adrift : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - adrift
Cytological map position -
Function - regulator of tracheal pathfinding of unknown function
Keywords - trachea
Symbol - aft
Genetic map position -
Classification - novel protein
Cellular location - nuclear
Developing neurons undergo complex migrations in the process of constructing a functional nervous system. An important area of neurobiological research is aimed at identifying and characterizing the positional cues that guide these migrations. The nervous system provides a highly diverse array of positional cues, many of which are presented by developing neurons and glial cells. This positional information potentially could be used by other migrating cells. Migrating tracheal cells in Drosophila contact specific neural and glial cells as they navigate to precise targets in the CNS, and a novel tracheal gene called adrift is required for the process. The Drosophila tracheal system is a branched network of epithelial tubes that transports oxygen throughout the body. It develops from 20 ectodermal cell clusters, each containing approximately 80 cells. Each cluster invaginates, forming an epithelial sac; then primary, secondary and terminal branches sequentially bud from each sac to generate the 20 tracheal hemisegments. Initially, six multicellular buds form and grow out along stereotyped paths to generate the six primary branches. One of these, the ganglionic branch (GB) targets the ventral nerve cord. The leading cell of the GB forms a unicellular tube, called a secondary branch, as do the leading cell or cells of other primary branches. These secondary branches grow toward different target tissues and later ramify into the terminal branches that supply the targets with oxygen (Englund, 1999).
The critical factor that controls the outgrowth of primary branches is the product of the branchless (bnl) gene, a homolog of vertebrate FGFs (Sutherland, 1996). bnl is expressed in discrete clusters of ectodermal and mesodermal cells arrayed around each tracheal sac. The secreted growth factor functions as a chemoattractant that activates the Breathless (Btl) FGF receptor on nearby tracheal cells and guides their migrations as they grow out and form primary branches. Branchless signaling also induces expression of genes, including pointed and DSRF, required for secondary and terminal branching in the leading cells of the primary branches. These cells go on to form secondary and subsequently terminal branches. Secondary ganglionic branches that grow into the ventral nerve cord navigate a remarkably complex and tortuous path, tracking along five different neural and glial substrata to reach their ultimate CNS target. Mutations in the gene adrift affect the first substratum switch causing failure of GBs to enter the CNS and misrouting along the ventral epidermis. Molecular characterization of the adrift gene demonstrates that it encodes a novel nuclear protein. Expression of the gene is induced in the lead cell of the migrating ganglionic branch by the bnl FGF pathway, whereupon it promotes the switch from one neural substratum to another (Englund, 1999).
Cell-specific tracheal and CNS markers were used to define the movements and contacts of the migrating tracheal ganglionic branches (GB) as they grow into the ventral nerve cord (VNC). There are twenty ganglionic branches, two in each Tr1 tracheal hemisgement and one in each hemisegment from Tr2 to Tr9. The navigation pathway of each GB is complex but highly stereotyped, involving interactions with two different nerves and several glial cell types along the way. The path followed by the 14 abdominal GBs is the same in each segment. The six thoracic GBs follow a similar path, except they turn dorsally and anteriorly as they approach the midline in the VNC (Englund, 1999).
The typical GB is an epithelial tube composed of seven cells (Samakovlis, 1996). As the GB buds and grows out, a single cell called GB1 at the end of the branch leads the migrations; the remaining six GB cells (GB2-GB7) follow in line behind. The GB migration path can be conceptually divided into five segments based on the direction of migration and the migration substratum and cell contacts. Initially, the GB tracks along the intersegmental nerve (ISN) toward the CNS (segment I). At the CNS entry point, the GB switches onto the segmental nerve (SN) and associates with exit glia nearby. In the CNS, the GB1 cell grows along the SN and segmental nerve root glia (segment II) and then the ventral longitudinal glia (segment III), approaching the midline. At the midline, it turns and extends dorsally along the dorsoventral channel surrounded by the channel glia (segment IV). Once it reaches the dorsal side of the neuropil, the GB1 cell turns back away from the midline, contacting the dorsal longitudinal glia and then continues to migrate posteriorly along the longitudinal connectives and surrounding glia toward the next hemisegment (segment V), to complete its embryonic migrations. During larval life, GB1 grows again, this time ramifying extensively into fine terminal branches that provide the ~230 neurons in each hemisegment with oxygen (Englund, 1999).
