BIOLOGICAL OVERVIEW

branchless is Drosophila's only known fibroblast growth factor (FGF), and as its name implies, is involved in branching morphogenesis. Branching morphogenesis is an essential part of development in organs such as the trachea and lungs. Before taking a closer look at the gene branchless, a brief overview of branching morphogenesis and the role of fibroblast growth factors in general: FGFs constitute a large family of peptide growth factors, with nine different mammalian FGF genes and four genes encoding FGF receptors (Johnson, 1993). FGFs function as mitogens, trophic factors and differentiation factors. FGFs are involved in several processes of branching morphogenesis: in lung morphogenesis, in branching of seminiferous tubules and as stimulators of angiogenesis.

The Drosophila FGF receptor Breathless (BTL), expressed in the developing tracheal system, promotes branching at several stages of tracheal development, and plays a permissive role in the formation of certain branches (Reichman-Fried, 1994 and 1995). A second FGF receptor, Fibroblast growth factor receptor 1, is necessary for heart formation and mesodermal cell migration (Beiman, 1996 and Gisselbrecht, 1996).

The Drosophila tracheal (respiratory) system is a tubular epithelial network that delivers oxygen to internal tissues. Trachea form from segmentally repeated clusters of tracheal precursor cells, which give rise to the tracheal system by cell migration and elongation. Each cluster invaginates from the ectoderm and forms an epithelial sac of about 80 cells. The six main (primary) branches of each sac begin to form as one or two lead cells from any of five or six positions migrate out in stereotyped directions, similar from segment to segment. A small number of cells follow the lead cells and organize into tubes as they migrate. Several hours later, secondary branches sprout from the primary branches. Subsequently secondary branches ramify into dozens of terminal branches, which are long cytoplasmic extension that form a lumen and transport oxygen directly to the fly's tissues. Each level of tracheal branching is controlled by a particular set of genes, which have provided molecular markers for the different branch types. For example, breathless is necessary for primary branching, pointed is required to form secondary branches (Samakovlis, 1996) and Serum response factor (Guillemin, 1996) regulates terminal branch formation (Sutherland, 1996).

In the absence of branchless, tracheal cells fail to migrate and branch. bnl is expressed not in the trachea but in ectodermal cells that overly the migrating and branching trachea. The striking feature of bnl expression is its spatial complexity, one of the most complex patterns known in the embryo. For example, at stage 11, just before tracheal branching begins, bnl expression appears in five small clusters of epidermal cells arrayed around the tracheal sac, at positions where the five primary tracheal branches will soon bud. As the primary branches grow by cell migration over the next 2 hours (stages 12 and 13), expression in the clusters decreases. This appears to occur in a specific spatial pattern: the bnl-expressing cells closest to or contacting the growing tracheal branches lose expression first, with the tracheal cells continuing to migrate toward the remaining bnl-expressing cells. Two more cell clusters begin expressing bnl as expression in the other clusters turns off, presaging the subsequent outgrowth of additional specific branches (Sutherland, 1996).

What regulates the complex expression pattern of bnl? The answer is not yet known, but most likely a conbination of segmentation and dorsal-ventral patterning genes act on the promoter sequence of bnl. The even more striking dynamic nature of bnl expression, with new patches developing bnl expression even as expression in older patches declines, suggests that there is a complex temporal scheme of bnl expression added on top of the spatial one, and that there is feedback between branching and bnl expression, resulting in the diminished expression overlying positions where branching has taken place (Sutherland, 1996).

In Drosophila, Branchless acts as a guidance molecule controlling tracheal cell migration, but does not act as a mitogen, a documented role of FGFs in vertebrates. As expected for a chemoattractant, the concentration of BNL is critical for migration: migration of tracheal branches is sensitive to the level of bnl expression. BNL acts not only on initial tracheal branching, but acts during formation of secondary and tertiary branching. At each stage in branching, different genes, each dependent on bnl function, are expressed in temporal sequence (Sutherland, 1996). What regulates the temporal progression of bnl dependent gene expression? As descrubed below, one of the critical regulatiors of bnl levels is the local oxygen supply.

