seven up


Evolution of nuclear receptors

A comparative tree of DNA-binding domain amino acid sequences reveals the evolutionary affinities of Drosophila nuclear receptor proteins. Knirps shows no close affinities to other nuclear receptor proteins. Drosophila Ecdysone receptor sequence is most similar to murine RIP14. Tailless has a close affinity to murine Tlx. Drosophila E78 and E75 fall in the same subclass as Rat Reverb alpha and beta, and C. elegans "CNR-14." Drosophila HR3 is in the same subclass as C. elegans "CNR-3." Drosophila HNF-4 is most closely related in sequence to Rat HNF-4. Drosophila Ftz-F1 and Mus ELP show sequence similarity to each other. Drosophila Seven up is closely related to Human COUP-TF. Drosophila Ultraspiracle is in the same subfamily as Human RXRalpha, Human RXRbeta, and Murine RXRgamma. The latter two groups, containing Ultraspiracle and Seven up, show a distant affinity to each other. Four other subfamilies show no close Drosophila affinities. These are: 1) C. elegans rhr-2, 2) Human RARalpha, beta and gamma, 3) Human thyroid hormone receptor alpha and beta, and 4) Human growth hormone receptor, glucocorticoid receptor, and progesterone receptor (Sluder, 1997).

From a database containing sequences of published nuclear hormone receptors (NRs), an alignment of the C, D and E domains of NR transcription factors was constructed. Using this alignment, tree reconstruction was performed using both distance matrix and parsimony analysis. The robustness of each branch was estimated using bootstrap resampling methods. The trees constructed by these two methods gave congruent topologies. From these analyses six NR subfamilies were derived: (I) a large clustering of thyroid hormone receptors (TRs), retinoic acid receptors (RARs), peroxisome proliferator-activated receptors (PPARs), vitamin D receptors (VDRs) and ecdysone receptors (EcRs) as well as numerous orphan receptors such as RORs or Rev-erbs; (II) retinoid X receptors (RXRs) together with COUP, HNF4, tailless, TR2 and TR4 orphan receptors; (III) steroid receptors; (IV) NGFIB orphan receptors; (V) FTZ-F1 orphan receptors; and finally (vi) only one gene (to date), the GCNF1 orphan receptor. The relationships between the six subfamilies are not known except for subfamilies I and IV, which appear to be related. Interestingly, most of the liganded receptors appear to be derived when compared with orphan receptors. This suggests that the ligand-binding ability of NRs has been gained by orphan receptors during the course of evolution to give rise to the presently known receptors. The distribution into six subfamilies correlates with the known abilities of the various NRs to bind to DNA as homo- or hetero-dimers. For example, receptors heterodimerizing efficiently with RXR belong to the first or the fourth subfamilies. It is suggested that the ability to heterodimerize evolved once, just before the separation of subfamilies I and IV and that the first NR was able to bind to DNA as a homodimer. From the study of NR sequences existing in vertebrates, arthropods and nematodes, two major steps of NR diversification have been defined: one that took place very early, probably during the multicellularization event leading to all the metazoan phyla, and a second occurring later on, corresponding to the advent of vertebrates. In vertebrate species, the various groups of NRs accumulated mutations at very different rates (Laudet, 1997).

Invertebrate Coup-TF family members

In a search for retinoid X receptor-like molecules in Drosophila, an additional member of the nuclear receptor superfamily, XR78E/F has been identified. In the DNA-binding domain, XR78E/F is closely related to the mammalian receptor TR2, as well as to the nuclear receptors Coup-TF and Seven-up. XR78E/F binds as a homodimer to direct repeats of the sequence AGGTCA. In transient transfection assays, XR78E/F represses ecdysone signaling in a DNA-binding-dependent fashion. XR78E/F has its highest expression in third-instar larvae and prepupae. These experiments suggest that XR78E/F may play a regulatory role in the transcriptional cascade triggered by the hormone ecdysone in Drosophila (Zelhof, 1995b).

The precisely defined sets of nerve and muscle cells responsible for locomotion in C. elegans allow genetic and cellular manipulations that provide a unique opportunity for investigating the generation of specific synaptic patterns. The sinuous forward and backward locomotion exhibited by C. elegans is produced by two neural circuits; one dedicated to forward movement and the other dedicated to backward movement. These two circuits converge on the dorsal and ventral body wall muscles and on two classes of inhibitory motor neurons: six dorsal D (DD) motor neurons (born embryonically) and 13 ventral (VD) motor neurons (born postembryonically). In adults, the DD and VD motor neurons form a cross-inhibitory network. A common genetic program is responsible for establishing the cellular morphology and maintaining the neurotransmitter profile (cholinergic inputs and GABAergic outputs) in both classes. However, none of the identified genes influences the synaptic patterns that distinguish the VD motor neurons from the DD motor neurons. Mutations in the gene unc-55 (uncoordinated) cause the VD motor neurons to adopt the synaptic pattern of the DD motor neurons and result in an asymmetric locomotive pattern when the animals move backward (Zhou, 1998 and references). unc-55 encodes a member of the nuclear hormone receptor gene family that is similar to the vertebrate chicken ovalbumin upstream promoter transcription factors. UNC-55 is expressed in the VD but not the DD motor neurons and functions to modify this genetic program and to create the synaptic pattern that distinguishes the two motor neuron classes from one another (Zhou, 1998).

In newly hatched animals the six embryonic DD motor neurons receive dorsal input and innervate ventral muscles, a synaptic pattern that is identical to the adult VD motor neurons. During the molt between the L1 and L2 stages when the VD motor neurons form their synapses, the DD motor neurons respecify their synapses and assume their adult innervation pattern. Thus both D motor neuron classes undergo synaptogenesis as the animal completes its first postembryonic molt, a period when the animal is immobile. This remodeling of the inhibitory neural circuits resembles the changes in identified neurons of certain holometabolous insects during metamorphosis. The insect molt is regulated by ecdysone, via the ecdysone receptor, which is also a member of the nuclear hormone receptor gene family. The reorganization of the inhibitory neural circuits coincides with the first molt in C. elegans. Particularly intriguing is the identification of a nuclear hormone receptor (UNC-55) that is in the same gene family as a number of Drosophila receptors that are involved in neural differentiation and are responsive to ecdysone. Although the hormone that initiates molting in C. elegans has not been identified, it is conceivable that UNC-55 in the VD motor neurons is activated by a hormone in conjunction with the molt cycle. Activation of these receptors could stabilize the synaptic pattern of the VD motor neurons by suppressing a response to a general (hormonal) signal that instructs the DD motor neurons to respecify their synaptic pattern and establish ventral inputs and dorsal outputs. In the absence of UNC-55 the VD motor neurons respond to the molt signal by default and form a synaptic pattern that is identical to the DD motor neurons. Specifically, it is proposed that activated UNC-55 receptors modify the expression of the common D motor neuron genetic program so that in the VD motor neurons proteins targeted for the presynaptic and postsynaptic processes are not redirected, whereas similar proteins in the DD motor neurons are redirected, thereby creating the synaptic pattern that distinguishes the two related classes of motor neurons (Zhou, 1998 and references therein).

