroepithelium located at the level of the ventral diencephalic sulcus. Cells that form the PVN remain near the ventricular zone, whereas those that form the SON migrate laterally to reach the surface of the hypothalamus. The bHLH-PAS transcription factor SIM1 (Drosophila homolog: Single minded) is expressed during the development of the hypothalamic-pituitary axis in three hypothalamic nuclei: the PVN, the anterior PVN (aPV), and the SON. To investigate Sim1 function in the hypothalamus, mice were produced carrying a null allele of Sim1 by gene targeting. Homozygous mutant mice die shortly after birth. Histological analysis shows that the PVN and the SON of these mice are hypocellular. At least five distinct types of secretory neurons, identified by the expression of oxytocin, vasopressin, thyrotropin-releasing hormone, corticotropin-releasing hormone, and somatostatin, are absent in the mutant PVN, aPV, and SON. Moreover, SIM1 controls the development of these secretory neurons at the final stages of their differentiation. A subset of these neuronal lineages in the PVN/SON are also missing in mice bearing a mutation in the POU transcription factor BRN2. Evidence is provided that, during development of the Sim1 mutant hypothalamus, the prospective PVN/SON region fails to express Brn2. These results strongly indicate that SIM1 functions upstream to maintain Brn2 expression, which in turn directs the terminal differentiation of specific neuroendocrine lineages within the PVN/SON (Michaud, 1998).

Drosophila Sim is a master regulator of the CNS midline. Loss of sim function results in the complete absence of midline development. Drifter, a POU domain transcription factor that binds the same DNA sequence as does BRN2, has also been implicated in controlling the development of CNS midline cells in the fly. Expression and phenotypic analysis have shown that Sim acts upstream of Drifter. In mice, SIM1 likewise acts upstream of a POU domain transcription factor BRN2. Specifically, Brn2 is down-regulated in a region of the prospective PVN/SON that continues to express the Sim1 mutant transcript, indicating that SIM1 and BRN2 function along the same pathway. The fact that Brn2 expression in the prospective hypothalamus of Sim1 mutant embryos is not altered until E12.5 suggests that Sim1 is not involved in initiating but in maintaining Brn2 expression. Whether SIM1 controls BRN2 transcription directly or indirectly remains an open question. Consistent with the conclusion that Sim1 functions upstream to maintain Brn2 expression, all the hypothalamic lineages that are reported to be affected by the loss of Brn2 function are also affected in Sim1-deficient mice; the loss of Brn2 function affects the development of vasopressin -, oxytocin-, and corticotropin-releasing hormone-producing cells, and the same cell types are affected by the loss of Sim1 function. In contrast, thyrotropin-releasing hormone- and somatostatin-producing cells are missing in Sim1 mutant but are present in Brn2 mutant PVN and aPV. This is consistent with the observation that thyrotropin-releasing hormone and BRN2 expression share minimal overlap in the PVN. Similarily, Brn2 is not expressed in the aPV, where somatostatin is produced abundantly (Michaud, 1998).

The loss of the five cell types studied here in Sim1 mutant mice raises the possibility that most, if not all, of the neuronal lineages constituting the PVN, SON, and aPV originate from the dorsal aspect of the prospective anterior hypothalamic Sim1 domain. This domain can be divided into an anterior region only expressing Sim1 and a posterior region expressing both Sim1 and Brn2. It is tempting to speculate that the corticotropin-releasing hormone (CRH), AVP, and OT lineages, which are affected in both Sim1 and Brn2 mutant mice, are derived from the posterior region, whereas the Thyrotropin-releasing hormone and somatostatin lineages, which are only affected in Sim1 mutant mice, are derived from the anterior region. This is consistent with the observation that in the newborn hypothalamus, Brn2 is not expressed in the aPV or in the anterior end of the PVN, where SS- and TRH-producing cells, respectively, are found. In Brn2 mutant mice, precursors of the PVN and SON survive up to E15.5 but fail to express the secreted neuropeptides. BRN2 binds and activates the CRH promotor, supporting a role for BRN2 in controlling the terminal stage of differentiation. The survival of the PVN/SON precursors up to E15.5 in both Sim1 and Brn2 mutant embryos and the down-regulation of Brn2 in Sim1 mutant embryos would suggest that the loss of Brn2 expression mediates the effect of the Sim1 mutant allele on the development of CRH, AVP, and OT neuroendocrine lineages. Whether Sim1 controls the differentiation of TRH- and SS-expressing cells directly or indirectly, through activation of another POU domain transcription factor, remains to be determined (Michaud, 1998).

