unplugged

EVOLUTIONARY HOMOLOGS

Cloning and expression patterns of Gbx-2 and related proteins

The homeobox gene Carp-Ovx1 shows similarity to vertebrate and invertebrate Ovx genes and to Drosophila unplugged. Its expression pattern was studied by in situ hybridization in carp embryos and juveniles. During segmentation, expression becomes gradually limited to the neural tube. In juveniles up to 9 weeks old, cells in the ventral telencephalon, the facial lobe and the vagal lobe show Ovx1 expression, confining expression to parts with chemosensory projections (Stroband, 1998).

In a search for homeobox genes expressed during early Xenopus development, a gene has been isolated which appears to be the Xenopus cognate of the mouse Gbx-2 gene. Expression of Xgbx-2 is first detectable by in situ hybridization at the midgastrula stage when it is predominantly expressed in the dorsolateral ectoderm, with a gap in expression at the dorsal midline. By the end of gastrulation and during neurulation, Xgbx-2 is expressed dorsolaterally in the neural ectoderm and laterally and ventrally in the epidermis, with sharp anterior expression borders in both tissues. The anteriormost expression in the neural ectoderm persists throughout the early stages of development, and was mapped to the region of rhombomere 1, with an anterior expression border in the region of the midbrain-hindbrain boundary. Thus, Xgbx-2 is expressed anterior to the Hox genes. Xgbx-2 expression is induced by retinoic acid (RA) in animal caps, and RA treatment of whole embryos expands and enhances Xgbx-2 expression in the ectoderm. It is suggested that Xgbx-2 plays a role in establishing the midbrain-hindbrain boundary, which appears to separate early neurectodermal regions expressing genes that are positively and negatively regulated by RA (von Bubnoff,1996).

The Gbx2 homeodomain is widely conserved in metazoans. The mouse Gbx2 locus was investigated by isolation and characterization of genomic clones and by physical localization to the genome. The Gbx2 gene contains a single intron that separates the proposed functional protein domains. This organization is conserved with human GBX2. Physical localization of Gbx2 to Chromosome 1C5-E1 indicates that the genomic relationship between the linked Gbx2 and En1 genes differs between mouse and human, making it unlikely to be functionally significant. The known expression pattern of Gbx2 has been extended beyond the gastrulation stage embryo and the developing CNS to pluripotent cells in vitro and in vivo. Gbx2 expression has been demonstrated in undifferentiated embryonic stem cells but is downregulated in differentiated cell populations. In the embryo, Gbx2 expression is detected before primitive streak formation, in the inner cell mass of the preimplantation embryo. Gbx2 is therefore a candidate control gene for cell pluripotency and differentiation in the embryo (Chapman, 1997).

The nested expression patterns of the paired-box containing transcription factors Pax2/5 and Pax6 demarcate the midbrain and forebrain primordium at the neural plate stage. In Pax2/5 deficient mice, the mesencephalon/metencephalon primordium is completely missing, resulting in a fusion of the forebrain to the hindbrain. Morphologically, in the alar plate the deletion is characterized by the substitution of the tectum (dorsal midbrain) and cerebellum (dorsal metencephalon) by the caudal diencephalon and in the basal plate by the replacement of the midbrain tegmentum by the ventral metencephalon (pons). Molecularly, the loss of the tectum is demonstrated by an expanded expression of Pax6, (the molecular determinant of posterior commissure), and a rostral shift of the territory of expression of Gbx2 and Otp (markers for the pons), toward the caudal diencephalon. These results suggest that an intact territory of expression of Pax2/5 in the neural plate, nested between the rostral and caudal territories of expression of Pax6, is necessary for defining the midbrain vesicle (Schwarz, 1999).

Polymerase chain reaction (PCR) was used to amplify portions of homeobox genes present in a human 11-week fetal brain cDNA library. One of these PCR products was determined by sequencing to be the gene Gastrulation and brain specific-2 (GBX2). Screening this human fetal brain cDNA library with probes specific for GBX2 led to the identification of a 2151-bp cDNA clone. The nucleotide sequence of the cDNA clone encodes for a protein of 347 amino acid residues. The amino acid sequence of the GBX2 homeodomain is identical (100%) to that of homologous gene, Gbx2, expressed in the developing mouse embryo, and is virtually identical (97%) to CHox7, a gene expressed in the developing chicken embryo. The 5' end of the GBX2 gene contains a CpG island in the untranslated region and a trinucleotide (CCG)8 repeat in the coding region. The amino-terminal end of the GBX2 protein is proline-rich, with 30 proline residues in one stretch of 120 amino acids. Using Northern analysis, a single 2.2-kb transcript was detected in the developing human CNS, as well as in other tissues. The human genomic clone for GBX2 was also isolated, characterized, and mapped to 2q36(d)-q37 by somatic cell hybrid analysis and fluorescence in situ hybridization. These studies provide a framework for designing future experiments that are needed to determine the functional significance of this gene in CNS development (Lin, 1996).