When challenged by the dietary soybean cysteine protease inhibitor scN, the cowpea bruchid (Callosobruchus maculatus) adapts to the inhibitory effects by readjusting the transcriptome of its digestive system, including the specific activation of a cathepsin B-like cysteine protease CmCatB. To understand the transcriptional regulation of CmCatB, a portion of its promoter was cloned in Drosophila and its activity was demonstrated in Drosophila cells using a chloramphenicol acetyltransferase reporter system. EMSAs detected differential DNA-binding activity between nuclear extracts of scN-adapted and -unadapted midguts. Two tandem chicken ovalbumin upstream promoter (COUP) elements were identified in the CmCatB promoter that specifically interacted with a protein factor unique to nuclear extracts of unadapted insect guts, where CmCatB expression was repressed. Seven-up (Svp) is a COUP-TF-related transcription factor that interacts with the COUP responsive element. Polyclonal anti-(mosquito Svp) serum abolished the specific DNA-binding activity in cowpea bruchid midgut extracts, suggesting that the protein factor is an Svp homolog. Subsequent cloning of a cowpea bruchid Svp (CmSvp) indicated that it shares a high degree of amino acid sequence similarity with COUP-TF/Svp orphan nuclear receptor family members from varied species. The protein was more abundant in scN-unadapted insect guts than scN-adapted guts, consistent with the observed DNA-binding activity. Furthermore, CmCatB expression was repressed when CmSvp was transiently expressed in Drosophila cells, most likely through COUP binding. These findings indicate that CmSvp may contribute to insect counter-defense, in part by inhibiting CmCatB expression under normal growth conditions, but releasing the inhibition when insects are challenged by dietary protease inhibitors (Ahn, 2007).

The nuclear receptor SpCOUP-TF is the highly conserved sea urchin homolog of the COUP family of transcription factors. SpCOUP-TF transcripts are localized in the egg and asymmetrically distributed in the early embryonic blastomeres. To examine the subcellular localization of SpCOUP-TF protein, polyclonal antibodies were separately raised against the divergent N-terminus as well as the conserved DNA-binding and ligand-binding domains. Immunohistochemical analyses suggest that SpCOUP-TF is a maternal protein residing in the cytoplasm of the unfertilized egg. After fertilization, and as soon as the two-cell-stage embryo, most of the receptor translocates from the cytoplasm to the cell nuclei. During the rapid embryonic cell division, SpCOUP-TF shuttles from the interphase nuclear periphery to the condensed chromosomes in mitosis, in a cell-cycle-dependent manner. In an attempt to confirm these observations, the subcellular localization of myc-tagged human COUP-TF I introduced into the sea urchin embryo by RNA injection of fertilized eggs was examined. The pattern of human COUP-TF I subcellular localization, detected with a monoclonal myc antibody, recapitulates the essential features described for the endogenous SpCOUP-TF trafficking. Replacement of the N-terminus of the human receptor with the unique sea urchin N-terminus enhances COUP-TF's localization to the nuclear rim during interphase. Deletion of the DNA-binding domain of human COUP-TF I results in loss of all aspects of nuclear periphery and chromosomal localization. Taken together these data suggest that SpCOUP-TF transcriptional activity is keyed on a cell-cycle-dependent mechanism that regulates chromosomal protein traffic (Vlahou, 2000).

It is reasonable to propose that both the nuclear envelope and the chromosomal associations of COUP-TF are coupled events, which may be mediated through shared protein-DNA and protein-protein interactions. These interactions may ensure that: (1) SpCOUP-TF remains in proximity to the DNA, and in this way, it can mediate transcriptional regulation of early genes in the interphase nuclei, and (2) following mitosis, SpCOUP-TF is readily redistributed to the nuclei of the daughter cells. The strict time constraints imposed by the rapid embryonic cell divisions may not allow the factor to enter rapidly into the daughter nuclei if released in the cytoplasm during prophase. For that reason, SpCOUP-TF may segregate with the fraction of lamins that remain chromosomal and, thus, use the chromosomes as a vehicle for its conveyance to the daughter nuclei. If this is the case, SpCOUP-TF might be considered a 'chromosomal passenger,' a term that has been used for the INCENP antigens. These have been shown to transiently bind the mitotic chromosomes in order to ensure their transfer to cytoskeletal elements of the mitotic spindle. Alternatively, it is also possible that SpCOUP-TF is actively involved and plays a regulatory role in the organization of chromatin during mitosis. The nuclear envelope and lamina provide structural support and play a coordinate role during the first steps of chromatin condensation. However, after the nuclear envelope and lamina are dismantled, this role is hypothesized to be undertaken by peripheral nuclear matrix proteins. If SpCOUP-TF acts as a bona fide nuclear matrix protein, it may contribute to the structural integrity of the chromosomes during mitosis (Vlahou, 2000 and references therein).

In bilaterians, COUP-TF nuclear receptors participate in neurogenesis and/or CNS patterning. In hydra, the nervous system is formed of sensory mechanoreceptor cells (nematocytes) and neuronal cells, both lineages deriving from a common stem cell. The hydra COUP-TF gene, hyCOUP-TF, which encodes highly conserved DNA-binding and ligand-binding domains, belongs to the monophyletic COUP-TFs orphan receptor family (NR2F). It is 44.6% homologous to Drosophila Seven up. In adult polyps, hyCOUP-TF is expressed in nematoblasts and a subset of neuronal cells. Comparative BrDU labeling analyses performed on cells expressing either hyCOUP-TF or the paired-like gene prdl-b show that prdl-b expression corresponds to early stages of proliferation, while hyCOUP-TF is detected slightly later. HyCOUP-TF and prdl-b expressing cells disappear in sf-1 mutants becoming 'nerve-free'. Moreover hyCOUP-TF and prdl-b expression was excluded from regions undergoing developmental processes. These data suggest that hyCOUP-TF and prdl-b belong to a genetic network that appeared together with neurogenesis during early metazoan evolution. The hyCOUP-TF protein specifically binds onto the evolutionarily conserved DR1 and DR5 response elements, and represses transactivation induced by RAR:RXR nuclear receptors in a dose-dependent manner when expressed in mammalian cells. Hence, a cnidarian transcription factor can be active in vertebrate cells, implying that functional interactions between COUP-TF and other nuclear receptors are evolutionarily conserved (Gauchat, 2004).

The type 1 SVP protein is a homolog of the human transcription factor COUP. Over the entire COUP protein 75% of the amino acids are identical to SVP. In the DNA binding domain, located from amino acids 200 to 267, homology is 94%, and in the putative ligand binding domain (C-terminal) there is 93% identity over 222 amino acids. The sequence of the type 2 cDNA fails to align with the ligand binding domain of members of the steroid receptor superfamily (Mlodzik, 1990).

Coup-TF family members in vertebrates

The protein encoded by the zebrafish gene svp[40] belongs to a distinct group within the steroid hormone receptor superfamily that includes Drosophila Seven-up and several vertebrate orphan receptors. Svp[40] shares a particularly high degree of amino acid sequence identity (approximately 86%) with the mammalian transcription factors ARP-1 and COUP. The gene is expressed in specific regional and segmental domains within the developing brain. During the early embryonic stages when hindbrain rhombomeres are formed, a segmental expression pattern is established as a step gradient, coinciding directly with the four anteriormost segments. This suggests a role in controlling rhombomere-specific expression of genes contributing to cell differentiation in the hindbrain. Treatment of zebrafish embryos with retinoic acid affects the svp[40] step gradient and causes an elimination of a regional expression domain in the retina. These observations are consistent with svp[40] being an integral part of the retinoid signaling network during hindbrain development (Fjose, 1995).