Development of the neuroendocrine hypothalamus is characterized by a precise series of morphogenetic milestones culminating in terminal differentiation of neurosecretory cell lineages. The homeobox-containing gene Orthopedia (Otp), is expressed in neurons giving rise to the paraventricular (PVN), supraoptic (SON), anterior periventricular (aPV), and arcuate (ARN) nuclei throughout their development. Homozygous Otp-/- mice die soon after birth and display progressive impairment of crucial neuroendocrine developmental events such as reduced cell proliferation, abnormal cell migration, and failure in terminal differentiation of the parvocellular and magnocellular neurons of the aPV, PVN, SON, and ARN. Moreover, the data provide evidence that two proteins, Otp and Sim1 (the latter a bHLH-PAS transcription factor that directs terminal differentiation of the PVN, SON, and aPV), act in parallel and are both required to maintain Brn2 expression, which, in turn, is required for neuronal cell lineages secreting oxytocin (OT), arginine vasopressin (AVP), and corticotropin-releasing hormone (CRH) (Acampora, 1999).

Analysis of Brn2 mutant mice reveals that it acts relatively late in neuroendocrine development, being required for terminal differentiation events of CRH, AVP, and OT cell lineages. Sim1 mutant mice show a more general effect, because they are impaired in terminal differentiation events leading to the activation of neuropeptides of the PVN and SON as well as the activation of SS in the aPV. Interestingly, from E12.5 onward, Sim1 minus mutants gradually lack Brn2 expression in the dorsal supraoptic/paraventricular (spv) primordium, indicating that Sim1 acts upstream of Brn2 and is required for maintenance of its expression. There is a striking similarity with the Sim1 mutant phenotype. Except in the ARN, Otp is fully coexpressed in time and space with Sim1, and is required for both terminal differentiation of parvocellular and magnocellular neurons of aPV, PVN, and SON and for maintenance of Brn2 expression. Noteworthy, at E11.5, Brn2 expression is slightly toned down and, at E12.5, disappears from the entire spv and adjacent territory in which it is coexpressed with Otp, thus suggesting that as compared with Sim1 minus phenotype, Otp may have a more generalized role in controlling Brn2 expression in post-mitotic neurons and may open the question as to whether Sim1 and Otp act in parallel, or is one downstream of the other with regard to the control of Brn2 expression? Interestingly, in Otp minus embryos, Sim1 expression is maintained in lacZ-positive cells in which Brn2 is lost and, in Sim1 minus embryos, Otp is expressed in the territory in which Brn2 disappears. These findings provide strong in vivo evidence that Otp and Sim1 act in parallel and are both required for proper expression of Brn2 in the spv and its derivatives, the PVN and SON (Acampora, 1999).

Class III POU factors and epidermal development

The roles of the POU domain genes Skin-1a/i (Skn-1a/i/Epoc/Oct-11) and Testes-1 (Tst-1/Oct-6/SCIP), respectively related to Drosophila pdm-1 and drifter) have been examined in epidermis where proliferating basal keratinocytes withdraw from the cell cycle, migrate suprabasally, and terminally differentiate to form a multilayered, stratified epithelium. The expression of the Skn-1a/i and Tst-1 genes is linked to keratinocyte differentiation in vivo and in vitro, whereas the ubiquitous POU domain factor Oct-1 is expressed highly in both proliferating and post-mitotic keratinocytes. Analysis of Skn-1a/i gene-deleted mice reveals that the Skn-1a/i gene modulates the pattern of expression of the terminal differentiation marker loricrin and inhibits expression of genes encoding markers of the epidermal keratinocyte wounding response. Although epidermis from Tst-1 gene-deleted mice develops normally, epidermis from mice deleted for both Skn-1a/i and Tst-1 is hyperplastic and fails to suppress expression of K14 and Spr-1 in suprabasal cells when transplanted onto athymic mice. This suggests that Skn-1a/i and Tst-1 serve redundant functions in epidermis. Therefore, at least two POU domain genes, Skn-1a/i and Tst-1, serve both distinct and overlapping functions to regulate differentiation of epidermal keratinocytes during normal development and wound healing (Andersen, 1997).

Brn1 and kidney development

A role for the POU transcription factor Brn1 in distal tubule formation and function in the mammalian kidney has been identified. Normal development of Henle's loop (HL), the distal convoluted tubule and the macula densa is severely retarded in Brn1-deficient mice. In particular, elongation and differentiation of the developing HL is affected. In the adult kidney, Brn1 is detected only in the thick ascending limb (TAL) of HL. In addition, the expression of a number of TAL-specific genes is reduced in the Brn1+/- kidney, including Umod, Nkcc2/Slc12a1, Bsnd, Kcnj1 and Ptger3. These results suggest that Brn1 is essential for both the development and function of the nephron in the kidney (Nakai, 2003).