The cDNA sequence of Stra7, a retinoic acid (RA)-inducible gene in P19 embryonal carcinoma (EC) cells, has been determined. The deduced Stra7 protein contains a homeodomain highly similar to that of chicken CHox7, and is highly conserved during evolution, from hemichordates to vertebrates. The mouse Stra7 cDNA corresponds to the full-length form of the 77 bp homeodomain-encoding cDNA fragment that was previously cloned and termed MMoxA or Gbx-2. Reverse-transcriptase-PCR analysis reveals the presence of Stra7/Gbx-2 transcripts in the adult brain, spleen, and female genital tract, whereas no expression is observed in heart, liver, lung, kidney, or testes. In situ hybridization analysis shows a restricted expression pattern of Stra7/Gbx-2 in the three primitive germ layers during gastrulation. Restricted expression is also detected in the pharyngeal arches. Subsequently, specific expression domains appear in the developing central nervous system, at the midbrain/hindbrain boundary and later in the cerebellum anlage, in certain rhombomeres, in dorsal regions of the spinal cord, and in the developing dorsal thalamus and corpus striatum (Bouillet, 1995).

Gbx-2 and subdivision of the brain

The expression patterns of four genes that are potential regulators of development were examined in the CNS of the embryonic day 12.5 mouse embryo. Three of the genes, Dlx-1, Dlx-2 (Tes-1), and Gbx-2, encode homeodomain-containing proteins; one gene, Wnt-3, encodes a putative secreted differentiation factor. These genes are expressed in spatially restricted transverse and longitudinal domains in the embryonic neural tube, and are also differentially expressed within the wall of the neural tube. Dlx-1 and Dlx-2 are expressed in two separate regions of the forebrain in an identical pattern. The Gbx-2 gene is expressed in four domains, two of which share sharp boundaries with the domains of the Dlx genes. One boundary is in the basal telencephalon between deep and superficial strata of the medial ganglionic eminence; the other boundary is in the diencephalon at the zona limitans intrathalamica. The Wnt-3 gene is expressed in a dorsal longitudinal zone extending from the hindbrain into the diencephalon, where its expression terminates at the zona limitans intrathalamica. Reciprocal patterns of expression are found within the dorsal thalamus for the Gbx-2 and Wnt-3 genes. These findings are consistent with neuromeric theories of forebrain development, and based upon them, a model for forebrain segmentation has been suggested (Bulfone, 1993).

The expression pattern of the GBX2 gene during chicken embryogenesis was examined. Initially, transcripts are found in the epiblast. With the onset of neurogenesis, transcripts mark the posterior neuroectoderm. Later on, expression is detectable in the isthmic region, the hindbrain and the neural tube. GBX2 transcripts, as well as the protein, mark the presumptive hindbrain region. After establishment of the brain vesicles GBX2 transcripts are also detected in distinct domains of the diencephalon. In addition to neural sites of expression, GBX2 is found in several domains including the otic vesicle, the somitic mesoderm, the lateral foregut endoderm, the ventral limb bud ectoderm and in the feather buds (Niss, 1998).

Gbx-2 is required for the normal development of the anterior hindbrain. Since much of the understanding of the normal development of this region derives from studies of avian embryos, a determination was made of the expression of Gbx-2 in chick embryos at stages relevant to the regionalization of the hindbrain. As the neural plate forms transcripts already have a clear anterior limit of expression and, subsequently, occupy a domain extending from the extreme posterior midbrain to the rhombomere 3/4 boundary. Subsequently, expression is restricted to the isthmus, a dorsal stripe of expression extending throughout the hindbrain in the ventricular region and the cells adjacent to rhombomere boundaries. Transcripts were also detected in pharyngeal endoderm, the otic placode and vesicle, pharyngeal arches and somites (Shamim, 1998).

Experimental studies in chick and analysis of mouse mutants have provided a framework for studying the early developmental processes involved in specifying the cerebellar anlage. Fate mapping studies in chick have shown that at early stages the cerebellum derives from cells in the mesencephalon and metencephalon (mes-met). Transplantation studies in chick have implicated the mes-met junction (isthmus) as a source of secreted factors that organize development of the entire mes-met, perhaps by stimulating proliferation and specifying positional values across the region. Fgf-8 has been implicated as a major factor involved in the isthmus organizing activity. Gene expression studies indicate that the anterior and posterior expression domains of the homeobox genes Otx-2 and Gbx-2, respectively, are the earliest indication of a division of the brain. The Otx-2/Gbx-2 expression border later resides at the mes-met junction. Genetic studies in mouse have shown that Otx-2 and Gbx-2 are required for normal development of cells on both sides of the border. Mutations affecting the secreted factor Wnt-1, which is expressed anterior to the Otx-2/Gbx-2 expression border, and the homeodomain transcription factors Engrailed-1,2 and Pax-2,5, which have broad overlapping expression domains in the mes-met, result in deletions of mes-met structures. Taken together, these studies suggest that specification of the cerebellar territory requires a hierarchy of complex cellular and genetic interactions that gradually subdivide the brain into smaller regions (Wassef, 1997).