At embryonic stage 13, the GB cells migrate ventrally toward the CNS, in close contact with the ISN. The GB cells are attracted toward the CNS by two ventrolateral clusters of epidermal cells expressing the bnl FGF. At stage 14, as the tip of the GB approaches the entry point into the VNC, expression of bnl ceases. The lead cell (GB1) turns posteriorly, breaks contact with the ISN and changes substratum to associate with the SN. During stage 15, GB1 migrates along the SN as it traverses laterally and ventrally inside the VNC: GB2 follows GB1 onto the SN. The other GB cells (GB3-GB7) also release contact with the ISN but they do not bind the SN or enter the VNC and they end up suspended in the body cavity. A group of CNS glial cells, termed exit glia, surround the point where the GB cells switch from the ISN to the SN, suggesting that they may play a role in the switching (Englund, 1999).
As the GB1 cell continues medially on the ventral side of the neuropil during stage 16, it comes in close contact with the ventral longitudinal glial cells. Just before reaching the midline glia, GB1 turns abruptly and migrates dorsally to reach the dorsal side of the neuropil. It then turns again and extends posteriorly on the dorsal longitudinal glial tracts toward the neighboring hemisegment. EM analysis of sagittal sections of the VNC of stage 16 wild-type embryos confirms the direct association between GB cells and various glial cells along the migration pathway. Inside the VNC, just beyond the point of the ISN to SN switch, a segmental nerve root glial cell (SNG) completely engulfs both the GB and the SN. In serial sections that allow one to follow the GB towards its tip, contacts between GB1 and longitudinal glia and dorsoventral channel glia are also seen (Englund, 1999).
One of the most challenging transitions on the GB migration path occurs at the entry into the CNS. At late stage 14, all seven cells of the branch have migrated ventrally in response to bnl signaling, and the lead cell, GB1, has been induced to express secondary and terminal branching genes. The GB1 cell leaves the ISN and associates with the SN. This substratum switch occurs at the CNS exit junction, which is composed of the two major nerve roots surrounded by a specialized group of exit glial cells. The junction is also a plexus region where motorneurons leaving the CNS switch pathways and selectively fasciculate into five major nerve branches destined for different regions of the musculature. Thus, the GB1 cell must not only release its old substratum but also select a new substratum from among several glial cells and neuronal projections. The GB2 cell makes a similar choice when it crosses the same junction ~1 hour later. In adrift mutants, most of the affected GBs migrate into the exit junction, release contact with the ISN, and extend posteriorly towards the SN but fail to attach to it or the surrounding glia. Instead, they continue migrating posteriorly along the ventral epidermis and muscle. This suggests that adrift functions in a signalling pathway that attracts the GB onto the SN or fixes on it once there. The expression pattern of the gene and mutant rescue by an adrift transgene expressed in migrating tracheal cells demonstrates that adrift is required in the cells that receive the branchless signal and not in the signaling cells or the SN cells to which the GB cells adhere (Englund, 1999).
What might be the source of the branchless signal? The most obvious candidates are the SN neurons or surrounding glial cells. Several axons including those of the RP1, RP3, RP4 and RP5 motoneurons switch from ISN to SN as they leave the CNS at the exit junction. These might express short-range attractive signals or adhesion molecules and provide a bridge for the growth of the GB from the ISN to SN. Other potential sources of the signal are the three or four exit glia cells and the segmental nerve root glia that are intimately associated with the GB at the exit junction. In the absence of mutants that selectively affect these glial subtypes, the roles of these glial cells in GB pathfinding could not be specifically assessed. However, preliminary analysis of the glial cells missing (gcm) mutant in which many glial cell types fail to differentiate properly, has revealed several GB pathfinding phenotypes, including misrouted branches that do not enter the CNS, supporting a role for glial cells in GB pathfinding events (Englund, 1999).
The adrift gene was identified by the phenotypic effect engendered by insertion of a transposable genetic marker into the gene. The original insertion, called the Pantip-4 marker, was isolated in the laboratory of M. Scott. In adrift mutants, GBs sporadically stall or are misrouted and fail to follow their stereotyped path into the CNS. In adrift 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. GBs in homozygous adrift mutant 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 to the posterior and continue to migrate along the ventral epidermis, forming a characteristic hook structure. The defect in GB guidance in adrift 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. 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 a target gene of branchless signaling, and 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).
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).
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).
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).
Search PubMed for articles about Drosophila adrift
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
Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.