InteractiveFly: GeneBrief

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


Gene name - unplugged

Synonyms -

Cytological map position - 45C1--45C9

Function - transcription factor

Keywords - trachea, CNS

Symbol - unpg

FlyBase ID:FBgn0015561

Genetic map position - 2-

Classification - homeodomain protein

Cellular location - presumably nuclear



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW
The unplugged (unpg) gene was identified by systematic screening of P element enhancer detector lines for beta-galactosidase expression patterns, suggestive of regulation by the homeotic gene Ultrabithorax (Ubx). Line f85, which carries a P element insertion in the unpg locus, expresses beta-galactosidase in a pattern restricted to the lateral ectoderm of the first thoracic segment, suggesting that Ubx may negatively regulate expression in more posterior segments. The f85 line also displays a segmentally repeated pattern of expression in neuroblasts and neurons. unplugged is required for formation of specific tracheal branches, including some segment-specific branches. The segmentally reiterated ganglionic branches fail to penetrate the CNS in the absence of unpg function. In addition, unpg function is specifically required for development of the cerebral branch, a tracheal branch uniquely derived from the first thoracic segment (Chiang, 1995).

The expression of unpg in founder cells of the cerebral branch within the first tracheal placode suggests an early role during branch development. unpg function, however, is most likely not involved in the initial commitment to founder cell fates, since expression of the lacZ reporter gene by the enhancer trap is maintained in unpg mutant embryos. Instead, unpg appears to be involved in branching morphogenesis by regulating cell migration or extension; in the absence of such function, the founder cells either die or adopt other branch patterns. This is consistent with the observation that in unpg mutant embryos the absence of the cerebral branch is occasionally accompanied by the presence of an ectopic branch in the first tracheal metamere. The appearance of this ectopic branch resembles that of the dorsal branch, as well as the dorsal cephalic branch, both of which, like the cerebral branch, originate from the first tracheal placode. In addition to the cerebral branch, unpg is also expressed in cells of the ganglionic branches, but here expression occurs much later, suggesting that unpg may have secondary functions during ganglionic branch development. Consistent with this view is the observation that the ganglionic branches develop but fail to extend consistently to the CNS in unpg mutant embryos. This phenotype is reminiscent of the hypormorphic alleles of pointed and breathless mutants. breathless encodes a Drosophila homolog of the fibroblast growth factor (FGF) receptor, and its expression in the developing tracheal system is required for the migration of tracheal cells. Thus, the observed unpg phenotype appears to be consistent with the role of unpg in the specification of tracheal cell migration or extension (Chiang, 1995).

Restricted expression of unpg in the cerebral branch founder cells requires normal function of genes in the Bithorax complex (BX-C). In the absence of these homeotic genes, the expression of unplugged expands more posteriorly to the abdominal segments. This is consistent with the notion that Ultrabithorax controls tracheal development by regulating the expression of target genes. Since unpg encodes a transcription factor and is required for cerebral branch development, it is suggested that normal restriction of cerebral branch development to T1 is mediated by Ubx repression of unpg. This repression is mediated by the 2.7 kb fragment located downstream of the unpg transcription unit (Chiang, 1995).

The orthodenticle-unplugged interface is positioned at the deutocerebral/tritocerebral boundary in Drosophila

Studies on expression and function of key developmental control genes suggest that the embryonic vertebrate brain has a tripartite ground plan that consists of a forebrain/midbrain, a hindbrain and an intervening midbrain/hindbrain boundary region, each of which are characterized by the specific expression of the Otx, Hox and Pax2/5/8 genes, respectively. The embryonic brain of Drosophila expresses all three sets of homologous genes in a similar tripartite pattern. Thus, a Pax2/5/8 expression domain is located at the interface of brain-specific otd/Otx2 and unpg/Gbx2 expression domains anterior to Hox expression regions. This territory is identified as the deutocerebral/tritocerebral boundary region in the embryonic Drosophila brain. Mutational inactivation of otd/Otx2 and unpg/Gbx2 result in the loss or misplacement of the brain-specific expression domains of Pax2/5/8 and Hox genes. In addition, otd/Otx2 and unpg/Gbx2 appear to negatively regulate each other at the interface of their brain-specific expression domains. These studies demonstrate that the deutocerebral/tritocerebral boundary (DTB) region in the embryonic Drosophila brain displays developmental genetic features similar to those observed for the midbrain/hindbrain boundary region in vertebrate brain development. This suggests that a tripartite organization of the embryonic brain was already established in the last common urbilaterian ancestor of protostomes and deuterostomes (Hirth, 2003).

In the embryonic CNS of vertebrates, the Pax2, Pax5 and Pax8 genes are expressed in specific domains that overlap in the presumptive MHB region. Drosophila has two Pax2/5/8 orthologs, Pox neuro (Poxn) and Pax2/Sparkling (Hirth, 2003).

The embryonic brain of Drosophila can be subdivided into the protocerebrum (PC or b1), deutocerebrum (DC or b2) and tritocerebrum (TC or b3) of the supra-esophageal ganglion and the mandibular (S1), maxillary (S2) and labial (S3) neuromeres of the sub-oesophageal ganglion. Expression of engrailed (en) delimits these subdivisions by marking their most posterior neurons. Because of morphogenetic processes, such as the beginning of head involution, the neuraxis of the embryonic brain curves dorsoposteriorly within the embryo. Accordingly, anteroposterior coordinates will here henceforth refer to the neuraxis rather than the embryonic body axis (Hirth, 2003).