Analysis of mouse embryos homozygous for a loss-of-function allele of Gbx2 demonstrates that this homeobox gene is required for normal development of the mid/hindbrain region. Gbx2 function appears to be necessary at the neural plate stage for the correct specification and normal proliferation or survival of anterior hindbrain precursors. It is also required to maintain normal patterns of expression at the mid/hindbrain boundary of Fgf8 and Wnt1, genes that encode signaling molecules thought to be key components of the mid/hindbrain (isthmic) organizer. In the absence of Gbx2 function, isthmic nuclei, the cerebellum, motor nerve V, and other derivatives of rhombomeres 1-3 fail to form. Additionally, the posterior midbrain in the mutant embryos appears to be extended caudally and displays abnormalities in anterior/posterior patterning. The failure of anterior hindbrain development is presumably due to the loss of Gbx2 function in the precursors of the anterior hindbrain. However, since Gbx2 expression is not detected in the midbrain it seems likely that the defects in midbrain anterior/posterior patterning result from an abnormal isthmic signaling center. These data provide genetic evidence for a link between patterning of the anterior hindbrain and the establishment of the mid/hindbrain organizer, and identify Gbx2 as a gene required for these processes to occur normally (Wassarman, 1997).

The RNA of the noncluster homeobox gene, Xgbx-2, is localized during neurulation to a narrow band of tissue at the midbrain hindbrain boundary (anterior hindbrain). The localized expression of Xgbx-2 within the nervous system prompted an assessment of its function during early development by injection of synthetic Xgbx-2 RNA into the animal pole region of both dorsal blastomeres at the four-cell stage. Injection of Xgbx-2 RNA leads to dose-dependent alterations in anterior dorsal structures. These defects include abnormal eye development, including reduced and missing eyes, reduced or missing cement glands, and abnormal brain development. Additionally, coinjection with lineage label (either beta-galactosidase or green fluorescent protein) shows there is a dose-dependent misplacement of cells. These misplaced cells can be found in such locations as the blastocoele, gastrocoele, or ventricles in the brain. In some spawnings, misplaced cells are expelled from the embryo into the periviteline space. In general, the phenotype of Xgbx-2 RNA-injected embryos is strikingly similar to the phenotypes observed when dominant-negative RNA constructs of Ca2+-dependent cell-adhesion molecules are injected into similar regions of early embryos. Xgbx-2 misexpression enhances the dissociation of animal hemisphere cells, and inhibits Ca2+-dependent cell adhesion in dissociated animal hemisphere cells in vitro. Additionally, when the expression of various calcium-dependent cadherins is tested, misexpression of Xgbx-2 prevents N-cadherin expression during early neurulation. These observations suggest that Xgbx-2 functions normally in the regionalization of the neural tube (specifically the anterior hindbrain) by regulating differential cell adhesion and subsequently cell identity (King, 1998).

The mid/hindbrain junction region, which expresses Fgf8, can act as an organizer to transform caudal forebrain or hindbrain tissue into midbrain or cerebellar structures, respectively. FGF8-soaked beads placed in the chick forebrain can similarly induce ectopic expression of mid/hindbrain genes and development of midbrain structures. In contrast, ectopic expression of Fgf8a in the mouse midbrain and caudal forebrain using a Wnt1 regulatory element produces no apparent patterning defects in the embryos examined. FGF8b-soaked beads can not only induce expression of the mid/hindbrain genes En1, En2 and Pax5 in mouse embryonic day 9.5 (E9.5) caudal forebrain explants, but also can induce the hindbrain gene Gbx2 and alter the expression of Wnt1 in both midbrain and caudal forebrain explants. FGF8b-soaked beads can repress Otx2 in midbrain explants. Furthermore, Wnt1-Fgf8b transgenic embryos in which the same Wnt1 regulatory element is used to express Fgf8b, have ectopic expression of En1, En2, Pax5 and Gbx2 in the dorsal hindbrain and spinal cord at E10.5, as well as exencephaly and abnormal spinal cord morphology. More strikingly, Fgf8b expression in more rostral brain regions appears to transform the midbrain and caudal forebrain into an anterior hindbrain fate through expansion of the Gbx2 domain and repression of Otx2 as early as the 7-somite stage. These findings suggest that normal Fgf8 expression in the anterior hindbrain not only functions to maintain development of the entire mid/hindbrain by regulating genes like En1, En2 and Pax5, but also might function to maintain a metencephalic identity by regulating Gbx2 and Otx2 expression (Liu, 1999).