There are two other zebrafish svp homologs: svp[44] is the zebrafish cognate of COUP, while svp[46] seems to be a novel member of the COUP/svp group. The CNS is a major site of expression for both genes. At early embryonic stages, both genes are expressed in domains corresponding to specific rhombomere primordia in the hindbrain. This suggests an involvement in hindbrain segmentation and/or rhombomere specification. Moreover, transcripts derived from both genes are detected within distinct areas of the eye rudiments, suggesting roles in eye patterning and/or cell differentiation (Fjose, 1993).

A Sonic hedgehog (See Drosophila Hedgehog) response element was identified in the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) promoter. The Shh response element binds to a factor distinct from Gli, a gene known to mediate Shh signaling. Although this binding activity is specifically stimulated by Shh-N (amino-terminal signaling domain), it can also be unmasked with protein phosphatase treatment in the mouse cell line P19, and induction by Shh-N can be blocked by phosphatase inhibitors. Thus, Shh-N signaling may result in dephosphorylation of a target factor that is required for activation of COUP-TFII-, Islet1-, and Gli response element-dependent gene expression. This finding identifies another step in the Shh-N signaling pathway. The phosphatase that mediates this dephosphorylation in response to Shh-N treatment is PP2A or is like PP2A (see Drosophila Twins). This particular response is channeled through a protein with DNA binding activity apparently unrelated to that of the Ci/Gli family. A similar protein phosphatase activity is also required in the Ci/Gli-mediate branch of the Drosophila Hh signaling pathway (Krishnan, unpublished result). Thus, activation of specific protein phosphatase activity appears to be a general feature of Hh signal transduction (Krishnan, 1997).

The COUP-TFs are highly expressed in the developing nervous systems of several species, indicating their possible involvement in neuronal development and differentiation. In the mouse, there are two very homologous COUP-TF genes (I and II); their expression patterns overlap extensively. To study the physiological function of mCOUP-TFI, a gene-targeting approach was undertaken. mCOUP-TFI null animals die perinataly. Mutant embryos display an altered morphogenesis of the ninth cranial ganglion and nerve. The aberrant formation of the ninth ganglion is most possibly attributable to extra cell death in the neuronal precursor cell population. In addition, at midgestation, aberrant nerve projection and arborization are observed in several other regions of mutant embryos. These results indicate that mCOUP-TFI is required for proper fetal development and is essential for postnatal development. mCOUP-TFI possesses vital physiological functions that are distinct from mCOUP-TFII, despite their high degree of homology and extensive overlapping expression patterns (Qui, 1997).

Members of the steroid/thyroid hormone receptor superfamily are involved in the control of cell identity and of pattern formation during embryonic development. Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) can act as regulators of various steroid/thyroid hormone receptor pathways. To begin to study the role of COUP-TFs during embryogenesis, a chicken COUP-TF (cCOUP-TF II) has been cloned that is highly homologous to human COUP-TF II. Northern analysis reveals high levels of cCOUP-TF II transcripts during organogenesis. Nuclear extracts from whole embryos and from embryonic spinal cords were used in electrophoretic mobility shift assays. These assays show that COUP-TF protein is present in these tissues and is capable of binding to a COUP element (a direct repeat of AGGTCA with one base pair spacing). Analysis of cCOUP-TF expression by in situ hybridization reveals high levels of cCOUP-TF II mRNA in the developing spinal motor neurons. Since the ventral properties of the spinal cord, including the development of motor neurons, is in part established by inductive signals from the notochord, an additional notochord was transplanted next to the dorsal region of the neural tube in order to induce ectopic motor neurons. An ectopic notochord induces cCOUP-TF II gene expression in the dorsal spinal cord in a region coextensive with ectopic domains of SC1 and Islet-1, two previously identified motor neuron markers. Collectively, these studies raise the possibility that cCOUP-TF II is involved in motor neuron development (Lutz, 1994).

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements that are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 were shown to bind specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, form DNA-protein complexes that have mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies are able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enables an eightfold increase in the hypoxic induction of a luciferase reporter construct that contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer are intact. The augmentation by HNF-4 in HeLa cells can be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene is significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. In contrast, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppresses the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role (Galson, 1995).

The 5'-region of the murine N-methyl-d-aspartate (NMDA) receptor channel subunit NR2C (GluRepsilon3) gene has been cloned and the cis- and trans-activating regulatory elements responsible for its tissue specific activity have been characterized. By using a native epsilon3-promoter/lacZ-construct and various 5'-deletion constructs, beta-galactosidase expression in non-neuronal NIH3T3 cells and in neuronal epsilon3-gene-expressing HT-4 cells were compared. Large parts of the epsilon3 promoter are shown to be responsible for the repression of the epsilon3 gene in non-neuronal cells. Deletion of exon 1 sequences leads to an enhancement of epsilon3 transcription, suggesting a role for the 5'-untranslated region in epsilon3 gene regulation. Sequence analysis of the promoter region reveals potential binding sites for the transcription factor Sp1, the murine fushi tarazu factor1 (FTZ-F1) homologs, embryonic LTR binding proteins (ELP1,2,3) and steroidogenic factor (SF-1), as well as for the chicken ovalbumin upstream promoter transcription-factor (COUP-TF). Electrophoretic mobility shift assays confirm specific binding of Sp1, SF-1 and COUP-TFI. Whereas point mutation studies indicate that, in neuronal HT-4 cells, Sp1 is apparently not critically involved in basal epsilon3 gene transcription, SF1 is a positive regulator. This was evident from a selective enhancement of epsilon3-promoter-driven reporter gene expression upon cotransfection of an SF1-expression vector, which was reverted by deletion and point mutation of the SF1 binding site (Pieri, 1999).

The Dax-1 gene encodes a protein that is structurally related to members of the orphan nuclear receptor superfamily. Dax-1 is coexpressed with another orphan nuclear receptor, steroidogenic factor-1 (SF-1), in the adrenal, gonads, hypothalamus, and pituitary gland. Mutations in Dax-1 cause adrenal hypoplasia congenita, a disorder that is characterized by adrenal insufficiency and hypogonadotropic hypogonadism. These developmental and endocrine abnormalities are similar to those caused by disruption of the murine Ftz-F1 gene (which encodes SF-1), suggesting that these nuclear receptors act along the same developmental cascade. Cloning of the murine Dax-1 gene revealed a candidate SF-1-binding site in the Dax-1 promoter. In transient expression assays in SF-1-deficient JEG-3 cells, SF-1 stimulates expression of the Dax-1 promoter. However, deletion or mutation of the consensus SF-1-binding site does not eliminate SF-1 stimulation. Further analyses have revealed the presence of a cryptic SF-1 site that creates an imperfect direct repeat of the SF-1 element. When linked to the minimal thymidine kinase promoter, each of the isolated SF-1 sites is sufficient to mediate transcriptional regulation by SF-1. Mutation of both SF-1 sites eliminates SF-1 binding and stimulation of the Dax-1 promoter. Unexpectedly, mutation of either half of the composite SF-1 sites increases basal activity in JEG-3 cells, suggesting interaction of a repressor protein. Gel shift analyses of the composite response element reveals an additional complex that is not supershifted by SF-1 antibodies. This complex was eliminated by mutation of either half-site, and it was supershifted by antibodies against chicken ovalbumin upstream promoter-transcription factor (COUP-TF). It is proposed that Dax-1 is stimulated by SF-1, and that SF-1 and COUP-TF provide antagonistic pathways that converge upon a common regulatory site (Yu, 1998).

