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

date revised: 10 August 2013

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