It is interesting that the phenotype observed in early Wnt1-Fgf8b transgenics is similar to that seen in Otx1+/-Otx2+/- or Otx1-/-Otx2+/- double mutants; an early induction of Gbx2 and repression of Otx2 in the midbrain and caudal forebrain. In Otx1-/-;Otx2+/- embryos, an anterior expansion of Fgf8 expression precedes an anterior shift of Wnt1 and En1 expression and an anterior retraction of Otx2 expression. The Otx mutant studies suggest a certain level of Otx2 expression is necessary to repress expression of Fgf8 in the midbrain and forebrain, and these results suggest that, in addition, expanded Fgf8 expression could contribute to repression of Otx2 expression in the midbrain. A reciprocal negative regulation between Otx2 and Fgf8 might therefore normally contribute to maintaining the Otx2 caudal boundary and positioning the organizer (Liu, 1999 and references therein).

Fgf8, which is expressed at the embryonic mid/hindbrain junction, is required for and sufficient to induce the formation of midbrain and cerebellar structures. To address the genetic pathways through which FGF8 acts, the epistatic relationships of mid/hindbrain genes that respond to FGF8 were examined, using a novel mouse brain explant culture system. En2 and Gbx2 are the first genes to be induced by FGF8 in wild-type E9.5 diencephalic and midbrain explants treated with FGF8-soaked beads. By examining gene expression in En1/2 double mutant mouse embryos, it was found that Fgf8, Wnt1 and Pax5 do not require the En genes for initiation of expression, but do for their maintenance, and Pax6 expression is expanded caudally into the midbrain in the absence of EN function. Since E9.5 En1/2 double mutants lack the mid/hindbrain region, forebrain mutant explants were treated with FGF8 and, significantly, the EN transcription factors were found to be required for induction of Pax5. Thus, FGF8-regulated expression of Pax5 is dependent on EN proteins, and a factor other than FGF8 could be involved in initiating normal Pax5 expression in the mesencephalon/metencephalon. The En genes also play an important, but not absolute, role in repression of Pax6 in forebrain explants by FGF8. Gbx2 gain-of-function studies have shown that misexpression of Gbx2 in the midbrain can lead to repression of Otx2. However, in the absence of Gbx2, FGF8 can nevertheless repress Otx2 expression in midbrain explants. In contrast, Wnt1 is initially broadly induced in Gbx2 mutant explants, as in wild-type explants, but not subsequently repressed in cells near FGF8 that normally express Gbx2. Thus GBX2 acts upstream of, or parallel to, FGF8 in repressing Otx2, and acts downstream of FGF8 in repression of Wnt1. This is the first such epistatic study performed in mouse that combines gain-of-function and loss-of-function approaches to reveal aspects of mouse gene regulation in the mesencephalon/metencephalon that have been difficult to address using either approach alone (Liu, 2001).

The homeobox gene Otx2 is expressed in the anterior neural tube with a sharp limit at the midbrain/hindbrain junction (the isthmic organizer). Otx2 inactivation experiments have shown that this gene is essential for the development of its expression domain. Using a knock-in strategy into the En1 locus, an investigation was carried out to see whether the caudal limit of Otx2 expression is instrumental in positioning the isthmic organizer and in specifying midbrain versus hindbrain fate by ectopically expressing Otx2 in the presumptive anterior hindbrain. Transgenic offspring display a cerebellar ataxia. Morphological and histological studies of adult transgenic brains reveal that most of the anterior cerebellar vermis is missing, whereas the inferior colliculus is complementarily enlarged. During early neural pattern formation expression of the midbrain markers Wnt1 and Ephrin-A5, the isthmic organizer markers Pax2 and Fgf-8 and the hindbrain marker Gbx2 are shifted caudally in the presumptive hindbrain territory. These findings show that the caudal limit of Otx2 expression is sufficient for positioning the isthmic organizer and encoding caudal midbrain fate within the mid/hindbrain domain (Broccoli, 1999).

The patterns of the Gbx2, Pax2, Wnt1, and Fgf8 gene expression were analyzed in the chick with respect to the caudal limit of the Otx2 anterior domain, taken as a landmark of the midbrain/hindbrain (MH) boundary. The Gbx2 anterior boundary is always concomitant with the Otx2 posterior boundary. The ring of Wnt1 expression is included within the Otx2 domain and Fgf8 transcripts included within the Gbx2 neuroepithelium. Pax2 expression is centered on the MH boundary with a double decreasing gradient. A new nomenclature is proposed to differentiate the vesicles and constrictions observed in the avian MH domain at stage HH10 and HH20, based on the localization of the Gbx2/Otx2 common boundary (Hidalgo-Snachez, 1999).

The mid/hindbrain (MHB) junction can act as an organizer to direct the development of the midbrain and anterior hindbrain. In mice, Otx2 is expressed in the forebrain and midbrain and Gbx2 is expressed in the anterior hindbrain, with a shared border at the level of the MHB organizer. In Gbx2-/- mutants, the earliest phenotype is a posterior expansion of the Otx2 domain during early somite stages. Furthermore, organizer genes are expressed at the shifted Otx2 border, but not in a normal spatial relationship. To test whether Gbx2 is sufficient to position the MHB organizer, Gbx2 was transiently expressed in the caudal Otx2 domain. The Otx2 caudal border indeed shifts rostrally and a normal appearing organizer forms at this new Otx2 border. Transgenic embryos show an expanded hindbrain and a reduced midbrain at embryonic day 9.5-10. It is proposed that formation of a normal MHB organizer depends on a sharp Otx2 caudal border and that Gbx2 is required to position and sharpen this border (Millet, 1999).