The polyomavirus enhancer binding protein 2alphaB (AML1/PEBP2alphaB/Cbfa2) plays a pivotal role in granulocyte colony-stimulating factor (G-CSF)-mediated differentiation of a myeloid progenitor cell line, 32Dc13. PEBP2alphaB interacting protein, Ear-2, is an orphan member of the nuclear hormone receptor superfamily that directly binds to and can inhibit the function of PEBP2alphaB. Ear-2 is expressed in proliferating 32Dc13 cells in the presence of interleukin 3 but is down-regulated during differentiation induced by G-CSF. Interestingly, AML1/ETO(MTG8), a leukemogenic chimeric protein can block the differentiation of 32Dc13 cells, which is accompanied by the sustained expression of ear-2. Overexpression of Ear-2 can prevent G-CSF-induced differentiation, strongly suggesting that ear-2 is a key negative regulator of granulocytic differentiation. These results indicate that a dynamic balance existing between PEBP2alphaB and Ear-2 appears to determine the choice between growth or differentiation for myeloid cells. Ear-2 is a homolog of Drosophila Seven-up, which is known to be negatively regulated by Lozenge, a Runt-domain-encoding homolog of AML1 (Ahn, 1998).

The embryonic expression of COUP-TFII, an orphan nuclear receptor, suggests that it may participate in mesenchymal-epithelial interactions required for organogenesis. Targeted deletion of the COUP-TFII gene results in embryonic lethality with defects in angiogenesis and heart development. COUP-TFII mutants are defective in remodeling the primitive capillary plexus into large and small microcapillaries. In the COUP-TFII mutant heart, the atria and sinus venosus fail to develop past the primitive tube stage. Reciprocal interactions between the endothelium and the mesenchyme in the vascular system and heart are essential for normal development of these systems. In fact, the expression of Angiopoietin-1, a proangiogenic soluble factor thought to mediate the mesenchymal-endothelial interactions during heart development and vascular remodeling, is down-regulated in COUP-TFII mutants. This down-regulation suggests that COUP-TFII may be required for bidirectional signaling between the endothelial and mesenchymal compartments essential for proper angiogenesis and heart development (Pereira, 1999).

Laminin expression and the subsequent deposition of a basement membrane by primitive endoderm cells is necessary for early mammalian development. The transcription factors COUP-TF I and II are up-regulated in primitive endoderm cells faster than LAMB1 and LAMC1, and either COUP-TF is sufficient to induce expression of these laminin genes (Murray, 2001).

COUP-TFII, an orphan member of the steroid receptor superfamily, has been implicated in mesenchymal-epithelial interaction during organogenesis. The generation of a lacZ knock-in allele in the COUP-TFII locus in mice allows use of X-gal staining to follow the expression of COUP-TFII in the developing stomach. COUP-TFII is expressed in the mesenchyme and the epithelium of the developing stomach. Conditional ablation of floxed COUP-TFII by Nkx3-2Cre recombinase in the gastric mesenchyme results in dysmorphogenesis of the developing stomach manifested by major patterning defects in posteriorization and radial patterning. The epithelial outgrowth, the expansion of the circular smooth muscle layer and enteric neurons as well as the posteriorization of the stomach resemble phenotypes exhibited by inhibition of hedgehog signaling pathways. Using organ cultures and cyclopamine treatment, downregulation of COUP-TFII level was shown in the stomach, suggesting COUP-TFII as a target of hedgehog signaling in the stomach. These results are consistent with a functional link between hedgehog proteins and COUP-TFII, factors that are vital for epithelial-mesenchymal interactions (Takamoto, 2005).

The nuclear hormone receptor Coup-TFII is required for the initiation and early maintenance of Prox1 expression in lymphatic endothelial cells

The homeobox gene Prox1 is crucial for mammalian lymphatic vascular development. In the absence of Prox1, lymphatic endothelial cells (LECs) are not specified. The maintenance of LEC identity also requires the constant expression of Prox1. However, the mechanisms controlling the expression of this gene in LECs remain poorly understood. The SRY-related gene Sox18 is required to induce Prox1 expression in venous LEC progenitors. Although Sox18 is also expressed in embryonic arteries, these vessels do not express Prox1, nor do they give rise to LECs. This finding suggests that some venous endothelial cell-specific factor is required for the activation of Prox1. This study demonstrates that the nuclear hormone receptor Coup-TFII is necessary for the activation of Prox1 in embryonic veins by directly binding a conserved DNA domain in the regulatory region of Prox1. In addition, it was shown that the direct interaction between nuclear hormone receptors and Prox1 is also necessary for the maintenance of Prox1 expression during early stages of LEC specification and differentiation (Srinivasan, 2010).

COUP-TF1 and the regionalization of the neocortex

Regionalization of the cerebral cortex is thought to involve two phases: an early regionalization phase and a later refinement phase. It has been shown that early regionalization of the neocortex does not require thalamic inputs and is regulated by intrinsic factors. Recently, two such intrinsic factors, Pax6 and Emx2, have been identified. In this study, COUP-TFI has been identified as a regulatory factor for early neocortical regionalization. The spatial and temporal expression pattern of COUP-TFI suggests a role in specification of the neocortex and in maintaining cortical identity. Altered region-specific expression of marker genes in the cortex as well as miswired area-specific connections between the cortex and the thalamus in COUP-TFI null mice indicate COUP-TFI plays a critical role in regulating early regionalization. These results substantiate that COUP-TFI, an intrinsic factor, may work in concert with Pax6 and Emx2 to specify neocortical identity (Zhou, 2001).

At E11.5, the onset of cerebral corticogenesis, COUP-TFI expression shows a graded pattern in the neocortex with high-lateral to low-medial expression in the frontal view, and high-caudal to low-rostral expression in the sagittal view. Section in situ hybridization indicates that the high caudolateral COUP-TFI expression gradient is maintained in the cortical plate even after birth. This COUP-TFI expression gradient in the superficial layers (layers 2/3) was also confirmed by whole-mount, in situ hybridization. This spatial and temporal expression pattern makes COUP-TFI a likely candidate for a regionalization regulatory factor (Zhou, 2001).

The potential role of COUP-TFI in early regionalization was examined by investigating the region-specific expression of marker genes including Id2, RORß, and Cadherin 8, which has been used to assess the regionalization of the neocortex in P3/P4 COUP-TFI mutant cortices. Id2, a helix-loop-helix transcription factor, is expressed in a region- and lamina-specific manner. Id2 transcripts are detected in the subplate and layers 6, 5, and 2/3. Id2 transcripts can be detected in the upper part of layer 5 and stop abruptly at a rostral boundary that has been suggested to lie between motor and somatosensory cortices. Id2 expression in layers 2/3 is restricted to the rostral part of the cortex with a boundary within the somatosensory area. However, the lamina-specific expression pattern is lost in COUP-TFI mutants, which may be because of the greatly reduced number of layer-4 neurons and subplate neurons in COUP-TFI mutants as well as the uniform expression of Id2 in layer 5. More importantly, the rostral boundary in layer 5 that separates motor and sensory cortices was not apparent, and Id2 also is expressed throughout the rostro-caudal axis in layers 2/3 in COUP-TFI mutants, suggesting that COUP-TFI is required for regional expression of Id2 in layers 5 and 2/3 neurons (Zhou, 2001).