The mode of Brn1 function in developing neocortical neurons is similar to its function in HL development, since Brn1 functions in both the proliferation and differentiation of precursors in both situations. In addition, Brn1 commonly plays an essential role in later phases of the developmental process. Nephrons require Brn1 function mainly during late development. The molecular pathways activated downstream of Brn1, however, are quite different between these two systems. In contrast to the activation of TAL-specific genes in developing HLs, Brn1 activates Dab1-dependent positioning processes in neocortical neurons. Therefore, the involvement of an additional molecule(s) specifying the pathways downstream of Brn1 is likely. Identification of such factors would aid the understanding of the molecular mechanisms underlying HL development (Nakai, 2003).

Brn-1 and Brn-2 function in production and positioning of mouse neocortical neurons

Formation of highly organized neocortical structure depends on the production and correct placement of the appropriate number and types of neurons. POU homeodomain proteins Brn-1 and Brn-2 are coexpressed in the developing neocortex, both in the late precursor cells and in the migrating neurons. Double disruption of both Brn-1 and Brn-2 genes in mice leads to abnormal formation of the neocortex with dramatically reduced production of layer IV-II neurons and defective migration of neurons unable to express mDab1. These data indicate that Brn-1 and Brn-2 share roles in the production and positioning of neocortical neuron development (Sugitani, 2002).

To explore their possible overlapping functions in neocortical development, Brn-1/Brn-2 double homozygous mutants were generated by intercrossing double heterozygotes that were healthy and fertile, with no apparent phenotype. Double homozygous mutants were born at the expected Mendelian ratio, but all of them died within 1 h after birth. In contrast to the limited abnormalities in Brn-1-/- or Brn-2-/- single mutants, Brn-1/Brn-2 double mutants suffered severe, broad brain defects. The olfactory bulb showed hypoplasia, and the cerebellum was less foliated, with loosely packed Purkinje cells. The neocortex was severely affected; its thickness was markedly reduced, and the stratification of the cortical neurons appeared to be disorganized (Sugitani, 2002).

The hypoplastic neocortex could be caused by reduced cell proliferation or accelerated cell death during embryonic corticogenesis. Because there is no evidence of increased apoptosis in Brn-1/Brn-2 double mutant cortex from embryonic day 14.5 (E14.5) to postnatal day 0 (P0), the proliferation of cortical progenitor cells was examined by bromodeoxyuridine (BrdU) labeling. In mice, most cortical plate neurons are produced in the ventricular zone (VZ) or in the subventricular zone (SVZ) from E12.5 to E16.5. Up to E13.5, there is no significant difference in the number of BrdU-labeled cells in the VZ of the double mutant embryos, compared with wild-type. Reduced cell proliferation in the VZ is observed at E14.5 and thereafter in Brn-1/Brn-2 mutant neocortex. Reduction in the number of BrdU-labeled cells is particularly severe in the cortical SVZ in the double mutant. Despite the hypoplasticity of the Brn-1/Brn-2 deficient cortex, expression of GAD67 and calbindin appears to be unaffected in the E19.0 neocortex, suggesting intact generation and migration of the cortical interneurons, most of which are derived from the ganglionic eminence. These results indicate that Brn-1 and Brn-2 share an essential role in the proliferation of cortical precursor cells within the VZ/SVZ from E14.5 onward, and that the reduction in subsequent cortical cell production could result in the hypoplastic neocortex seen in the double mutant neonate. Analysis of the temporal expression pattern for Brn-1 and Brn-2 proteins in the developing wild-type neocortex has revealed that their expression in the VZ is initiated at ~E14.5 and is prominent thereafter in the VZ/SVZ, with a pattern that corresponds with the period of reduced cell proliferation in the neocortex of double mutant embryos. These results suggest that Brn-1 and Brn-2 may function in the proliferation of late cortical progenitor cells in a cell-autonomous manner (Sugitani, 2002).