There is a long-standing controversy regarding the mechanisms that generate the functional subdivisions of the cerebral neocortex. One model proposes that thalamic axonal input specifies these subdivisions; the competing model postulates that patterning mechanisms intrinsic to the dorsal telencephalon generate neocortical regions. Gbx-2 mutant mice, whose thalamic differentiation is disrupted, were investigated. Despite the lack of cortical innervation by thalamic axons, neocortical region-specific gene expression (Cadherin-6, EphA-7, Id-2, and RZR-beta) develops normally. This provides evidence that patterning mechanisms intrinsic to the neocortex specify the basic organization of its functional subdivisions (Miyashita-Lin, 1999).

The anatomical and functional organization of dorsal thalamus (dTh) and ventral thalamus (vTh), two major regions of the diencephalon, is characterized by their parcellation into distinct cell groups, or nuclei, that can be histologically defined in postnatal animals. However, because of the complexity of dTh and vTh and difficulties in histologically defining nuclei at early developmental stages, understanding of the mechanisms that control the parcellation of dTh and vTh and the differentiation of nuclei is limited. A set of regulatory genes, which include five LIM-homeodomain transcription factors (Isl1, Lhx1, Lhx2, Lhx5, and Lhx9) and three other genes (Gbx2, Ngn2, and Pax6), have been defined that are differentially expressed in dTh and vTh of early postnatal mice in distinct but overlapping patterns that mark nuclei or subsets of nuclei. These genes exhibit differential expression patterns in dTh and vTh as early as embryonic day 10.5, when neurogenesis begins; the expression of most of them is detected as progenitor cells exit the cell cycle. Soon thereafter, their expression patterns are very similar to those observed postnatally, indicating that unique combinations of these genes mark specific cell groups from the time they are generated to their later differentiation into nuclei. These findings suggest that these genes act in a combinatorial manner to control the specification of nuclei-specific properties of thalamic cells and the differentiation of nuclei within dTh and vTh. These genes may also influence the pathfinding and targeting of thalamocortical axons through both cell-autonomous and non-autonomous mechanisms (Nakagawa, 2001).

The dTh is parcellated into over one dozen nuclei. The principal sensory nuclei, dorsal lateral geniculate (dLG), ventroposterior (VP), and ventral medial geniculate (MGv), relay sensory information from the periphery to primary sensory areas of the neocortex, visual, somatosensory, and auditory, respectively, via thalamocortical axons (TCAs). Other nuclei, such as posterior (Po) and lateral posterior (LP), project broadly to cortex. The vTh has three major nuclei: reticular (RT), zona incerta (ZI), and ventral lateral geniculate (vLG). Different domains of embryonic vTh are required for TCA pathfinding (Nakagawa, 2001 and references therein).

The vTh and dTh have been defined as adjacent domains of the embryonic diencephalic alar plate based on expression of the homeodomain transcription factors Dlx2 and Gbx2, respectively, and restrictions in cell movement. However, little is known about the organization of embryonic dTh and vTh into discrete cell groups that presage their differentiation into nuclei, because the morphology and connections that define nuclei emerge late in development. The LIM-homeodomain (LIM-HD) family of transcription factors, as well as Gbx2, Pax6, and Neurogenin2, are candidates to be differentially expressed within dTh and vTh and control their parcellation. The LIM-HD genes Lhx1 and Lhx5 are expressed in early embryonic diencephalon, Lhx2 and Lhx9 in embryonic dTh, and Isl1 in adult RT. LIM-HD genes are intriguing because their unique combinations mark subsets of spinal neurons and specify their phenotypes, including axonal projections. Gbx2 is expressed broadly early in dTh and later in a subset of nuclei that require it for their differentiation, as well as for the development of the TCA projection. Pax6, a paired-box transcription factor, is expressed broadly early in vTh, later more discretely, and is required for development of RT, ZI, and vLG and TCA pathfinding. Ngn2, a basic helix-loop-helix transcription factor expressed in a subset of progenitor cells in dTh, is required for sensory neuron differentiation and dorsoventral patterning of the telencephalon. These regulatory genes are expressed in distinct yet often overlapping patterns, suggesting that they cooperate to control the specification and differentiation of thalamic nuclei and cell types (Nakagawa, 2001 and references therein).