RORß, which encodes an orphan nuclear receptor, is specifically expressed in layer 4 neurons. RORß expression is greatly reduced at P0 in COUP-TFI mutants because of the death of layer 4 neurons. Since the purpose of this investigation was to study the regional expression of RORß rather than compare expression levels, images of RORß transcript were taken in P3 COUP-TFI mutants at a much longer exposure time (about twice as long for mutants). RORß expression in P3 control-mouse cortex displays a distinct regional pattern with a caudal boundary within the somatosensory cortex and a region of much reduced expression in a more rostral position. However, in mutant cortex, uniform expression of RORß is detected from rostral to caudal cortex with expanded caudal expression indicating that COUP-TFI controls the region-specific expression of RORß (Zhou, 2001).

Cadherin 8 belongs to the type II class of cadherin, a family of cell adhesion molecules that are important for morphogenesis of the central nervous system. At P3, in situ hybridization assay reveals that Cadherin 8 is uniformly expressed in layer 5 while its expression in layers 2/3 and 4 is restricted to the rostral part of the neocortex. However, in P3 mutant cortex, Cadherin 8 expression in layers 2/3 and 4 loses its rostral restriction and can be detected throughout the rostral-caudal axis, while expression in layer 5 is unchanged. The expression of Cadherin 8 in superficial layers (layers 2/3) is also detected. Cadherin 8 is expressed in the frontal area of the neocortex with a boundary around the limit of motor cortex in P0 control brains. The rostral Cadherin 8 expression boundary in superficial layers is shifted caudally, especially in the medial region, in P0 mutant. Therefore, the loss of restricted rostral expression of Cadherin 8 in layers 2/3 and 4 indicates that COUP-TFI is required for region-specific expression of Cadherin 8. Taken together, the region-specific expression of many genes including Id2, RORß, and Cadherin 8 around P4, when cortical lamination is close to completion, is altered in COUP-TFI mutants. This indicates that normal neocortical regionalization requires COUP-TFI. Since expression of these marker genes is regulated by intrinsic mechanisms and is independent of thalamocortical innervation, these findings establish COUP-TFI as one of the intrinsic regulatory factors that control the specification of the neocortex (Zhou, 2001).

Interestingly, COUP-TFI itself also is expressed in a region-specific manner at P4. Although COUP-TFI transcripts are detected throughout the cortex, a higher expression in the rostral part of layers 2/3 and 4 is observed and it correlates with Cadherin 8 expression in layers 2/3 and 4. In addition, COUP-TFI is highly expressed in layer 4 neurons with several boundaries that resemble the expression pattern of RORß. Although the expression pattern of COUP-TFI in the postnatal cortex correlates with components of some marker genes, it differs considerably from other expression patterns, suggesting that COUP-TFI is important for maintaining region-specific gene expression of some but not all of the markers examined (Zhou, 2001).

Because expression of COUP-TFI displays a region-specific pattern very early (it can be detected at E11.5) during corticogenesis, the expression patterns of Id2, RORß, and Cadherin 8 were examined in late embryonic development. E17.5 was chosen because this is a time when generation of cortical plate neurons has been completed (around early E17) and massive innervation of the cortical plate by thalamic axons has not yet started. However, at this stage, most cortical plate neurons still are migrating along radial glia, so layer-specific expression cannot be identified. Id2 expression displays a high-caudal to low-rostral gradient in control cortex, including the cortical plate and the ventricular zone. Its expression in mutants, though, shows a uniform rostral-to-caudal expression throughout the cortex. RORß expression in control cortex shows a high-rostral to low-caudal gradient in the cortical plate (layer 4 neurons), which is interrupted in the future somatosensory cortex. Mutation of COUP-TFI results in the uniform expression of RORß, and in it having a more caudal boundary (Zhou, 2001).

Expression of Cadherin 8 in control cortical plate shows a high-medial to low-lateral pattern, while its transcripts display a high-lateral to low-medial pattern in the mutant cortices. Taken together, COUP-TFI regulates region-specific expression of Id2, RORß, and Cadherin 8 before E17.5. The fact that graded or region-specific expression of these marker genes is lost at E17.5, a time before thalamic axons innervate the cortical plate, again confirms that early regionalization of the neocortex is independent of thalamic innervation. Therefore, specification of the neocortex by COUP-TFI occurs shortly after the fate of cortical plate neurons is determined and before the innervation of the cortical plate by thalamic axons takes place (Zhou, 2001).

The intrinsic factors, Pax6 and Emx2 also regulate the region-specific expression of Cadherin 6 and Cadherin 8. To elucidate the possible link between COUP-TFI and Pax6/Emx2, the graded expression of Pax6 and Emx2 was investigated in COUP-TFI mutants. In a pattern similar to that observed in the control cortices, Pax6 transcripts are detected in a high-rostral to low-caudal gradient in sagittal sections and in a high-lateral to low-medial gradient in coronal sections in E13.5 mutant cortices. The gradient expression of Emx2, which normally is in a high-caudal to low-rostral and high-medial to low-lateral pattern, also is maintained in COUP-TFI mutant cortices at E13.5. The unchanged, graded expression of Pax6 and Emx2 in COUP-TFI mutant cortices indicates that COUP-TFI, Pax6, and Emx2 might function independently in regulating early regionalization of the neocortex. In addition, the fact that COUP-TFI is expressed not only in the ventricular zone but also in the cortical plate, while Pax6 and Emx2 are expressed mainly in the ventricular zone with complementary expression gradients suggests that COUP-TFI does not act downstream of Pax6 or Emx2 in the cortical neurons. Additionally, the difference in altered expression profiles (e.g., Cadherin 8) between COUP-TFI null mice and Pax6 and Emx2 mutants further suggests that COUP-TFI and Pax6/Emx2 act through different pathways and these intrinsic factors work in concert to establish the identity of each subdivision of the neocortex (Zhou, 2001).

While the graded expression of Pax6 and Emx2 is lost around E15.5 and E16.5, respectively, the graded COUP-TFI expression is maintained in the neocortex even after birth. Furthermore, COUP-TFI transcript can be detected throughout the cortex during corticogenesis while Pax6 and Emx2 are mainly confined to the ventricular zone. In addition, COUP-TFI expression at P4 also is region-specific and certain domains correlate well with those of Cadherin 8 and RORß. These findings suggest that COUP-TFI is not only required for initiating the early regionalization of the neocortex but also may play a role in maintaining such regional identity, while Pax6 and Emx2 mainly function in initiation of the early regionalization (Zhou, 2001).

The molecular mechanisms behind how intrinsic factors such as COUP-TFI, Pax6, and Emx2 regulate region-specific gene expression still is largely unknown. It is not known if intrinsic factors act directly or indirectly in regulating transcription of marker genes. The complexity and diversity of the expression patterns suggest that early regional specification is achieved through the cooperation of multiple intrinsic factors. As a transcription factor, COUP-TFI regulates gene transcription through both transrepression and transactivation mechanisms. It will be interesting to determine how COUP-TFI controls the region-specific expression of those marker genes. Moreover, understanding the mechanism whereby the graded expression of COUP-TFI, Pax6, and Emx2 is established will greatly enhance understanding of how neocortical regionalization takes place (Zhou, 2001).