It is important to note that the DTB is located anterior to the expression domain of the Drosophila Hox1 ortholog labial (lab), which is expressed in the posterior tritocerebrum. Moreover, the DTB is located posterior to the expression domain of the Drosophila Otx orthologue orthodenticle (otd) in the protocerebrum and anterior deutocerebrum. Thus, in Drosophila as in vertebrates, a Pax2/Poxn (Pax2/5/8) expression domain is located between the anterior otd/Otx2 and the posterior Hox-expressing regions. This raises the question of whether the DTB in the embryonic Drosophila brain might have developmental genetic features similar to those observed for the MHB in vertebrate brain development (Hirth, 2003).

In the embryonic vertebrate brain, Otx2 is expressed anterior to and abutting Gbx2. The future MHB as well as the overlapping domains of Pax2, Pax5 and Pax8 expression are positioned at this Otx2-Gbx2 interface. To investigate if comparable expression patterns are found in the embryonic fly brain, the brain-specific expression of the Drosophila Gbx2 ortholog unplugged (unpg) was determined in relation to that of otd, using immunolabelling and an unpg-lacZ reporter gene that expresses ß-galactosidase like endogenous unpg. The otd gene is expressed in the protocerebrum and anterior deutocerebrum of the embryonic brain, as well as in midline cells in more posterior regions of the CNS. Expression of unpg-lacZ in the embryonic CNS is first detected at stage 8 in neuroectodermal and mesectodermal cells at the ventral midline, with an anterior limit of expression at the cephalic furrow. Subsequently, the unpg expression domains in the CNS widen and have their most anterior border in the posterior deutocerebrum. Double immunolabelling of Otd and ß-galactosidase reveal that the posterior border of the brain-specific otd expression domain coincides with the anteriormost border of the unpg expression domains along the anteroposterior neuraxis. There is no overlap of otd and unpg expression in the brain or in more posterior regions of the CNS (Hirth, 2003).

These findings indicate that the otd-unpg interface is positioned at the anterior border of the DTB. This was confirmed by additional immunolabelling studies examining unpg-lacZ, otd, Poxn and en expression in the protocerebral/deutocerebral region of the embryonic brain. Thus, double immunolabelling of Otd and En confirms that the posterior border of otd expression extends beyond the protocerebral en-b1 stripe into the anterior deutocerebral domain. Labelling Otd and Poxn confirms that the Poxn expression domain of the DTB is posterior to this deutocerebral otd expression boundary. Labelling En and ß-galactosidase (indicative of unpg expression), confirms that the anteriormost unpg expression domain overlaps with the en-b2 stripe. Finally, labelling ß-galactosidase and Poxn confirms that this anteriormost unpg expression domain overlaps with the Poxn expression domain of the DTB. Therefore, in terms of overall gene expression patterns, it is found that a transversal domain of adjacent Pax2/Poxn expression defines the DTB region of the embryonic Drosophila brain. Furthermore, this region is located between an anterior otd expression domain and a posterior Hox expression domain. Moreover, it is located abutting and posterior to the interface of otd and unpg expression along the anteroposterior neuraxis (Hirth, 2003).

In mammalian brain development, homozygous Otx2-null mutant embryos lack the rostral brain, including the MHB-specific Pax2/5/8 expression domain, whereas Gbx2 null mutants misexpress Otx2 and Hoxb1 in the brain. Moreover, Otx2 and Gbx2 negatively regulate each other at the interface of their expression domains. To test if similar regulatory interactions occur in the embryonic brain of Drosophila, the expression of the corresponding orthologs was analyzed in otd and unpg mutant embryos. In otd-null mutant embryos, the protocerebrum is absent because protocerebral neuroblasts are not specified. Analysis of unpg, en and Poxn expression in otd-null mutant embryos reveals that the anteriormost border of unpg expression shifts anteriorly into the anterior deutocerebrum, while Poxn fails to be expressed in the deutocerebrum. In contrast to inactivation of otd, inactivation of unpg does not result in a loss of cells in the mutant domain of the embryonic brain, as is evident from the expression of an unpg-lacZ reporter construct in unpg-null mutant embryos. Analysis of otd expression in unpg-null mutants shows that the posterior limit of brain-specific otd expression shifts posteriorly into the posterior deutocerebrum, thus extending into the DTB. This was confirmed by additional immunolabelling studies examining otd, Poxn and en expression in the protocerebral/deutocerebral region of the embryonic brain in unpg-null mutants. Double immunolabelling of Otd and En in unpg-null mutants confirms that the posterior border of brain-specific otd expression extends posteriorly to the deutocerebral en-b2 stripe into the posterior deutocerebrum. In addition, double immunolabelling of Otd and Poxn in unpg-null mutants confirms that the posterior border of brain-specific otd expression extends posteriorly into the Poxn expression domain of the DTB. Moreover, analysis of lab expression in unpg-null mutants shows that brain-specific lab expression shifts anteriorly into the anterior tritocerebrum. Thus, in both Drosophila and mammals, mutational inactivation of otd/Otx2 and unpg/Gbx2 results in the loss or misplacement of the brain-specific expression domains of orthologous Pax and Hox genes. Moreover, otd and unpg appear to negatively regulate each other at the interface of their expression domains (Hirth, 2003).