Lineage analyses and birthdating studies suggest that common cortical precursor cells first produce neurons of layer VI and then layer V (at E11.5-E15.5) and, even later, generate neurons destined for layers IV-II (at E14.5-E17.0) by successive cell division. From the late embryonic neurogenesis stage, glial progenitor cells also proliferate and increase their numbers, differentiating into astrocytes or oligodendrocytes during a postnatal stage. The finding that Brn-1 and Brn-2 function in cell proliferation, specifically at the late neurogenesis stage, prompted an examination of whether Brn-1 and Brn-2 function in the production of upper-layer neurons and/or in the generation/expansion of glial progenitor cells. The formation of each cortical layer and the status of gliogenesis in the double mutant cortex at E19.0 or E18.5 were assessed using the following markers for different layers and glial progenitors: Tbr-1 for layer VI, subplate and SVZ; Wnt7b for layer VI, ER81 for layer V; RORß for layer IV, mSorLA or Svet1 for layers II/III and SVZ cells, Olg-1 for oligodendrocyte progenitors, B-FABP/BLBP for immature astrocytes and radial glial cells, and CR-50 for Cajal-Retzius neurons in the marginal zone (MZ). The marker studies indicate that the initial step of gliogenesis seem to be unaffected in Brn-1/Brn-2 mutant neocortex, whereas the numbers of RORß-positive, mSorLA-positive, or Svet1-positive neurons are dramatically reduced in Brn-1/Brn-2 mutant neocortex with mSorLA-expressing or Svet1-expressing SVZ cells lining the entire surface of the enlarged lateral ventricles of the mutant brains. These results suggest that Brn-1 and Brn-2 are essential for proper production of neocortical neurons destined for layers VI-II (Sugitani, 2002).

Molecular marker analysis also revealed abnormal layering of the remaining cortical neurons in Brn-1/Brn-2-deficient neocortex, in which the majority of ER81-positive layer V neurons, normally laminated above the Tbr-1-positive or Wnt7b-positive layer VI, were found beneath the Tbr-1-positive or Wnt7b-positive layer. It has been well documented that the laminar structure of the neocortex is built by migration of successively produced neurons in an inside-to-outside fashion, such that neurons born earlier reside in deeper layers, and those born later occupy more superficial layers within the cortical plate (CP) between the MZ and the subplate (SP). Thus, the largely inverted packing pattern of layer V and VI neurons in Brn-1/Brn-2 mutant cortex can be caused by either abnormal cell migration or cell fate defects such that the timing of layer VI and layer V neuronogenesis is inverted. To distinguish between the two possibilities, embryos were labelled with BrdU at E12.5, E13.5, and E14.5; during this time span, layer VI-V neuronogenesis is at a peak. The localization of BrdU-positive cortical neurons was examined in E19.0 embryos. If the abnormal lamination is caused by cell fate defects, BrdU-labeled neurons should appear in comparable positions in the wild-type and Brn-1/Brn-2 mutant cortices. Conversely, if neuronal migration is affected, neurons labeled at the same time should occupy different positions in wild-type and mutant mice. In E19.0 wild-type cortex, cells born on E12.5 occupied the SP and the deepest part of layer VI, and most of the cells at E13.5 predominantly occupied layer VI above the E12.5-born cohort. The relative positions of E13.5-born to E12.5-born neurons in the Brn-1/Brn-2-deficient cortex at E19.0 were comparable with those in their wild-type littermates. The positioning of E14.5-born neurons, however, was significantly altered. E14.5-born cells in wild-type cortex occupied layers V and IV in a superficial region of the CP, whereas those in Brn-1/Brn-2-deficient cortex remained in the intermediate zone (IZ), beneath the cohort of E12.5-born cells. Together with the abnormal localization of the layer V neurons in the IZ of Brn-1/Brn-2 mutant cortex, these BrdU neural birthdating experiments suggest abnormal migration of the layer V neurons born after E13.5 in Brn-1/Brn-2 mutant cortex (Sugitani, 2002).

Correct neuronal migration requires both radial glial fibers as guiding scaffolds for migrating neurons and Cajal-Retzius neurons that play a key role in neuronal lamination by producing the secreted Reelin protein. The alignment and density of radial glial fibers, labeled with antibodies against B-FABP or Nestin, were not altered. Furthermore, neither the number of Cajal-Retzius neurons nor their immunolabeling intensity for Reelin was changed in the Brn-1/Brn-2-deficient cortex. In fact, Cajal-Retzius neurons in the wild-type cortex expressed neither Brn-1 nor Brn-2 at E16.5 and E18.5 cortex. Thus, the migration defects in the Brn-1/Brn-2-deficient cortex do not seem to be a consequence of a disrupted radial glial fiber system or a loss of Reelin-expressing Cajal-Retzius neurons. Given Brn-1/Brn-2 coexpression in migrating neurons both in the IZ and CP, the altered migration of Brn-1/Brn-2-deficient cortical neurons can be a result of cell-autonomous defects (Sugitani, 2002).