The sets of genes that are expressed in dTh and vTh are distinct from one another and similar to those expressed in dorsal and ventral spinal cord, respectively. This similarity suggests that the expression patterns in thalamus might be established by mechanisms similar to those in spinal cord. In spinal cord, inductive signals from the roof plate and floor plate control neuronal fate along the dorsoventral axis. Signals from the roof plate, such as TGFß family members, are required in dorsal spinal cord for the induction of Lhx2 and Lhx9, which define D1A and D1B interneurons, respectively. In ventral spinal cord, distinct classes of motor neurons and ventral interneurons are generated by a graded signaling activity of Shh. Shh controls these neural fates by establishing different progenitor cell populations defined by their expression of Pax6 and Nkx2.2. Pax6 establishes distinct populations of ventral progenitor cells and controls the identity of motor neurons and V1 and V2 interneurons, whereas Nkx2.2 specifies the identity of V3 interneurons at a more ventral location. These genes appear to be essential intermediaries for Shh to regulate the differential expression of LIM-HD proteins, including Lhx1, Lhx3, Lhx4, Lhx5, Isl1, and Isl2. In diencephalon, Shh is transiently expressed as early as E9.5 in the zona limitans intrathalamica , which at this stage is a narrow cell domain interposed between prospective dTh and vTh. Similar to ventral spinal cord, Nkx2.2 and Pax6 are also expressed in progenitor cells in vTh. Shh induces in vitro the expression of Isl1 in chick forebrain explants and neuroepithelial cells from rat forebrain. Therefore, ZLI-derived Shh may specify progenitor cell types in vTh to produce different neuronal subtypes, which are determined by the subset of LIM-HD and other transcription factors expressed by these neurons. Interestingly, dTh, which is adjacent to the ZLI, does not express any of the LIM-HD genes induced by Shh and expressed in vTh. Ngn2, which is expressed by progenitor cells of dTh but not vTh, could act to limit the responsiveness of dTh to an Shh-mediated induction of vTh-type LIM-HD genes, which may be a crucial step in regionalization of the diencephalon (Nakagawa, 2001 and references therein).

Otx2 and Gbx2 are among the earliest genes expressed in the neuroectoderm, dividing it into anterior and posterior domains with a common border that marks the mid-hindbrain junction. Otx2 is required for development of the forebrain and midbrain, and Gbx2 for the anterior hindbrain. Furthermore, opposing interactions between Otx2 and Gbx2 play an important role in positioning the mid-hindbrain boundary, where an organizer forms that regulates midbrain and cerebellum development. The expression domains of Otx2 and Gbx2 are initially established independently of each other at the early headfold stage, and then their expression rapidly becomes interdependent by the late headfold stage. Since the repression of Otx2 by retinoic acid is dependent on an induction of Gbx2 in the anterior brain, molecules other than retinoic acid must regulate the initial expression of Otx2 in vivo. In contrast to previous suggestions that an interaction between Otx2- and Gbx2-expressing cells may be essential for induction of mid-hindbrain organizer factors such as Fgf8, it has been found that Fgf8 and other essential mid-hindbrain genes are induced in a correct temporal manner in mouse embryos deficient for both Otx2 and Gbx2. However, expression of these genes is abnormally co-localized in a broad anterior region of the neuroectoderm. By removing Otx2 function, development of rhombomere 3 is rescued in Gbx2^–/– embryos, showing that Gbx2 plays a permissive, not instructive, role in rhombomere 3 development. These results provide new insights into induction and maintenance of the mid-hindbrain genetic cascade by showing that a mid-hindbrain competence region is initially established independent of the division of the neuroectoderm into an anterior Otx2-positive domain and posterior Gbx2-positive domain. Furthermore, Otx2 and Gbx2 are required to suppress hindbrain and midbrain development, respectively, and thus allow establishment of the normal spatial domains of Fgf8 and other genes (Li, 2001).

Whether Gbx2 is required after embryonic day 9 (E9) to repress Otx2 in the cerebellar anlage and position the midbrain/hindbrain organizer was examined. In contrast to Gbx2 null mutants, mice lacking Gbx2 in rhombomere 1 (r1) after E9 (Gbx2-CKO) are viable and develop a cerebellum. A Gbx2-independent pathway can repress Otx2 in r1 after E9. Mid/hindbrain organizer gene expression, however, continues to be dependent on Gbx2. Fgf8 expression normally correlates with the isthmus where cells undergo low proliferation and in Gbx2-CKO mutants this domain is expanded. It is proposed that Fgf8 permits lateral cerebellar development through repression of Otx2 and also suppresses medial cerebellar growth in Gbx2-CKO embryos. This work has uncovered distinct requirements for Gbx2 during cerebellum formation and provides a model for how a transcription factor can play multiple roles during development (Li, 2002).

In Gbx2-CKO embryos, the juxtaposition of the Wnt1 and Fgf8 expression domains is present at the 8 somite stage, but, consistent with previous studies showing that an interaction between Otx2 and Gbx2 positions the mid/hindbrain organizer, the border is shifted posteriorly to the new Otx2/Gbx2 border. In contrast, at E9.5 when Gbx2 transcripts are no longer detected in r1, Wnt1 and Fgf8 were broadly coexpressed in the alar plate of r1. The derepression of Wnt1 in the alar plate of r1 where Gbx2 is normally expressed demonstrates a cell-autonomous requirement for Gbx2 in repression of Wnt1 expression after E9.5, in agreement with previous studies. Since ectopic expression of Wnt1 in r1 can induce Fgf8 in chick embryos, derepression of Wnt1 in r1 cells in Gbx2-CKO embryos could contribute to the expansion of Fgf8 expression in this region. Furthermore, the expression domain of Pax2 in the isthmus is expanded posteriorly in Gbx2-CKO embryos from E9.5 and largely overlaps with that of Fgf8, consistent with the observation that Pax2 is essential for induction of Fgf8. These experiments show that Gbx2 is required from E8.5 onward to repress Wnt1 expression in r1 and maintain the normal relative expression domains of Wnt1 and Fgf8 (Li, 2002).