In addition to remarkable similarities in orthologous gene expression between insects and chordates, this study also shows that several functional interactions among key developmental control genes involved in establishing the Pax2/5/8-expressing MHB region of the vertebrate brain are also conserved in insects. Thus, in the embryonic brains of both fly and mouse, the intermediate boundary regions, DTB and MHB, are positioned at the interface of otd/Otx2 and unpg/Gbx2 expression domains. These boundary regions are deleted in otd/Otx2-null mutants and mispositioned in unpg/Gbx2-null mutants. Moreover, otd/Otx2 and unpg/Gbx2 genes engage in crossregulatory interactions, and appear to act as mutual repressors at the interface of their brain-specific expression domains. However, not all of the functional interactions among genes involved in MHB formation in the mouse appear to be conserved at the Drosophila DTB. Thus, in the embryonic Drosophila brain, no patterning defects are observed in null mutants of Pax2, Poxn, en or bnl. It remains to be seen if these genes play a role in the postembryonic development of the Drosophila brain (Hirth, 2003).

It is conceivable that the similarities of orthologous gene expression patterns and functional interactions in brain development evolved independently in insects and vertebrates. However, a more reasonable explanation is that an evolutionary conserved genetic program underlies brain development in all bilaterians. This would imply that the generation of structural diversity in the embryonic brain is based on positional information that has been invented only once during evolution and is provided by genes such as otd/Otx2, unpg/Gbx2, Pax2/5/8 and Hox, conferring on all bilaterians a common basic principle of brain development. If this is the case, comparable orthologous gene expression and function should also characterize embryonic brain development in other invertebrate lineages such as the lophotrochozoans. This prediction can now be tested in lophotrochozoan model systems such as Platynereis or Dugesia (Hirth, 2003).

Taken together, these results indicate that the tripartite ground plan that characterizes the developing chordate brain is also present in the developing insect brain. This implies that a corresponding tripartite organization already existed in the brain of the last common urbilaterian ancestor of insects and chordates. Therefore, an urbilaterian origin of the tripartite brain is proposed (Hirth, 2003).

The Drosophila brain develops from the procephalic neurogenic region of the ectoderm. About 100 neural precursor cells (neuroblasts) delaminate from this region on either side in a reproducible spatiotemporal pattern. Neuroblast maps have been prepared from different stages of the early embryo (stages 9, 10 and 11, when the entire population of neuroblasts has formed), in which about 40 molecular markers representing the expression patterns of 34 different genes are linked to individual neuroblasts. In particular, a detailed description is presented of the spatiotemporal patterns of expression in the procephalic neuroectoderm and in the neuroblast layer of the gap genes empty spiracles, hunchback, huckebein, sloppy paired 1 and tailless; the homeotic gene labial; the early eye genes dachshund, eyeless and twin of eyeless; and several other marker genes (including castor, pdm1, fasciclin 2, klumpfuss, ladybird, runt and unplugged). Based on the combination of genes expressed, each brain neuroblast acquires a unique identity, and it is possible to follow the fate of individual neuroblasts through early neurogenesis. Furthermore, despite the highly derived patterns of expression in the procephalic segments, the co-expression of specific molecular markers discloses the existence of serially homologous neuroblasts in neuromeres of the ventral nerve cord and the brain. Taking into consideration that all brain neuroblasts are now assigned to particular neuromeres and individually identified by their unique gene expression, and that the genes found to be expressed are likely candidates for controlling the development of the respective neuroblasts, these data provide a basic framework for studying the mechanisms leading to pattern and cell diversity in the Drosophila brain, and for addressing those mechanisms that make the brain different from the truncal CNS (Urbach, 2003).

Expression of the homeodomain gene unplugged (unpg) in the trunk starts at stage 8 in the ventral midline and becomes detectable in NBs of the ventral nerve cord at late stage 11. Using an unpg-lacZ line, unpg expression is observed in the head at stage 9 in a large domain encompassing the intercalary, antennal and most of the ocular ectoderm. Until stage 11, the expression is gradually lost in the intercalary ectoderm, but upregulated in the dorsal part of the antennal and adjacent ocular ectoderm. In contrast to trunk NBs, which have already divided several times before expressing unpg at late stage 11, unpg-lacZ is weakly expressed already at stage 9 in all deutocerebral and almost all protocerebral NBs. At late stage 11, it is strongly expressed in almost all deutocerebral NBs (except for some ventral ones), and in some ocular NBs close to the deutocerebral/ocular border. Until the end of embryogenesis, unpg expression is observed in the putative progeny cells of the unpg-lacZ-positive deuto- and protocerebral NBs (Urbach, 2003).


REGULATION
Promoter Structure

To identify cis-regulatory regions that are responsible for the normal unpg expression in embryos, six restriction fragments encompassing 20 kb of unpg genomic sequence were tested for their ability to direct expression of a lacZ reporter gene containing a minimal hsp70 promoter. The only region capable of driving a lacZ reporter gene expression similar to the unpg protein distribution is located at the 3' end of the unpg transcription unit. The 2.7 kb fragment gives characteristic unpg-like expression in the CNS and the cerebral and ganglionic branches of the tracheal system during embryogenesis. This fragment also gives unpg-like expression in the cells around the first tracheal pit at the germband extended stage. Thus a 2.7 kb fragment of unpg 3' flanking sequence contains most of the cis-regulatory elements for the normal unpg expression in embryos (Chiang, 1995)