Cells migrate via diverse pathways and in different modes to reach their final destinations during development. Tangential migration has been shown to contribute significantly to the generation of neuronal diversity in the mammalian telencephalon. GABAergic interneurons are the best-characterized neurons that migrate tangentially, from the ventral telencephalon, dorsally into the cortex. However, the molecular mechanisms and nature of these migratory pathways are only just beginning to be unravelled. A novel dorsal-to-ventral migratory route has been identified, in which cells migrate from the interganglionic sulcus, located in the basal telencephalon between the lateral and medial ganglionic eminences, towards the pre-optic area and anterior hypothalamus in the diencephalon. With the help of transplantations and gain-of-function studies in organotypic cultures, it was shown that COUP-TFI and COUP-TFII are expressed in distinct and non-overlapping migratory routes. Ectopic expression of COUP-TFs induces an increased rate of cell migration and cell dispersal, suggesting roles in cellular adhesion and migration processes. Moreover, cells follow a distinct migratory path, dorsal versus ventral, which is dependent on the expression of COUP-TFI or COUP-TFII, suggesting an intrinsic role of COUP-TFs in guiding migrating neurons towards their target regions. Therefore, it is proposed that COUP-TFs are directly involved in tangential cell migration in the developing brain, through the regulation of short- and long-range guidance cues (Tripodi, 2005).

This report provides the first insights into the functional roles of COUP-TFI and COUP-TFII in neuronal migration in vivo. It has been shown that COUP-TFI can regulate cell adhesion mechanisms required for the differentiation of embryonal carcinoma cells. Because of the substrates on which cells were plated, it was suggested that COUP-TFI could modify the synthesis of ECM molecules, making cells autonomous for migration and spreading. The expression data in vivo and functional data in organotypic cultures support these results in a context in which cell migration assumes a fundamental role in achieving neuronal diversity. Expression of COUP-TFs has been demonstrated in highly motile neurons, such as GABAergic and reelin-positive cells; the co-localization of COUP-TFI in migrating neurons has been demonstrated in a series of grafting experiments, and a role of COUP-TFs in modulating cell migration has been directly demonstrated in a gain-of-function approach. The data also demonstrate that COUP-TFs can control directional migration. Indeed, cells expressing higher levels of COUP-TFs (and thus GFP) not only tend to spread and lose their aspect of aggregates, but also follow specific migratory pathways (dorsal and/or ventral) that are intrinsic to cells expressing COUP-TFs (Tripodi, 2005).

Cortex-specific deletion of the transcription factor gene COUP-TFI (also known as Nr2f1) in mice to demonstrate previously unknown fundamental roles for it in patterning mammalian neocortex into areas. The highest COUP-TFI expression is observed in the cortical progenitors and progeny in parietal and occipital cortex that form sensory areas, and the lowest expression was observed in frontal cortex that includes motor areas. Cortical deletion of COUP-TFI resulted in massive expansion of frontal areas, including motor, to occupy most of neocortex, paralleled by marked compression of sensory areas to caudal occipital cortex. These area patterning changes are preceded and paralleled by corresponding changes in molecular markers of area identity and altered axonal projections to maintain patterned area-specific input and output connections. It is concluded that COUP-TFI is required for balancing patterning of neocortex into frontal/motor and sensory areas by acting in its expression domain to repress frontal/motor area identities and to specify sensory area identities (Armentano, 2007).

COUP-TFI promotes radial migration and proper morphology of callosal projection neurons by repressing Rnd2 expression

During corticogenesis, late-born callosal projection neurons (CPNs), a diverse population of neocortical projection neurons that connect the two hemispheres of the cerebral cortex via the corpus callosum, acquire their laminar position through glia-guided radial migration and then undergo final differentiation. However, the mechanisms controlling radial migration and final morphology of CPNs are poorly defined. This study shows that in COUP-TFI mutant mice CPNs are correctly specified, but are delayed in reaching the cortical plate and have morphological defects during migration. Interestingly, it was observed that the rate of neuronal migration to the cortical plate normally follows a low-rostral to high-caudal gradient, similar to that described for COUP-TFI. This gradient is strongly impaired in COUP-TFI-/- brains. Moreover, the expression of the Rho-GTPase Rnd2, a modulator of radial migration, is complementary to both these gradients and strongly increases in the absence of COUP-TFI function. COUP-TFI directly represses Rnd2 expression at the post-mitotic level along the rostrocaudal axis of the neocortex. Restoring correct Rnd2 levels in COUP-TFI-/- brains cell-autonomously rescues neuron radial migration and morphological transitions. Impairments were also observed in axonal elongation and dendritic arborization of COUP-TFI-deficient CPNs, which were rescued by lowering Rnd2 expression levels. Thus, these data demonstrate that COUP-TFI modulates late-born neuron migration and favours proper differentiation of CPNs by finely regulating Rnd2 expression levels (Alfano, 2011).

Subcortical origins of human and monkey neocortical interneurons

Cortical GABAergic inhibitory interneurons have crucial roles in the development and function of the cerebral cortex. In rodents, nearly all neocortical interneurons are generated from the subcortical ganglionic eminences. In humans and nonhuman primates, however, the developmental origin of neocortical GABAergic interneurons remains unclear. This study shows that the expression patterns of several key transcription factors in the developing primate telencephalon are very similar to those in rodents, delineating the three main subcortical progenitor domains (the medial, lateral and caudal ganglionic eminences) and the interneurons tangentially migrating from them. On the basis of the continuity of Sox6, COUP-TFII and Sp8 transcription factor expression and evidence from cell migration and cell fate analyses, it is proposed that the majority of primate neocortical GABAergic interneurons originate from ganglionic eminences of the ventral telencephalon. These findings reveal that the mammalian neocortex shares basic rules for interneuron development, substantially reshaping understanding of the origin and classification of primate neocortical interneurons (Ma, 2013).

The basic organization of cortical microcircuits is conserved across mammals but also shows species differences. This study provides strong evidence that the majority of primate neocortical interneurons that express Sox6, Sp8 or COUP-TFII are derived from the subcortical ganglionic eminences, as they are in rodents. Although the results cannot eliminate the possibility that a small population of neocortical interneurons is generated from the human neocortical ventricular zone and SVZ, it is concluded that it is the subpallium, not the pallium, the major source of neocortical GABAergic interneurons in humans and nonhuman primates. Thus, genetic analyses of rodent cortical interneuron development and function should provide direct insights into the genetic and molecular mechanisms that can underlie human neuropsychiatric disorders involving interneuron dysfunction (Ma, 2013).

Using DiI to label ganglionic eminence cells in human fetal brain slices, previous studies have shown that a subpopulation of cells migrate into the neocortex from the ganglionic eminence. In the present study, it was also found that some adenoGFP-labeled cells migrated tangentially from ganglionic eminences toward the neocortex in E55 monkey brain slice cultures. Thus, tangential migration of interneurons from the subpallium to the pallium in humans, and perhaps all vertebrates, appears to be a shared trait. In rodents, nearly all cortical interneurons originate from the MGE, the dLGE and CGE and the preoptic area. However, in the human and monkey neocortex, the proportion of subcortical-derived interneurons is not clear. Indeed, it has been reported that Ascl1-expressing progenitors in the human neocortical proliferative zone give rise to two-thirds of neocortical GABAergic interneurons; however, it is not known into which subpopulation of interneurons they differentiate. By contrast, this study shows that Sox6+, Sp8+ and COUP-TFII+ interneurons that comprise the majority of primate neocortical interneurons are generated from ganglionic eminences. Previous studies have clearly identified that Ascl1 is expressed by progenitors for human neocortical glutamatergic projection neurons at GW15; similar findings have been obtained in mice. Thus, Ascl1 is not a specific marker of GABAergic lineages. Likewise, in the current study strong expression was found of Ascl1 in the ganglionic eminences; the majority of Ascl1+ cells in the cortical ventricular zone and SVZ expressed Pax6 and/or Tbr2 in E80 monkey brains and GW18 and GW24 human fetal brains. Moreover, none of these Ascl1+ cells in the neocortical ventricular zone and SVZ expressed GABA, GAD65 or GAD67. Thus, in rodents and primates, Ascl1 is a marker for progenitors of projection neurons in the pallium only. In the subpallium, Ascl1 is a marker for the progenitors of interneurons (Ma, 2013).