Development of the CNS involves highly combinatorial actions of transcription factors. Gbx2 is initially required to repress Otx2 before E8.5 to allow specification of the cerebellar primordium. After E8.5, Gbx2 is not essential for the repression of Otx2 because a second pathway is induced that can repress Otx2. Gbx2 is nevertheless still required for maintenance of normal expression of Wnt1 and Fgf8. The temporal changing requirement for Gbx2 during cerebellar development demonstrated in this work provides a different paradigm for how the same transcription factor can control sequential events during a single developmental process (Li, 2002).

The mouse homeobox gene Gbx2 is first expressed throughout the posterior region of the embryo during gastrulation, and becomes restricted to rhombomeres 1-3 (r1-3) by embryonic day 8.5 (E8.5). Previous studies have shown that r1-3 do not develop in Gbx2 mutants and that there is an early caudal expansion of the midbrain gene Otx2 to the anterior border of r4. Furthermore, expression of Wnt1 and Fgf8, two crucial components of the isthmic organizer, is no longer segregated to adjacent domains in Gbx2 mutants. In this study, the phenotypic analysis of Gbx2 mutants has been extended by showing that Gbx2 is not only required for development of r1-3, but also for normal gene expression in r4-6. To determine whether Gbx2 can alter hindbrain development, Hoxb1-Gbx2 (HG) transgenic mice were generated in whichGbx2 is ectopically expressed in r4. Gbx2 was shown to be insufficient to induce r1-3 development in r4. To test whether an Otx2/Gbx2 interface can induce r1-3 development, the HG transgene was introduced onto a Gbx2-null mutant background and a new Otx2/Gbx2 border was recreated in the anterior hindbrain. Development of r3, but not r1 and r2, is rescued in Gbx2^–/–; HG embryos. In addition, the normal spatial relationship of Wnt1 and Fgf8 is established at the new Otx2/Gbx2 border, demonstrating that an interaction between Otx2 and Gbx2 is sufficient to produce the normal pattern of Wnt1 and Fgf8 expression. However, the expression domains of Fgf8 and Spry1, a downstream target of Fgf8, are greatly reduced in mid/hindbrain junction area of Gbx2^–/–; HG embryos and the posterior midbrain is truncated because of abnormal cell death. Interestingly, it was shown that increased cell death and a partial loss of the midbrain are associated with increased expression of Fgf8 and Spry1 in Gbx2 conditional mutants that lack Gbx2 in r1 after E9.0. These results together suggest that cell survival in the posterior midbrain is positively or negatively regulated by Fgf8, depending on Fgf8 expression level. These studies provide new insights into the regulatory interactions that maintain isthmic organizer gene expression and the consequences of altered levels of organizer gene expression on cell survival (Li, 2005).

The organizing center located at the midbrain-hindbrain boundary (MHB) patterns the midbrain and hindbrain primordia of the neural plate. Studies in several vertebrates have shown that the interface between cells expressing Otx and Gbx transcription factors marks the location in the neural plate where the organizer forms, but it is unclear how this location is set up. Using mutant analyses and shield ablation experiments in zebrafish, it has been found that axial mesendoderm, as a candidate tissue, has only a minor role in positioning the MHB. Instead, the blastoderm margin of the gastrula embryo acts as a source of signal(s) involved in this process. Positioning of the MHB organizer is tightly linked to overall neuroectodermal posteriorization, and specifically depends on Wnt8 signaling emanating from lateral mesendodermal precursors. Wnt8 is required for the initial subdivision of the neuroectoderm, including onset of posterior gbx1 expression and establishment of the posterior border of otx2 expression. Cell transplantation experiments further show that Wnt8 signaling acts directly and non-cell-autonomously. Consistent with these findings, a GFP-Wnt8 fusion protein travels from donor cells through early neural plate tissue. These findings argue that graded Wnt8 activity mediates overall neuroectodermal posteriorization and thus determines the location of the MHB organize (Rhinn, 2005).