Transcriptional Regulation

unplugged expression occurs in portions of the tracheal system that penetrate the CNS, including the cerebral branch specific to T1. To test the possibility that genes in the BX-C play a role in regulating unpg expression, the distribution of unpg transcript was examined in Ultrabithorax and abdominal-A mutants, and in Ubx, abd-A, Abd-B triple mutants. In Ubx mutant embryos additional unpg expression is observed in cells surrounding the tracheal pits of T2 and T3, indicative of a role for Ubx in repression of unpg in the posterior segments and consistent with homeotic transformation in Ubx mutants of posterior T2 and T3 toward a T1 identity. In abd-A mutants embryos extra patches of unpg-expressing cells around the tracheal pits extend posteriorly to A7, indicating a role for Abd-A in the repression of unpg expression in the abdominal segments. The homeotic gene Abdominal-B probably contributes to the repression of unpg expression in A7, since slightly elevated expression in A7 is observed in the triple mutants (Chiang, 1995).

The specific branching defects observed in unplugged mutant embryos prompted an examination of the relationship between unpg and pointed (pnt), a gene encoding a member of the ets family of transcription factors; pnt is expressed in tracheal placodes and in the developing tracheal branches. In the absence of pnt gene function, tracheal cells fail to migrate and branches do not extend to target tissues. In particular, stalling of the ganglionic branches at the ventral oblique musculature in hypomorphic pnt embryos is reminiscent of the most extreme phenotype observed in unpg embryos. To determine the regulatory relationship between pnt and unpg genes, pnt homozygous embryos were immunostained with Unplugged-specific antibody, in order to follow the fate of the ganglionic branches. At stage 12.5, ganglionic branch precursor cells migrating toward the CNS are clearly visible in wild-type embryos. In pnt mutant embryos, no migratory cells that are expressing Unpg protein can be detected; however, a few precursor cells accumulating low levels of Unpg protein occasionally can be identified. By stage 14, the ganglionic branches of normal embryos are well developed, as is evident from a group of 8 to 9 unpg-expressing cells along the ventrolateral region of each hemisegment. In pnt mutant embryos, only 3 to 4 unpg-expressing cells can be detected; these unpg-expressing cells are clustered in a group suggesting that the precursor cells remain immobile and fail to extend from the tracheal pits. These results suggest that the ganglionic branch phenotype in pointed embryos may be in part due to a loss or reduction of unpg gene expression and failure of unpg-expressing cells to extend into the CNS (Chiang, 1995). It would be of interest to determine if unpg functions downstream of pnt in the ventral midline of the CNS.


DEVELOPMENTAL BIOLOGY

Embryonic

unplugged expression first appears at stage 8 (3-3.5 hours of development) in the midline of the central nervous system (CNS) (Chiang, 1995). At midstage 11 (S4 neuroblast delamination stage), unpg expression is detected in neuroblasts NB 4-1, NB 5-3, NB 6-2 and NB 7-2 (Cui, 1995). These neuroblasts divide during germband extension to generate sibling neuroblasts and neurons that largely correspond to engrailed-expressing cells within the CNS. As the germband retracts [Images], midline CNS expression begins to fade, and by stage 14, the CNS expression is restricted to a few cells in each segment. Outside the CNS, unpg expression is first observed in two clusters of ectodermal cells located laterally within the labial and first thoracic (T1) segments of stage 9 embryos. During germband extension unpg expression continues in T1 and rapidly diminishes in the labial segment. By stage 11, the lateral cells are recognizable as 15-20 unpg- expressing cells around the anterior part of the first tracheal pit. As the germband retracts, these cells begin to migrate anterodorsally with expression restricted to 5-6 cells. By stage 13, the expression is detected in a few cells close to the dorsal midline of the embryos; these cells appear to form long cytoplasmic connections that prefigure the cerebral branches of the tracheal system. As the germband retracts, a new expression domain within the invaginated tracheal pits appears on each side of the CNS in segments T1-A7. Expression in this domain is restricted to a few cells per hemisegment, which may represent the precursors of the ganglionic branches of the tracheal system. During germband retraction, these precursor cells extend ventrally and dorsally. By stage 14, the ganglionic branch in each hemisegment consists of 7-9 unpg-expressing cells whose cell bodies appear to form a continuous chain that penetrates the CNS of stage 14 embryos. No RNA or protein expression of unpg outside the CNS can be detected in later stage embryos (Chiang, 1995).

To determine the tissue types of cells expressing unpg outside the CNS, double labeling experiments were performed using Unpg-specific antiserum and other antibodies that recognize different tissue types in the embryo. The elongated morphology of Unpg-expressing cells resembles the morphology of cells in the developing tracheal system. Indeed, double-labelling with Unpg-specific antiserum and 2A12, a monoclonal antibody that specifically highlights the lumen of the tracheal system, demonstrates that most Unpg-expressing cells outside the CNS also express the 2A12 antigen. On the ventrolateral side of each hemisegment, the Unpg protein accumulates in the nuclei of 7-9 cells overlapping with the 2A12 antigen in the ganglionic and lateral branches of the tracheal system. The organization of ganglionic branches differs between thoracic and abdominal segments, and this difference is reflected by the unpg expression pattern. On the dorsal side of stage 13 embryos, Unpg protein accumulates in 5-6 nuclei overlapping with 2A12 antigen in the cerebral branch of the first tracheal metamere. By stage 14, the cerebral branch courses posteriorly and medially so that it lies close to the dorsal midline of T2. Thus, unpg expression outside of the CNS is restricted to cells of the cerebral and ganglionic branches of the tracheal system during embryonic development (Chiang, 1995).

