unplugged

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.

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).

Effects of Mutation or Deletion

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).

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date revised: 10 August 2003  

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