To investigate the molecular mechanisms underlying the neuronal migration defects in Brn-1/Brn-2 mutant cortex, an RT-PCR analysis was performed on various genes involved in neuronal migration. mDab1, VLDLR/ApoER2, and alpha3-integrin have been shown to function in positioning cortical neurons by mediating Reelin signal transduction. CDK5, p35 (one of the CDK5 activator subunits), Lis1 (Pafah1b1), and Doublecortin are also thought to affect neuronal migration in the developing cortex. Among all these tested genes, only mdab1 expression was clearly affected in the Brn-1/Brn-2 double mutant cortex at E16.5. Therefore, the spatial distribution of the mdab1 mRNA in the cortex of Brn-1/Brn-2 mutant embryos and wild-type littermates was examined by RNA in situ hybridization. In the wild-type cortex at E16.5, mdab1 mRNA is expressed throughout the cortical wall, except for the MZ and SP. High levels of mdab1 mRNA are detected in the upper regions of the IZ and in the CP. In the Brn-1/Brn-2-deficient cortex at E16.5, mdab1 mRNA expression is significantly reduced throughout the cortical wall and, in particular, is undetectable in the upper region of the IZ just beneath the chondroitin sulfate proteoglycans (CSPG)-positive SP, in which p35-highly expressing late-born neurons are abnormally congested. Therefore, the slight reduction in p35 mRNA levels in the E16.5 mutant cortex detected by RT-PCR analysis might be caused by decreased numbers of p35-expressing neurons produced from E14.5 onward. Furthermore, quantitative RT-PCR analysis showed that mdab1 expression is reduced also in Brn-1/Brn-2 double heterozygotes, which show no histological defects in their neocortex. RNA in situ hybridization also showed that precipitously graded reduction of mdab1 mRNA levels correlates well with Brn-1/Brn-2 gene dosages. These results imply that Brn-1 and Brn-2 act genetically upstream to activate mDab1-dependent positioning processes in cortical neurons. The early-born neurons lacking Brn-1 and Brn-2, however, migrate and split the preplate into the MZ and SP properly; such mobility is not seen in the mdab1 mutant cortex. In yotari and scrambler, mutant mice carrying loss-of-function mutations in the mdab1 gene, cortical neurons fail to split the preplate to form the CP between the MZ and SP. The maintenance of integrity of preplate splitting in Brn-1/Brn-2 mutant E16.5 cortex could be caused by the redundant function of another class III POU factor, Brn-4, that also shares high homology in its primary structure with Brn-1 and Brn-2. In wild-type as well as double-mutant cortex, Brn-4 expression is also detected in the migrating neurons at ~E15.5, but is reduced after then. In Brn-1/Brn-2 mutant cortex, mDab1 expression is detected until E15.5 but is hardly detectable at E16.5. Therefore, Brn-4, like Brn-1 and Brn-2, might also be able to activate mDab1-dependent processes in the positioning of early-born neurons (Sugitani, 2002).

This study has shown that there are two distinct types of the expression pattern of Brn-1/Brn-2 proteins in developing neocortex. Brn-1/Brn-2 expression in the precursor cells is restricted to a late pool of neural precursors, and Brn-1/Brn-2 is also expressed in a wide range of the postmitotic neurons, including Tbr-1-positive cortical plate neurons. Double disruption of both Brn-1 and Brn-2 genes in mice leads to two types of abnormalities during the neocortical development: selective loss of the neurons positive for layer IV-II markers (RORß, mSorLA, and Svet1), and significantly reduced mDab1 expression in all remaining neurons at late phase, independently of Brn-1/Brn-2 expression in their precursors (Sugitani, 2002).

Several lines of evidence suggest that mDab1 functions downstream of Reelin in a signaling pathway that controls cell positioning in the developing cortex. However, it is not yet clear how these molecules dictate the spatial position of cortical neurons, including subplate neurons. Interestingly, in the Brn-1/Brn-2-deficient cortex, mDab1 expression is severely reduced only at a late stage, when most of the E14.5-born neurons migrate through the IZ, but do not reach the MZ, remaining congested just beneath the SP. Therefore, these results imply that mDab1 may be necessary for CP neurons to migrate through the SP. Alternatively, Brn-1 and Brn-2 could also regulate expression of other molecules that may be essential in this process. However, the hypoplasticity of the Brn-1/Brn-2-deficient cortex cannot be explained by an inability to express mdab1, because reduced cell proliferation has not been reported in mdab1 mutant cortex, and loss of RORß-expressing or mSorLA-expressing neurons was not observed in yotari. tailless and pax6 expression, which are known to be essential for proper generation of cortical neurons, were examined. However, no changes were found in their expression in Brn-1/Brn-2 mutant cortex (Sugitani, 2002).