Four genes, ming, even-skipped, unplugged and achaete, are expressed in specific neuroblast sublineages. These neuroblasts can be identified in embryos lacking both neuroblast cytokinesis and cell cycle progression (string mutants) and in embryos lacking only neuroblast cytokinesis (pebble mutants). unplugged and achaete genes are expressed normally in string and pebble mutant embryos, indicating that temporal control is independent of neuroblast cytokinesis or counting cell cycles. In contrast, neuroblasts require cytokinesis to activate sublineage castor expression (while a single, identified neuroblast requires cell cycle progression to activate even-skipped expression). This suggests that neuroblasts have an intrinsic gene regulatory hierarchy controlling unplugged and achaete expression, but that mechanisms dependent on cell cycle or cytokinesis are required for castor and eve CNS expression (Cui, 1995).

For more information on Drosophila neuroblast lineages, see Linking neuroblasts to their corresponding lineage, a site carried by Flybrain, an online atlas and database of the Drosophila nervous system.


EFFECTS OF MUTATION

To study the function of the unpg gene, the 1912 line carrying a P element insertion in the first intron of unpg was exposed to transposase to generate mutations for phenotypic analysis. Of approximately 230 excision events, 12 were associated with homozygous lethality. The DNA lesion associated with unpg27 begins in the 5' end of the P element and extends to the region close to a SpeI restriction site in the third exon. Thus, the unpg r37 deletion removes all of exon 2 and part of exon 3, including the entire homeodomain sequence. Interestingly, the mutation still retains lacZ expression in embryos, consistent with the findings that the major regulatory sequences for unpg expression are located downstream of the unpg transcription unit. Specific expression of unpg in neural branches of the tracheal system suggests that unpg may play a role in tracheal development. Indeed, tracheal staining of unpb r37 homozygous mutant embryos with antibody 2A12 reveals the absence of the entire cerebral branch , with occasional ectopic branches in the first tracheal metamere. Also absent is the cerebral anastomosis, which normally is associated with the cerebral branch. A specific defect is also observed in the ganglionic branches, which in most cases extend only partially and fail to penetrate the CNS. Similar effects on the cerebral branch and anastomosis and on ganglionic branches are observed with the unpg r225 and unpg r1 alleles. The specific defects observed in the unpg mutants are consistent with the unpg protein distribution and suggest a specific role for unpg in the formation of tracheal branches that penetrate the CNS. Despite these tracheal defects, about 3-5% of homozygous unpg r37 flies, under uncrowded culture conditions, eclose to adulthood; these escapers exhibit an upheld wing phenotype (Chiang, 1995).


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).

The posteriorizing gene Gbx2 is a direct target of Wnt signalling and the earliest factor in neural crest induction

Wnt signalling is required for neural crest (NC) induction; however, the direct targets of the Wnt pathway during NC induction remain unknown. This study shows that the homeobox gene Gbx2 is essential in this process and is directly activated by Wnt/beta-catenin signalling. By ChIP and transgenesis analysis it was shown that Gbx2 regulatory elements that drive expression in the NC respond directly to Wnt/beta-catenin signalling. Gbx2 has previously been implicated in posteriorization of the neural plate. This study unveils a new role for this gene in neural fold patterning. Loss-of-function experiments using antisense morpholinos against Gbx2 inhibit NC and expand the preplacodal domain, whereas Gbx2 overexpression leads to transformation of the preplacodal domain into NC cells. The NC specifier activity of Gbx2 is dependent on the interaction with Zic1 and the inhibition of preplacodal genes such as Six1. In addition, that Gbx2 is upstream of the neural fold specifiers Pax3 and Msx1. These results place Gbx2 as the earliest factor in the NC genetic cascade being directly regulated by the inductive molecules, and support the notion that posteriorization of the neural folds is an essential step in NC specification. A new genetic cascade is proposed that operates in the distinction between anterior placodal and NC territories (Li, 2009).

Tbx1 controls cardiac neural crest cell migration during arch artery development by regulating Gbx2 expression in the pharyngeal ectoderm

Elucidating the gene regulatory networks that govern pharyngeal arch artery (PAA) development is an important goal, as such knowledge can help to identify new genes involved in cardiovascular disease. The transcription factor Tbx1 plays a vital role in PAA development and is a major contributor to cardiovascular disease associated with DiGeorge syndrome. This study used various genetic approaches to reveal part of a signalling network by which Tbx1 controls PAA development in mice. The crucial role played by the homeobox-containing transcription factor Gbx2 downstream of Tbx1 was investigated. PAA formation requires the pharyngeal surface ectoderm as a key signalling center from which Gbx2, in response to Tbx1, triggers essential directional cues to the adjacent cardiac neural crest cells (cNCCs) en route to the caudal PAAs. Abrogation of this signal generates cNCC patterning defects leading to PAA abnormalities. Finally, it was shown that the Slit/Robo signalling pathway is activated during cNCC migration and that components of this pathway are affected in Gbx2 and Tbx1 mutant embryos at the time of PAA development. It is proposed that the spatiotemporal control of this tightly orchestrated network of genes participates in crucial aspects of PAA development (Calmont, 2009).

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).

