adrift

Transcriptional Regulation

adrift is expressed in the leading cells of growing tracheal branches, near clusters of branchless FGF-expressing cells and in a pattern very similar to that of several known branchless-induced genes including pointed, DSRF/pruned and sprouty. This suggested that adrift expression might also be induced by the bnl signaling pathway. Expression of an adrift lacZ reporter was examined in embryos mutant for four components of the branchless pathway: bnl, breathless, pnt and pruned. Initial expression of the adrift reporter in stage 11 tracheal cells is normal in all four mutants, but subsequent expression in the leading cells of the branches is absent in bnl, btl and pnt mutants. Expression in pruned mutants is unaffected. In a complementary experiment in which bnl was misexpressed under the control of the hsp70 promoter, expression of the adrift reporter expands to include additional cells in each branch. Thus, the Branchless FGF pathway induces adrift expression in the leading cells of tracheal branches, and this induction requires the bnl FGF, the btl FGF receptor and the pointed ETS domain transcription factor (Englund, 1999).

DEVELOPMENTAL BIOLOGY

Embryonic

Immunolocalization studies in wild-type embryos detect Adrift protein in the nuclei of all cells at cellular blastoderm stage, presumably derived from the abundant maternal Adrift mRNA, and also in nuclei of the gonads, epidermis and brain lobes of older embryos. Unfortunately, the antisera are not sensitive enough to reproducibly detect the endogenous tracheal expression of the gene. However, when adrift is overexpressed in the developing tracheal system using the UAS/GAL4 system, Adrift antigen was readily detected and is predominantly nuclear (Englund, 1999).

The pattern of adrift transcription during embryonic development was determined by Northern blot analysis, whole-mount in situ hybridization and analysis of an adrift lacZ reporter (Pantip-4). The gene is maternally expressed and transcripts are evenly distributed during the syncytial blastoderm stage; most of the maternal transcripts are degraded during early embryogenesis. Zygotic transcription is first detected by in situ hybridization in mid-embryogenesis in the developing tracheal system and later in the developing gonad. Tracheal expression is highly dynamic as revealed by both in situ hybridization and expression of the adrift lacZ reporter. At stage 11, the gene is expressed weakly in all tracheal cells. As the primary branches bud and grow out during stages 12 and 13, the gene is preferentially expressed in the leading cells of the GB and other growing primary branches. During stages 14 and 15, expression becomes further restricted to just the GB1 terminal cell and other terminal cells that lead the migrations toward the CNS and other target tissues. No tracheal expression is detected in adrift mutant embryos. Maternal and gonadal expression, however, is detected in the mutants, indicating that adrift alleles selectively affect tracheal expression of the gene. Analysis of the molecular lesions in the alleles has identified alterations at the P-element insertion site upstream of the coding region, suggesting that this may be a cis-regulatory region important for tracheal expression of the gene (Englund, 1999).

Effects of Mutation or Deletion

The adrift gene was identified by the phenotypic effect engendered by insertion of a transposable genetic marker into the gene. The Pantip-4 P(lacZ, w+) transposon is located at chromosomal position 54F and expresses the beta-galactosidase marker in the lead cells of the GB and other growing primary branches (Samakovlis, 1996). The original insertion, generated in the laboratory of M. Scott, causes weak, sporadic defects in GB outgrowth. By introducing a source of transposase, 170 w- transposon excision alleles were obtained. Two homozygous viable alleles (excisions 28 and 70) display a similar tracheal phenotype and fail to complement for this function. These alleles are referred to as adrift₁ and adrift₂ because of their tracheal phenotypes. Molecular analysis indicates that adrift₁ represents the zygotic null condition for the tracheal function of the gene and thus became the focus of the phenotypic analysis. In adrift mutants, GBs sporadically stall or are misrouted and fail to follow their stereotyped path into the CNS. In adrift₁ mutants, 18% of GBs miss the entry point into the VNC and continue to migrate along the ventral epidermis. An additional 6% of GBs stall at or before reaching the VNC. Neither defect is seen in wild-type controls. Double labeling with tracheal and neural markers demonstrates that GBs in homozygous adrift₁ embryos grow normally over the ISN during the initial phase of their ventral migration but sporadically fail to make the switch to the SN and associate with the exit glial cells. Most of the affected branches instead turn posteriorly and continue to migrate along the ventral epidermis, forming a characteristic ‘hook’ structure (Englund, 1999).

The defect in GB guidance in adrift₁ mutants does not result from grossly aberrant differentiation of the tracheal cells. The cells retain their ability to migrate and express all of the appropriate secondary and terminal branch markers tested including pointed, sprouty, Pantip-4 lacZ and DSRF. Furthermore, the structure of the nerves and glial cells normally contacted by the growing GB are unaffected in the mutant. It is concluded that adrift is specifically required to promote recognition and association of the GB1 cell with the SN and glial cells at the first guidepost in its navigation into the CNS (Englund, 1999).

Englund, C., et al. (1999). adrift, a novel bnl-induced Drosophila gene, required for tracheal pathfinding into the CNS. Development 126: 1505-1514. Medline abstract: 10068643

Loo, S., Laurenson, P., Foss, M., Dillin, A. and Rine, J. (1995). Roles of ABF1, NPL3, and YCL54 in silencing in Saccharomyces cerevisiae. Genetics 141: 889-902. Medline abstract: 8582634

Samakovlis, C., Hacohen, N., Manning, G., Sutherland, D., Guillemin, K. and Krasnow, M. A. (1996). Development of the Drosophila tracheal system occurs by a series of morphologically distinct but genetically coupled branching events. Development 122: 1395-1407. Medline abstract: 8625828

Sutherland, D., Samakovlis, C. and Krasnow, M. A. (1996). branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and pattern of branching. Cell 87: 1891-1101. Medline abstract: 8978613

date revised: 23 March 99

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