Gene name - unplugged
Cytological map position - 45C1--45C9
Function - transcription factor
Symbol - unpg
Genetic map position - 2-
Classification - homeodomain protein
Cellular location - presumably nuclear
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).
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).
Direct comparison between the unpg cDNA and genomic sequences reveals that the transcription unit of unpg is organized in 3 exons (each 3.5 kb) and separated by two introns (each 62 bp). Sequence analysis of the first intron of unpg also reveals that it contains a transposable element; this 1360 bp element is moderately repetitive and has copy numbers ranging from 25 to 30 within the Drosophila genome. Whether presence of the 1360 bp element in the unpg locus has any functional significance is unknown (Chiang, 1995).
Bases in 5' UTR - 125
Exons - 3
Bases in 3' UTR - 196
The Unpg protein contains a homeodomain (residues 319-378) belonging to a family that includes several vertebrate homeobox genes. The homeodomain shares amino acid identities ranging between 90% and 93% for homeodomains of the CHox7 gene in chicken, the HOX7Q and GBX2 genes in human, partial sequence of the MMoxA gene in mouse, the XlHox7a and XlHox7b gene in Xenopus, the G9 gene in goldfish, and the Hrox7 gene in abalone. Little is known about the expression and function of these vertebrate homologs; however, the MMoxA and G9 genes were initially isolated from brain libraries and thus may be involved in brain development or function. One of two introns in the unpg transcription unit interrupts homeodomain coding sequences at a location first noted in labial-class homeobox genes. This location, between Gln 44 and Val 45, is conserved for introns of many other homeodomain genes of different species, but is distinct from the location of the intron that interrupts homeodomain coding sequences in engrailed and related genes. Outside of the homeodomain, unpg shares no significant homology with other known proteins in the data base. However, Pro/Gln-rich regions are found amino terminal to the homeodomain, with one particular region from residues 111 to 142 comprising 52% proline and glutamine residues. Pro/Gln-rich regions are found in many proteins that are capable of transcriptional activation (Chiang, 1995).
date revised: 8 April 98
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