Relatively little is known about the development of the thalamus, especially its differentiation into distinct nuclei. This study demonstrate that Gbx2-expressing cells in mouse diencephalon contribute to the entire thalamic nuclear complex. However, the neuronal precursors for different thalamic nuclei display temporally distinct Gbx2 expression patterns. Gbx2-expressing cells and their descendents form sharp lineage-restriction boundaries delineating the thalamus from the pretectum, epithalamus and prethalamus, revealing multiple compartmental boundaries within the mouse diencephalon. Without Gbx2, cells originating from the thalamus abnormally contribute to the epithalamus and pretectum. This abnormality does not result from an overt defect in patterning or cell-fate specification in Gbx2 mutants. Chimeric and genetic mosaic analysis demonstrate that Gbx2 plays a cell-nonautonomous role in controlling segregation of postmitotic thalamic neurons from the neighboring brain structures that do not express Gbx2. It is proposed that, within the developing thalamus, the dynamic and differential expression of Gbx2 may be involved in the specific segregation of thalamic neurons, leading to partition of the thalamus into different nuclei (Chen, 2009).

Studies in mouse, Xenopus and chicken have shown that Otx2 and Gbx2 expression domains are fundamental for positioning the midbrain-hindbrain boundary (MHB) organizer. Of the two zebrafish gbx genes, gbx1 is a likely candidate to participate in this event because its early expression is similar to that reported for Gbx2 in other species. Zebrafish gbx2, in contrast, acts relatively late at the MHB. To investigate the function of zebrafish gbx1 within the early neural plate, a combination of gain- and loss-of-function experiments was used. Ectopic gbx1 expression in the anterior neural plate reduces forebrain and midbrain, represses otx2 expression and repositions the MHB to a more anterior position at the new gbx1/otx2 border. In the case of gbx1 loss-of-function, the initially robust otx2 domain shifts slightly posterior at a given stage (70% epiboly), as does MHB marker expression. Ectopic juxtaposition of otx2 and gbx1 leads to ectopic activation of MHB markers fgf8, pax2.1 and eng2. This indicates that, in zebrafish, an interaction between otx2 and gbx1 determines the site of MHB development. This work also highlights a novel requirement for gbx1 in hindbrain development. Using cell-tracing experiments, gbx1 was found to cell-autonomously transform anterior neural tissue into posterior. Previous studies have shown that gbx1 is a target of Wnt8 graded activity in the early neural plate. Consistent with this, it was shown that gbx1 can partially restore hindbrain patterning in cases of Wnt8 loss-of-function. It is proposed that in addition to its role at the MHB, gbx1 acts at the transcriptional level to mediate Wnt8 posteriorizing signals that pattern the developing hindbrain. These results provide evidence that zebrafish gbx1 is involved in positioning the MHB in the early neural plate by refining the otx2 expression domain. In addition to its role in MHB formation, gbx1 is a novel mediator of Wnt8 signaling during hindbrain patterning (Rhinn, 2009).

Cerebellar development in the absence of Gbx function in zebrafish

The midbrain-hindbrain boundary (MHB) is a well-known organizing center during vertebrate brain development. The MHB forms at the expression boundary of Otx2 and Gbx2, mutually repressive homeodomain transcription factors expressed in the midbrain/forebrain and anterior hindbrain, respectively. The genetic hierarchy of gene expression at the MHB is complex, involving multiple positive and negative feedback loops that result in the establishment of non-overlapping domains of Wnt1 and Fgf8 on either side of the boundary and the consequent specification of the cerebellum. The cerebellum derives from the dorsal part of the anterior-most hindbrain segment, rhombomere 1 (r1), which undergoes a distinctive morphogenesis to give rise to the cerebellar primordium within which the various cerebellar neuron types are specified. Previous studies in the mouse have shown that Gbx2 is essential for cerebellar development. Using zebrafish mutants this study shows that in the zebrafish gbx1 and gbx2 are required redundantly for morphogenesis of the cerebellar primordium and subsequent cerebellar differentiation, but that this requirement is alleviated by knocking down Otx. Expression of fgf8, wnt1 and the entire MHB genetic program is progressively lost in gbx1-;gbx2- double mutants but is rescued by Otx knock-down. This rescue of the MHB genetic program depends on rescued Fgf signaling, however the rescue of cerebellar primordium morphogenesis is independent of both Gbx and Fgf. Based on these findings a revised model is proprosed for the role of Gbx in cerebellar development (Su, 2013).

Mutual repression between Gbx2 and Otx2 in sensory placodes reveals a general mechanism for ectodermal patterning

In the vertebrate head, central and peripheral components of the sensory nervous system have different embryonic origins, the neural plate and sensory placodes. This raises the question of how they develop in register to form functional sense organs and sensory circuits. This study shows that mutual repression between the homeobox transcription factors Gbx2 and Otx2 patterns the placode territory by influencing regional identity and by segregating inner ear and trigeminal progenitors. Activation of Otx2 targets is necessary for anterior olfactory, lens and trigeminal character, while Gbx2 function is required for the formation of the posterior otic placode. Thus, like in the neural plate antagonistic interaction between Otx2 and Gbx2 establishes positional information thus providing a general mechanism for rostro-caudal patterning of the ectoderm. These findings support the idea that the Otx/Gbx boundary has an ancient evolutionary origin to which different modules were recruited to specify cells of different fates (Steventon, 2012).