It is worth noting that cultured neocortical ventricular zone and SVZ progenitors from GW15 human fetal brains did not produce GABA+ interneurons. In this study, E55 monkey neocortical slices and dissociated cells were cultured with BrdU, and cortical progenitors were found to produce very few BrdU+GABA+ cells. Thus, cell fate analyses in brain slices and cell cultures further suggest that embryonic primate neocortical progenitors do not generate a large proportion of neocortical GABAergic interneurons (Ma, 2013).

In the early development of the mouse telencephalon, both Sox6 and Sp8 are expressed by the progenitors of glutamatergic projection neurons in the pallial ventricular zone and SVZ, in which they control pallial progenitor identity and regulate cortical patterning. In the subpallium, Sox6 is expressed mainly in the SVZ but not the ventricular zone of the MGE, although a small population of Sox6+ cells in the MGE SVZ are mitotic. In contrast, Sp8 is expressed mainly in the SVZ of the dLGE and CGE but not the MGE; many Sp8+ cells in the dLGE and CGE are capable of proliferation. Accordingly, Sox6 and Sp8 are expressed by nearly all MGE-derived and a large number of dLGE- and CGE-derived mouse cortical interneurons, respectively, from migrating to mature stages. The transcription factor COUP-TFII is expressed mainly in the mouse CGE, but a small population of COUP-TFII+ cells is also observed in the MGE and LGE. In the adult mouse neocortex, the majority of CGE- and dLGE-derived and a subset of MGE-derived interneurons continuously express COUP-TFII. It is also noted that Sox6+, COUP-TFII+ and Sp8+ cells account for more than 90% of the neocortical interneurons in adult mouse brains. In this study, it was observed that the expression patterns of Sox6, COUP-TFII and Sp8 in developing and adult human and macaque monkey brains are very similar to those in mice. Furthermore, expression of Gsx2 and Nkx2-1, fundamental features of mouse subpallial progenitors, was observed only in the subpallium of developing human and macaque monkey brains. It is therefore proposed that the majority of neocortical interneurons in monkey and human brains originate from subcortical ganglionic eminences of the ventral telencephalon. It was recently found that the numbers of SOM+, NPY+ and nNOS+ neocortical interneurons are substantially reduced in fetal and infant cases of human holoprosencephaly with severe ventral forebrain hypoplasia, further indicating their subcortical developmental origins (Ma, 2013).

In the adult monkey and human neocortex, MGE-derived interneurons (Sox6+) account for about 60% of all cortical interneurons, although the proportions in different cortical areas are different. Sp8 is expressed extensively from the rostral to caudal extent of the telencephalon, including primarily the olfactory bulb, RMS, dLGE and CGE. However, few Sp8+COUP-TFII+ cells were observed in the monkey and human RMS and olfactory bulb. This suggests that Sp8+COUP-TFII+ cells in the primate dLGE and CGE migrate mainly into the cortex. On the basis of the expression pattern of COUP-TFII in the LGE and CGE (COUP-TFII is expressed in increasing rostral-to-caudal gradients in the subpallium) and the presence of COUP-TFII+ interneurons in the adult neocortex, it is estimated that the number of CGE-derived interneurons appeared to be more prominent than those derived from the LGE. Thus, as in rodents, although it is difficult to identify the boundary between the LGE and CGE, human and monkey LGE-derived cortical interneurons may contribute less substantially to the populations of cortical interneurons than MGE- and CGE-derived populations (Ma, 2013).

The classification of cortical interneurons in primates in the present study was based largely on the continuity of Sox6, COUP-TFII and Sp8 transcription factor expression. This analysis revealed that, similarly to mouse mature neocortical interneurons, PV+ and SOM+ cortical interneurons in primates were derived from the MGE, whereas CR+ cortical interneurons are derived mainly from the CGE (nearly all CR+ interneurons expressed COUP-TFII and/or Sp8). Moreover, the laminar distribution of inhibitory interneurons in the mouse, monkey and human neocortex also share common features. For example, LGE- and CGE-derived COUP-TFII+ and/or Sp8+ interneurons preferentially occupy superficial cortical layers. This further suggests conserved molecular mechanisms that regulate the development of cortical interneurons in the mammalian brain (Ma, 2013).

COUP-TF and Retinal Development

Determination of the dorso-ventral dimension of the vertebrate retina is known to involve retinoic acid (RA): high RA activates expression of a ventral retinaldehyde dehydrogenase and low RA of a dorsal dehydrogenase. In the early eye vesicle of the mouse embryo, expression of the dorsal dehydrogenase is preceded by, and transiently overlaps with, the RA-degrading oxidase CYP26. Subsequently in the embryonic retina, CYP26 forms a narrow horizontal boundary between the dorsal and ventral dehydrogenases, creating a trough between very high ventral and moderately high dorsal RA levels. Most of the RA receptors are expressed uniformly throughout the retina except for the RA-sensitive RARbeta, which is down-regulated in the CYP26 stripe. The orphan receptor COUP-TFII, which modulates RA responses, colocalizes with the dorsal dehydrogenase. The organization of the embryonic vertebrate retina into dorsal and ventral territories divided by a horizontal boundary has parallels to the division of the Drosophila eye disc into dorsal, equatorial and ventral zones, indicating that the similarities in eye morphogenesis extend beyond single molecules to topographical patterns (McCaffery, 1999).

Transcriptional networks, which are initiated by secreted proteins, cooperate with each other to orchestrate eye development. The establishment of dorsal/ventral polarity, especially dorsal specification in the optic vesicle, is poorly understood at a molecular and cellular level. This study shows that COUP-TFI (Nr2f1) and COUP-TFII (Nr2f2) are highly expressed in the progenitor cells in the developing murine eye. Phenotype analysis of COUP-TFI and COUP-TFII single-gene conditional knockout mouse models suggests that COUP-TFs compensate for each other to maintain morphogenesis of the eye. However, in eye-specific COUP-TFI/TFII double-knockout mice, progenitor cells at the dorso-distal optic vesicle fail to differentiate appropriately, causing the retinal pigmented epithelium cells to adopt a neural retina fate and abnormal differentiation of the dorsal optic stalk; the development of proximo-ventral identities, neural retina and ventral optic stalk is also compromised. These cellular defects in turn lead to congenital ocular colobomata and microphthalmia. Immunohistochemical and in situ hybridization assays reveal that the expression of several regulatory genes essential for early optic vesicle development, including Pax6, Otx2, Mitf, Pax2 and Vax1/2, is altered in the corresponding compartments of the mutant eye. Use of ChIP assay, siRNA treatment and transient transfection in ARPE-19 cells in vitro, demonstrates that Pax6 and Otx2 are directly regulated by COUP-TFs. Taken together, these findings reveal novel and distinct cell-intrinsic mechanisms mediated by COUP-TF genes to direct the specification and differentiation of progenitor cells, and that COUP-TFs are crucial for dorsalization of the eye (Tang, 2010).