Previous reports have indicated that the earliest events of cell class specification within each cortical layer occur in coordination with neuronogenesis within the proliferating zone. At later stages, when superficial layers are being generated, the progenitors become restricted to an upper-layer fate. A subpopulation of SVZ cells derived from the VZ has been shown to represents neuronal progenitors committed to upper-layer neurons. Because Brn-1 and Brn-2 are specifically expressed in late precursor cells within the cortical VZ/SVZ and function in the proliferation of these cells both in the VZ and especially in the SVZ, these factors might share an intrinsic role in the production of fate-committed neuronal precursors and/or cortical neurons destined for the upper layers. Further analysis on these overlapping mutants would provide insight into the developmental mechanisms of the mammalian neocortex with its great diversity of cortical neurons (Sugitani, 2002).

Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif

Proneural proteins play a central role in vertebrate neurogenesis, but little is known of the genes that they regulate and of the factors that interact with proneural proteins to activate a neurogenic program. The proneural protein Mash1 and the POU proteins Brn1 and Brn2 interact on the promoter of the Notch ligand Delta1 and synergistically activate Delta1 transcription, a key step in neurogenesis. Overexpression experiments in vivo indicate that Brn2, like Mash1, regulates additional aspects of neurogenesis, including the division of progenitors and the differentiation and migration of neurons. This study identifies, by in silico screening, a number of additional candidate target genes that are recognized by Mash1 and Brn proteins through a DNA-binding motif similar to that found in the Delta1 gene and present a broad range of activities. It is thus proposed that Mash1 synergizes with Brn factors to regulate multiple steps of neurogenesis (Castro, 2006).

Delta1 is a common target of the proneural genes Mash1 and Neurogenin1/2 in mouse embryos. To determine whether Mash1 and Neurogenin1/2 directly transcribe Delta1, the regulatory sequences of this gene were analyzed. Two evolutionarily conserved enhancers active in different CNS regions have been identified in the Delta1 gene. To determine whether these enhancers mediate the regulation of Delta1 by proneural genes, transgenic mouse lines were generated. A transgene containing the full-length 4.3 kb mouse Delta1 promoter driving lacZ was expressed broadly in the embryonic brain and spinal cord at E11.5, in a pattern similar to that of endogenous Delta1. A transgene containing the proximal Delta1 neural enhancer (hereafter called DeltaM) and a minimal promoter driving lacZ was, in contrast, only expressed in parts of the Delta1 expression domain, including the dorsal spinal cord and ventral telencephalon, which also express Mash1. A transgene containing the distal Delta1 enhancer (hereafter called DeltaN) driving lacZ was expressed in a complementary manner in the neural tube, including the ventral spinal cord and dorsal telencephalon, which also express Ngn1 and Ngn2. To test whether these enhancers are regulated by proneural genes, the transgenic lines were bred with proneural null mutant mice. On a Mash1 null mutant background, the DeltaM-lacZ transgene was not expressed in the CNS at E11.5, demonstrating that the DeltaM enhancer requires Mash1 function for its activation. On a Ngn1;Ngn2 double mutant background, the DeltaN-lacZ transgene showed reduced expression in the spinal cord and was not expressed in the brain, except near the hindbrain border, showing that the DeltaN enhancer is activated by Neurogenins (Castro, 2006).

It was next asked whether proneural proteins directly interact with the Delta1 enhancers in the embryonic CNS by performing chromatin immune precipitation (ChIP) experiments. An antibody to Mash1 coprecipitated the DeltaM sequence in chromatin prepared from E12.5 wild-type telencephalon, but not from Mash1 mutant telencephalon, and the antibody did not precipitate the DeltaN sequence or the Delta1 coding sequence. Conversely, an antibody to Ngn2 coprecipitated the DeltaN sequence from wild-type, but not from Ngn2 null mutant telencephalon, and it did not precipitate the DeltaM or Delta1 coding sequences. Therefore, Mash1 and Ngn2 specifically bind in vivo to the DeltaM and DeltaN enhancers, respectively (Castro, 2006).

To further examine the interaction of Mash1 with the DeltaM enhancer, transcription assays were performed in P19 cells. It was first verified that Mash1 induces Delta1 transcription in these cells. After transfection of P19 cells with a Mash1 expression vector, the Mash1 transcript level increased in less than 4 hr, while the Delta1 transcript level increased less than 3 hr later, suggesting that Delta1 is directly transcribed by Mash1 in this system. By performing ChIP experiments with the Mash1 antibody on Mash1-transfected and mock-transfected P19 cells, it was shown that Mash1 specifically binds to the DeltaM sequence, indicating that Mash1 uses the same enhancer element to activate Delta1 in P19 cells and in the embryo. To examine the regulation of DeltaM by Mash1, the DeltaM sequence was inserted in a luciferase reporter vector and its transcriptional activity was tested in P19 cells. The DeltaM reporter was strongly activated when cotransfected with a Mash1 expression construct (Castro, 2006).