To form a functional nervous system its peripheral and central components must develop in register. In the head, the olfactory bulb, the retina and the targets and proximal parts of the sensory ganglia are derived from the central nervous system, while the olfactory epithelium, the lens, inner ear and distal cranial ganglia arise in the non-neural ectoderm from specialized structures, the sensory placodes. How is anterior-posterior patterning between both territories integrated? During development sensory placode precursors originate in the pre-placodal region, where cells of different fates are initially intermingled. Over time, they acquire distinct rostro-caudal identity leading to the alignment with their central counterparts suggesting that a global patterning mechanism imparts positional information to the entire ectoderm. This study shows that the transcription factors Otx2 and Gbx2 are important components of such a mechanism. In the pre-placodal region (PPR), they segregate otic and trigeminal progenitors, while they establish a compartment boundary at the midbrain-hindbrain boundary (MHB) in the neural plate and prevent mixing of cells with different fates. In both regions, Otx2 and Gbx2 seem to play a dual role: they repress each other to endow cells with unique identities and to suppress the alternative fate (trigeminal vs otic; midbrain vs rhombomere1), while simultaneously mediating sorting. Initially, both genes partially overlap and mutual repression at the transcriptional level is likely to form a gene expression the boundary. Subsequently, cell sorting ensures compartmentalization to restrict cells of the same fate to a contiguous domain. Accordingly, in the brain, Otx2 deficient cells segregate from wild type neighbors as do cells expressing exogenous Otx2 in rhombomere. Likewise, the results show that Otx2 and Gbx2 expressing cells sort out in the non-neural ectoderm. The degree of cell mixing in the placode territory is still under debate with more cell mixing observed in chick than in Xenopus. As fate maps may introduce some error due to variability between different embryos, ultimately live imaging over long time periods will be required to resolve this question. Nevertheless, together with previous studies on neural and neural crest cells the current findings establish cross-regulatory interactions between Otx2 and Gbx2 as key components for global ectodermal patterning. Both factors establish anterior-posterior identity across the embryonic ectoderm and mediate cell sorting to segregate cells of different fates (Steventon, 2012).

These observations also suggest that signals that establish anterior-posterior identity not only pattern the neural plate, but the entire ectoderm with transcription factors like Otx2 and Gbx2 as a read-out. Among these Fgfs, Wnts, Retinoic Acid, Nodals and BMPs provide posteriorizing factors, while their antagonists protect anterior identity. Indeed, elevated Wnt activity in zebrafish leads to an expansion of posterior neural and placodal fates. Wnt signaling also promotes derivatives of the posterior neural plate border, neural crest cells, and Gbx2, a direct Wnt target, mediates its activity. In addition to such global patterning mechanisms local signaling and downstream transcriptional networks subsequently fine tune allocation of different cell fates (Steventon, 2012).

In the PPR, the Otx2/Gbx2 boundary roughly separates prospective otic and trigeminal fates suggesting that olfactory, lens and trigeminal precursors receive different transcriptional inputs from otic progenitors. The transcriptional regulation of the PPR marker Six1 supports this idea. Although Six1 is expressed in a contiguous domain containing all sensory progenitors, different enhancers control its expression along the anterior-posterior axis. Cells from the anterior Six1 domain contribute to the olfactory, lens and trigeminal placodes, but not to the otic. These findings suggest that one of the first subdivisions of the placode territory occurs between trigeminal and otic precursors clearly grouping trigeminal precursors together with other anterior placodes unlike an earlier suggestion to group profundal and trigeminal placodes in Xenopus with posterior progenitors. Shortly thereafter, the PPR begins to express other transcription factors in nested domains to subdivide this territory further (Steventon, 2012).

Otx2 and Gbx2 are already expressed at gastrula stagesand act early during placode specification. Gbx2 is required for the onset of otic-specific genes, where it appears to act as transcriptional activator: the constitutive repressor Gbx2-EnR mimics MO-mediated knock-down. This is in contrast to its earlier role as repressor during boundary formation suggesting that the availability of cofactors determines the final outcome as observed for other homeobox factors. After initial specification, otic development is Gbx2 independent, although it is later involved in ear morphogenesis. The lack of an early ear phenotype in Gbx2 mutant mice is likely due to functional redundancy with Gbx1. In contrast, Otx2 is necessary for both formation and maintenance of lens and olfactory identity consistent with its continued expression in both placodes. In the trigeminal placode, Otx2 is downregulated shortly after its specification probably due to repression by Pax3, which also inhibits Pax6 in this territory. Like Gbx2, Otx2 switches from a transcriptional repressor at early stages to an activator later. In summary, like in the neural plate, in the PPR Otx2 and Gbx2 are among the earliest factors that subdivide a contiguous territory along the anterior-posterior axis (Steventon, 2012).

Although Otx2 and Gbx2 are required for early placode specification, neither factor alone is sufficient to endow cells with new regional character or to induce ectopic placodes. This appears to differ considerably from their activity in the neural tube, where ectopic expression of either factor respecifies anterior-posterior identity . However, here Otx2 and Gbx2 mainly function to position the MHB, an organizer region that itself patterns the brain. Thus, changes in regional identity are likely to be a consequence of MHB induction. Whether a similar organizing center forms at the Otx2/Gbx2 boundary in the PPR remains to be established, however, so far the results argue against this notion. The finding that neither Gbx2 nor Otx2 is sufficient to induce ectopic placodes suggests that additional factors cooperate to control the expression of placode-specific downstream targets. This is indeed the case in the lens, where Otx2 directly binds to the lens-specific FoxE3 enhancer and together with intracellular effectors of Notch signaling activates its transcription (Steventon, 2012).