Requirement for COUP-TFI and II in the temporal specification of neural stem cells in CNS development

In the developing CNS, subtypes of neurons and glial cells are generated according to a schedule that is defined by cell-intrinsic mechanisms that function at the progenitor-cell level. However, no critical molecular switch for the temporal specification of CNS progenitor cells has been identified. This study found that chicken ovalbumin upstream promoter-transcription factor I and II (Coup-tfI and Coup-tfII, also known as Nr2f1 and Nr2f2) are required for the temporal specification of neural stem/progenitor cells (NSPCs), including their acquisition of gliogenic competence, as demonstrated by their responsiveness to gliogenic cytokines. COUP-TFI and II are transiently co-expressed in the ventricular zone of the early embryonic CNS. The double knockdown of Coup-tfI/II in embryonic stem cell (ESC)-derived NSPCs and the developing mouse forebrain caused sustained neurogenesis and the prolonged generation of early-born neurons. These findings reveal a part of the timer mechanisms for generating diverse types of neurons and glial cells during CNS development (Naka, 2008).

Three mechanisms have been proposed so far for the temporally regulated specification of NSPCs for the switch from neurogenesis to gliogenesis. One is the attenuation of neurogenesis by reduced expression levels of proneural genes, such as Neurogenins. Another is the induction of gliogenesis through the activation of proglial genes, such as Nuclear Factor I family or NFI (Drosophila homolog Nuclear Factor I). Finally, it has been suggested that temporally regulated epigenetic modifications of glia-specific genes regulate their response to gliogenic cytokines. The current data suggest the existence of an additional step that requires the function of Coup-tfI/II for NSPCs to acquire gliogenic competency, which occurs in parallel with or upstream of the other processes (Naka, 2008).

This proposal is supported by the following evidence. First, expression of COUP-TFI/II was transiently upregulated in the early neurogenic period in NSPCs and markedly decreased before the onset of gliogenesis. Second, Coup-tfI/II knockdown resulted in the maintenance of epigenetic silencing at the Gfap gene. Third, only limited neurogenic phenotypes were induced by Coup-tfI/II knockdown in advanced-stage neurospheres, which mainly generate glia. Finally, Coup-tfI/II knockdown inhibited the neurogenesis-to-gliogenesis transition without substantially changing the expression dynamics of most known genes associated with neurogenesis and gliogenesis. This study also revealed an involvement of Coup-tfI/II in the timing of neurogenesis in various brain regions, at least in a certain time window (Naka, 2008).

With regard to gliogenesis in Drosophila embryonic CNS, although one type of neuroglioblast divides to simultaneously generate precursors with restricted potential that give rise to either glial cells or neurons44, another type of neuroglioblast sequentially gives rise to neurogenic intermediate precursors and precursors generating both neurons and glia45. Thus, it would be interesting to know whether Drosophila SVP could also be involved in the sequential neurogenesis/gliogenesis switch in this context, in a similar manner to its mammalian homolog. It is still unclear whether the temporal identity transition for the change of neuropotency and the acquisition of gliogenic competency in developing NSPCs is regulated by a single mechanism or two independent ones that are used by COUP-TFI/II. The absence of linear correlation between the extents of enhanced neurogenesis and the production of early-born neurons in response to Coup-tfI/II knockdown in vitro may support the two-mechanism possibility. Identification of the target genes of Coup-tfI/II will be necessary to further understanding of this issue (Naka, 2008).

A model is proposed for the role of Coup-tfI/II in the sequentially regulated temporal specification of NSPCs in mouse cortical development. At the first stage of temporal specification (E10-11), only neurons, including preplate Cajal-Retzius neurons, are generated. At the second stage (E12-14), Coup-tfI and II are transiently upregulated to move from early neurogenesis to late neurogenesis and to confer gliogenic competency on NSPCs. Consequently, NSPCs become able to differentiate into astrocytes in response to gliogenic cytokines in vitro, but gliogenesis does not begin with the transient increase in Coup-tfI/II expression in vivo. At the third stage (E15-18), NSPCs start to express markers for immature glia, such as glutamine synthetase, but little GFAP expression is detected. During this time, the expression levels of the Coup-tfI/II in NSPCs quickly drop and neurogenesis tapers off. At the final stage (after birth), astrocyte differentiation, defined by GFAP and S100b expression, accelerates. This study has uncovered a portion of the molecular machinery involved in regulating the temporal specification of NSPCs in CNS development. Further study of the mechanisms underlying the temporal specification of NSPCs should suggest the means for the controlled production of any desired type of neuron from stemcells both in vitro and in vivo, which should greatly contribute to the development and advancement of regenerative medicine of the CNS (Naka, 2008).

Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI

Transcription factors with gradients of expression in neocortical progenitors give rise to distinct motor and sensory cortical areas by controlling the area-specific differentiation of distinct neuronal subtypes. However, the molecular mechanisms underlying this area-restricted control are still unclear. Here, we show that COUP-TFI controls the timing of birth and specification of corticospinal motor neurons (CSMN) in somatosensory cortex via repression of a CSMN differentiation program. Loss of COUP-TFI function causes an area-specific premature generation of neurons with cardinal features of CSMN, which project to subcerebral structures, including the spinal cord. Concurrently, genuine CSMN differentiate imprecisely and do not project beyond the pons, together resulting in impaired skilled motor function in adult mice with cortical COUP-TFI loss-of-function. These findings indicate that COUP-TFI exerts critical areal and temporal control over the precise differentiation of CSMN during corticogenesis, thereby enabling the area-specific functional features of motor and sensory areas to arise (Tomassy, 2010).

COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons

COUP-TFI is an orphan nuclear receptor acting as a strong transcriptional regulator in different aspects of forebrain embryonic development. In this study, COUP-TFI expression and function were investigated in the mouse olfactory bulb (OB), a highly plastic telencephalic region in which continuous integration of newly generated inhibitory interneurons occurs throughout life. OB interneurons belong to different populations that originate from distinct progenitor lineages. This study shows that COUP-TFI is highly expressed in tyrosine hydroxylase (TH)-positive dopaminergic interneurons in the adult OB glomerular layer (GL). Odour deprivation, which is known to downregulate TH expression in the OB, also downregulates COUP-TFI in dopaminergic cells, indicating a possible correlation between TH- and COUP-TFI-activity-dependent action. Moreover, conditional inactivation of COUP-TFI in the EMX1 lineage results in a significant reduction of both TH and ZIF268 expression in the GL. Finally, lentiviral vector-mediated COUP-TFI deletion in adult-generated interneurons confirmed that COUP-TFI acts cell-autonomously in the control of TH and ZIF268 expression. These data indicate that COUP-TFI regulates TH expression in OB cells through an activity-dependent mechanism involving ZIF268 induction and strongly argue for a maintenance rather than establishment function of COUP-TFI in dopaminergic commitment. This study reveals a previously unknown role for COUP-TFI in the adult brain as a key regulator in the control of sensory-dependent plasticity in olfactory dopaminergic neurons (Bovetti, 2013)

seven up: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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