Two E-boxes (hereafter called E1-box and E2-box) were identified in the DeltaM sequence that are completely conserved in the human, mouse, chick, and zebrafish Delta1/DeltaD gene. To test whether these motifs mediate the direct binding of Mash1 to DeltaM, the two E-boxes were mutated either separately or together and examined the activity of the resulting DeltaM mutants. Mutation of each E-box separately or the two E-boxes together abolished activation of DeltaM by Mash1 and severely reduced the capacity of Mash1 to activate the full-length Delta1 promoter in P19 cells. A shorter version of DeltaM that mostly contains the 2 E-boxes and the 17 nucleotides in between (DeltaM short was activated by Mash1 as efficiently as DeltaM (Castro, 2006).

To determine whether activation of DeltaM by Mash1 involves motifs other than the two E-boxes, the DeltaM short element was mutated further. Interestingly, a perfect evolutionarily conserved consensus binding site for the POU family of homeodomain proteins, or octamer, is present in this element, one nucleotide 5′ from the E2-box. Point mutations in each half-site of the octamer motif, octT64G and octC68G, known to disrupt the interaction of the octamer with the homeodomain (POUH) and the POU-specific domain (POUS) of POU factors, respectively, abolished activation of DeltaM short by Mash1, and the same mutations introduced in the complete DeltaM element or the full-length Delta1 promoter also severely reduced their activation by Mash1. This raised the possibility that activation of Delta1 by Mash1 requires binding of both Mash1 and a POU protein to adjacent motifs in the DeltaM enhancer (Castro, 2006).

The proximity of the two sites suggested that binding of Mash1 to the E2-box might be influenced by binding of a putative POU protein to the adjacent octamer. To address this possibility, a luciferase reporter vector containing the multimerized E2 sequence and a minimal promoter (E26 construct) was generated, so that the interaction of Mash1 with E2 could be analyzed in P19 cells independently of the rest of the DeltaM element. It was asked whether the octamer sequence adjacent to E2 had an effect on the Mash1::E2 interaction by generating a reporter construct containing three copies of a sequence containing both the octamer and the E2-box in the same configuration as in DeltaM ([oct+E2]3). Mash1 activated this construct more efficiently. Moreover, a mutation in the octamer that interferes with POU protein binding (octT64G abolished activation of (octmut+E2)3 by Mash1, thus suggesting that binding of a POU protein to the octamer sequence increases the efficiency of the Mash1::E2 interaction (Castro, 2006).

This study shows that the activation of Delta1 expression by Mash1, a key aspect of its proneural function, involves a functional synergy between Mash1 and the POU genes Brn1 and Brn2. The synergistic activation of Delta1 by Mash1 and Brn1/2 likely reflects recruitment of Mash1 by a Brn protein to the DeltaM enhancer. Brn1/2 proteins on their own bind strongly to the consensus octamer sequence present in this enhancer, while Mash1 alone binds only poorly to the adjacent E2-box, but Mash1 efficiently forms a complex with Brn proteins on the octamer-E2 motif. The configuration of this binding motif plays an essential role in the recruitment process, since increasing the distance between the octamer and the E2-box by just one nucleotide is sufficient to abolish Mash1 recruitment and enhancer activity. The importance of keeping the two DNA-binding sites in close proximity strongly suggests that Mash1-E47 and Brn1/2 physically interact when bound to DNA (Castro, 2006).

The interaction of Mash1 and Brn proteins may also enhance the transcriptional activity of the complex. This is another well-documented mechanism of functional synergy, operating, for example, in the interaction between NeuroM, Isl1, and Lhx3 on the HB9 promoter. Although the primary mechanism underlying the functional synergy of Mash1 and Brn1/2 on the Delta1 promoter is cooperative binding to DNA, the role of Brn proteins does not appear to be restricted to Mash1 recruitment. Indeed, direct binding of Mash1 to the Mash1/Brn motif in the absence of Brn protein binding, e.g., when the low-affinity E2-box sequence in the Mash1/Brn motif is converted to a high-affinity one, is not sufficient to activate a Delta1 reporter construct. This suggests that Brn1/2 also potentiate the transcriptional activity of Mash1, perhaps by recruiting an essential coactivator or by initiating a conformational change that exposes the Mash1 activation domain (Castro, 2006).