The development of cranial sensory placodes and neural crest is considered to be a key step in the evolution of the vertebrate head. Like in vertebrates Gbx and Otx form a boundary within the Amphioxus ectoderm raising the question whether, at an early stage of their evolution, neural crest and placodes co-opted an already existing gene expression boundary to position themselves along the anterior-posterior axis. Despite Gbx2/Otx2 apposition in Amphioxus, MHB specific genes such as En, Wnt1, FGF8/17/18 and Pax2/5/8 are not restricted to this boundary, indicating that MHB organizer genes were recruited to the Otx/Gbx border in early vertebrates. A Gbx/Otx boundary appears to have been present in the early bilaterian ancestor as Unpg/Gbx and Otd/Otx also negatively regulate one another to form a boundary that positions En and Pax2/5/8 in Drosophila. In addition, Gbx2 and Otx2 form a boundary in the annelid Platynereis dumerilii that corresponds to a band of En expression. Together these findings raise the possibility that Otx2 and Gbx2 form an ancient boundary of gene expression responsible for anterior-posterior patterning of both the neural plate and neural plate border. However, this boundary has been utilized differently in each territory: to position an organizing region at the MHB, and to specify placodal fates in the PPR (Steventon, 2012).

Gbx2 directly restricts Otx2 expression to forebrain and midbrain, competing with class III POU factors

Otx2 plays essential roles in rostral brain development, and its counteraction with Gbx2 has been suggested to determine the midbrain-hindbrain boundary (MHB) in vertebrates. The FM enhancer has been identified that is conserved among vertebrates and drives Otx2 transcription in forebrain/midbrain from the early somite stage. This study found that the POU homeodomain of class III POU factors (Brn1, Brn2, Brn4, and Oct6) associates with noncanonical target sequence TAATTA in the FM enhancer. MicroRNA-mediated knockdown of Brn2 and Oct6 diminished the FM enhancer activity in anterior neural progenitor cells (NPCs) differentiated from P19 cells. The class III POU factors associate with the FM enhancer in forebrain and midbrain but not in hindbrain. It was also demonstrated that the Gbx2 homeodomain recognizes the same target TAATTA in the FM enhancer, and Gbx2 associates with the FM enhancer in hindbrain. Gbx2 misexpression in the anterior NPCs represses the FM enhancer activity and inhibits Brn2 association with the enhancer, whereas Gbx2 knockdown caused ectopic Brn2 association in the posterior NPCs. These results suggest that class III POU factors and Gbx2 share the same target site, TAATTA, in the FM enhancer and that their region-specific binding restricts Otx2 expression at the MHB (Inoue, 2012).

Gbx2 regulates thalamocortical axon guidance by modifying the LIM and Robo codes

Combinatorial expression of transcription factors forms transcriptional codes to confer neuronal identities and connectivity. However, how these intrinsic factors orchestrate the spatiotemporal expression of guidance molecules to dictate the responsiveness of axons to guidance cues is less understood. Thalamocortical axons (TCAs) represent the major input to the neocortex and modulate cognitive functions, consciousness and alertness. TCAs travel a long distance and make multiple target choices en route to the cortex. The homeodomain transcription factor Gbx2 is essential for TCA development, as loss of Gbx2 abolishes TCAs in mice. Using a novel TCA-specific reporter, this study has discovered that thalamic axons are mostly misrouted to the ventral midbrain and dorsal midline of the diencephalon in Gbx2-deficient mice. Furthermore, conditionally deleting Gbx2 at different embryonic stages has revealed a sustained role of Gbx2 in regulating TCA navigation and targeting. Using explant culture and mosaic analyses, it was demonstrated that Gbx2 controls the intrinsic responsiveness of TCAs to guidance cues. The guidance defects of Gbx2-deficient TCAs are associated with abnormal expression of guidance receptors Robo1 and Robo2. Finally, Gbx2 was demonstrated to control Robo expression by regulating LIM-domain transcription factors through three different mechanisms: Gbx2 and Lhx2 compete for binding to the Lmo3 promoter and exert opposing effects on its transcription; repressing Lmo3 by Gbx2 is essential for Lhx2 activity to induce Robo2; and Gbx2 represses Lhx9 transcription, which in turn induces Robo1. These findings illustrate the transcriptional control of differential expression of Robo1 and Robo2, which may play an important role in establishing the topography of TCAs (Chatterjee, 2012).

Gbx2, a LIF/Stat3 target, promotes reprogramming to and retention of the pluripotent ground state

Activation of signal transducer and activator of transcription 3 (Stat3) by leukemia inhibitory factor (LIF) maintains mouse embryonic stem cell (mESC) self-renewal and also facilitates reprogramming to ground state pluripotency. Exactly how LIF/Stat3 signaling exerts these effects, however, remains elusive. This study identified gastrulation brain homeobox 2 (Gbx2) as a LIF/Stat3 downstream target that, when overexpressed, allows long-term expansion of undifferentiated mESCs in the absence of LIF/Stat3 signaling. Elevated Gbx2 expression also enhanced reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. Moreover, overexpression of Gbx2 was sufficient to reprogram epiblast stem cells to ground state ESCs. Thee results reveal a novel function of Gbx2 in mESC reprogramming and LIF/Stat3-mediated self-renewal (Tai, 2013).

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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Biological Overview

date revised: 10 August 2013

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