How does Wnt8 participate in positioning of the MHB organizer? wnt8 is expressed in the marginal cells and hypoblast and two receptors, fz8c and fz9, are detected in both hypoblast and epiblast. Conceivably, Wnt8 is transmitted in a planar fashion through the neuroectoderm. This idea is supported by the clonal analysis of wnt8 overexpressing cells: gbx1 is activated in the host tissue one or two cells distant from the transplanted cells, and otx2 is repressed four or five cells distant from the transplanted cells. In unmanipulated neuroectoderm, the onset of gbx1 expression occurs close to the wnt8 domain with little or no overlap, and the otx2 expression domain is situated eight to ten cell diameters away from the wnt8 domain at 60% of epiboly. Thus, the wnt8 expression domain is appropriately located to generate a graded morphogenetic Wnt8 signal that regulates the expression of gbx1 and otx2 genes in vivo. This finding is more generally consistent with the ability of Wnt molecules to form gradients and to activate target genes in a concentration-dependent manner, as in the Drosophila wing imaginal disc, where expression of wingless target genes like neuralized, distalless and vestigial depends on the distance from wingless-expressing cells. Similarly, in the unmanipulated zebrafish neuroectoderm, the otx2 and the gbx1 domains are located at different distances from the Wnt8 source at the lateral blastoderm margin. Following global misexpression experiments, different Wnt8 doses can differentially regulate otx2 and gbx1 expression: wnt8 ectopic expression can induce gbx1 expression at low/intermediate doses, but represses at high doses. Conversely, otx2 is increasingly repressed with increasing wnt8 concentration. Similarly, around wnt8-expressing clones, gbx1 is induced at a distance of one or two cells around the clone, whereas otx2 is repressed at a distance of four or five cells. This suggests that a lower Wnt8 concentration is needed to repress otx2 than to induce gbx1. Altogether, these observations suggest that Wnt8 has properties of a morphogen whose activity is required to correctly position the otx2/gbx1 interface, and probably other target genes in the forming neural plate. The observation of secreted Wnt8-GFP protein emanating from clones of producing cells is generally consistent with this possibility. Distribution of another signaling molecule in the early neural plate, Fgf8, is carefully controlled by endocytosis. It will be interesting to determine if Wnt8 protein is indeed distributed in a graded fashion, and which mechanisms control this distribution. In mice, Wnt8 is expressed in the posterior epiblast of early primitive streak-stage embryos; although its function is unknown, Wnt8 may therefore serve a similar function as proposed in this study (Rhinn, 2005).

Gbx-2 and Transformation

The homeobox gene GBX2 was identified as a target gene of the v-Myb oncoprotein encoded by the avian myeloblastosis virus (AMV). GBX2 activation by c-Myb requires signal transduction emanating from the cell surface while the leukemogenic AMV v-Myb constitutively induces the GBX2 gene. Mutations in the DNA binding domain of AMV-Myb render it independent of signaling events and concomitantly abrogate the collaboration between Myb and CCAAT Enhancer Binding Proteins (C/EBP), which are involved in granulocyte differentiation. Ectopic expression of GBX2 in growth factor-dependent myeloblasts induces monocytic features and independence from exogenous cytokines, reflecting distinct features of AMV-transformed cells. These results suggest that Myb or factors it interacts with contribute to hematopoietic lineage choice and differentiation in a signal transduction-dependent fashion (Kowenz-Leutz, 1997).

GBX genes, a homeobox-containing human family of DNA-binding transcription factors consisting of GBX1 and GBX2, are overexpressed in a panel of human prostatic cancer cell lines (ie., TSU-pr1, PC3, DU145, and LNCaP) when compared to normal prostate. Specific primer sets have been designed for reverse transcription-PCR detection of the expression of GBX1 versus GBX2 in human prostate cancer. These studies demonstrate that the GBX2 gene, but not the GBX1 gene, is consistently overexpressed in this panel of human prostate cancer cell lines when compared to normal human prostate. To examine the importance of GBX2 expression for prostate cancer malignancy, GBX2-overexpressing TSU-pr1 and PC3 human prostatic cancer cells were transfected with a eukaryotic expression vector containing an antisense GBX2 homeobox domain cDNA. Stable transfectant clones were obtained with five- to ten-fold decreased levels of GBX2 mRNA expression. When tested in vitro, the clonogenic ability of the GBX2 antisense transfectants was reduced by approximately 50% in both cell lines. When implanted subcutaneously into nude mice, the tumorigenicity of the antisense GBX2 transfectants from both human prostatic cancer cell lines was inhibited by more than 70% when compared to the parental cells. These results suggest that expression of GBX2 gene is required for malignant growth of human prostate cells (Gao, 1998).

The most studied secondary neural organizer is the isthmic organizer, which is localized at the mid-hindbrain transition of the neural tube and controls the anterior hindbrain and midbrain regionalization. Otx2 and Gbx2 expressions are fundamental for positioning the organizer and the establishment of molecular interactions that induce Fgf8. Evidence in this study demonstrates that Otx2 and Gbx2 have an overlapping expression in the isthmic region. This area is the transversal domain where expression of Fgf8 is induced. The Fgf8 protein produced in the isthmus stabilizes and up-regulates Gbx2 expression, which, in turn, down-regulates Otx2 expression. The inductive effect of the Gbx2/Otx2 limit keeps Fgf8 expression stable and thus maintains its positive role in the expression of Pax2, En1,2 and Wnt1 (Garda, 2001).

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