An evolutionarily conserved Mash1/Brn-binding motif was found in the vicinity of 21 mouse genes. Six of them are components of the Notch pathway, which, together with the finding that a dominant-negative Brn construct blocks Notch activity in the chick neural tube, suggests that Mash1/Brn protein complexes play a major role in regulating Notch signaling in the CNS. Other genes associated with a Mash1/Brn motif also have important roles in neural development but act independently of Notch signaling. This is notably the case of Dcamkl1 or doublecortin-like kinase, a microtubule-associated protein that has recently been implicated in multiple aspects of development of the cerebral cortex, including cell cycle progression, neuronal commitment, neuronal migration, and axon growth (Castro, 2006).

Some of the other genes associated with a Mash1/Brn motif have not been previously studied in the developing nervous system, but studies in other systems suggest that they may also have varied functions during neurogenesis downstream of Mash1 and Brn1/2. The zinc finger transcription factor Insm1 is regulated by the bHLH gene Neurogenin3 in the pancreas, where it promotes neuroendocrine cell differentiation. Fbw7 is an ubiquitin ligase with an important role in promoting cell cycle arrest in G1/G0 through degradation of cyclin E, c-myc, and c-jun. Fbw7 has also been implicated in degradation of Notch1 (Castro, 2006).

These data thus support the idea that Mash1 acts in synergy with Brn proteins to activate a genetic program that controls multiple steps of neurogenesis, including precursor selection through Notch activation, cell cycle exit, neuronal differentiation, and migration. Analysis of Brn1/Brn2 double mutant mice has shown that these two genes regulate neuronal migration and the proliferation of subventricular zone precursors in the cerebral cortex, a region where neurogenesis is primarily regulated by the proneural gene Ngn2. Whether Brn1/Brn2 mutant mice also display neurogenesis defects in regions where Mash1 is the main proneural gene remains to be analyzed (Castro, 2006).

An important question raised by these results is whether Mash1 regulates aspects of neurogenesis independently of Brn proteins. In support of this notion, additional direct targets of Mash1 have been identified in the brain that are not associated with a conserved Mash1/Brn motif. Moreover, a study of Mash1 function in a neuroendocrine prostate cell line has revealed a number of putative direct targets in this tissue that are not associated with a conserved Mash1/Brn motif, and some of these genes are also regulated by Mash1 in the telencephalon in overexpression experiments. These different findings thus support a model whereby Mash1 interacts with different DNA-binding cofactors to activate different subprograms of neurogenesis, similar to the regulation of different subprograms of myogenesis by MyoD (Castro, 2006).

Identification and characterisation of the early differentiating cells in neural differentiation of human embryonic stem cells

One of the challenges in studying early differentiation of human embryonic stem cells (hESCs) is being able to discriminate the initial differentiated cells from the original pluripotent stem cells and their committed progenies. It remains unclear how a pluripotent stem cell becomes a lineage-specific cell type during early development, and how, or if, pluripotent genes, such as Oct4 and Sox2, play a role in this transition. Here, by studying the dynamic changes in the expression of embryonic surface antigens, this study identified the sequential loss of the markers Tra-1-81 and SSEA4 during hESC neural differentiation and isolated a transient Tra-1-81(-)/SSEA4(+) (TR-/S4+) cell population in the early stage of neural differentiation. These cells are distinct from both undifferentiated hESCs and their committed neural progenitor cells (NPCs) in their gene expression profiles and response to extracellular signalling; they co-express both the pluripotent gene Oct4 and the neural marker Pax6. Furthermore, these TR-/S4+ cells are able to produce cells of both neural and non-neural lineages, depending on their environmental cues. The results demonstrate that expression of the pluripotent factor Oct4 is progressively downregulated and is accompanied by the gradual upregulation of neural genes, whereas the pluripotent factor Sox2 is consistently expressed at high levels, indicating that these pluripotent factors may play different roles in the regulation of neural differentiation. The identification of TR-S4+ cells provides a cell model for further elucidation of the molecular mechanisms underlying hESC neural differentiation (Noisa, 2012).

SOX2 co-occupies distal enhancer elements with distinct POU factors in ESCs and NPCs to specify cell state

SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, there is no detailed understanding of how SOX2 controls these distinct stem cell populations. This study shows by genome-wide analysis that, while SOX2 binds to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 binds the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. Similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupies a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs leads to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. These findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors (Lodato, 2013).

Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons

Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. This study revealed a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an 'on-target' pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myelin transcription factor 1 (Myt1l) zinc finger transcription factor do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, this study identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types (Wapinski, 2013).

drifter: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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