Sprouting of the major tracheal branches is stereotyped and controlled by hard-wired developmental cues. Ramification of the fine terminal branches is variable and regulated by oxygen, and this process is controlled by a local signal or signals produced by oxygen-starved cells. Evidence is provided that the critical signal is Branchless (Bnl) FGF, the same growth factor that patterns the major branches during embryogenesis. During larval life, oxygen deprivation stimulates expression of Bnl, and the secreted growth factor functions as a chemoattractant that guides new terminal branches to the expressing cells. Thus, a single growth factor is reiteratively used to pattern each level of airway branching, and the change in branch patterning results from a switch from developmental to physiological control of its expression (Jarecki, 1999).

There were 68% more cytoplasmic extensions and mature branches in first instar larvae grown for 20 hr under 5% O2 compared to siblings grown under normal atmospheric oxygen (21% O2). Conversely, larvae grown under high oxygen tension (60% O2) had fewer branches than normoxic controls. Similar effects are observed at each larval stage and at different positions in the animal. The morphology of the branches is also affected by oxygen. Under high oxygen tension the terminal branches are shorter and straight with few side branches, whereas under the low oxygen condition they are long and tortuous with many side branches. Thus, oxygen is an important regulator of terminal branching, influencing the initial budding and the final number and morphology of the branches (Jarecki, 1999).

To determine if the effects of oxygen on terminal branching are globally regulated or mediated by local signals produced by oxygen-starved tissues, small regions of the larvae were deprived of their normal oxygen supply. This was accomplished by generating clones of blistered- tracheal cells, which are unable to form terminal branches, leaving the surrounding region without its normal tracheation. The blistered+ tracheal cells in neighboring segments grow into the detracheated region, sprouting 40% more terminal branches than normal, whereas blistered+ tracheal cells not bordering the clone are unaffected. To determine if it was the absence of terminal branches or just the absence of an oxygen supply that induced neighboring branches to grow, clones of synaptobrevin- tracheal cells that extend terminal branches but are unable to form a lumen and deliver oxygen to the target were tested. Even in the presence of the nonfunctional synaptobrevin- terminal branches, neighboring wild-type tracheal cells grow into the oxygen-starved region. This implies that it is lack of oxygen delivery to the region, not some other function of missing terminal branches, that causes neighboring tracheal cells to respond. It is concluded that terminal branching is regulated by local oxygen need, and that oxygen-starved cells produce a signal or signals that can attract tracheal branches from as far as one segment away (Jarecki, 1999).

When bnl expression is examined by in situ hybridization at larval stages of development, it is found that the gene turns back on by the first larval instar and continues to be expressed throughout larval life. However, in contrast to its highly restricted expression pattern in the embryo, the gene is broadly expressed in the larva, including all tissues that become heavily tracheated with terminal branches. In particular, the three most highly tracheated tissuesógut, muscles, and central nervous system (CNS)óall show generalized expression of bnl during the first and second larval instars when terminal branches are sprouting. Thus, bnl is expressed in the appropriate tissues at the right time to regulate terminal branching in the larva. Several tissues that are not tracheated, including the epidermis and salivary gland, do not express bnl, strengthening the correlation between bnl expression and terminal branching. The correlation is not absolute though, as several other tissues with few or no branches, including the imaginal discs, heart, and fat body, do express significant levels of BNL mRNA. However, in no case does a terminally tracheated tissue not express bnl (Jarecki, 1999).

The arborization pattern of terminal branches is remarkably complex. Each terminal tracheal cell sprouts dozens of branches that spread out and contact nearly every cell in the target. At first glance, the complex and variable nature of the branch pattern suggests that the process is highly random, with the constraint only that branches fill the available space. The results presented here support a model in which the final pattern, although variable, is not at all random. Instead, branching is precisely controlled during development to meet the oxygen needs of the target cells. A model is proposed in which each cell experiencing an oxygen debt senses the impending crisis and responds by upregulating expression of Bnl. Bnl FGF diffuses to nearby tracheal cells and stimulates new tracheal branches to form and grow toward each signaling source. This supplies oxygen to the hypoxic cells and shuts off the signal. The process is dynamic and repeats itself many times due to the constantly changing balance in cell oxygen need and supply. Over the course of development, most cells would become hypoxic, serve as an FGF signaling center, and receive an appropriate tracheal supply. Thus, the ultimate pattern of tracheal branches would reflect the complex history of the oxygen needs and Bnl expression pattern of the tissues (Jarecki, 1999).

GENE STRUCTURE

Genomic length - 28 kb

Bases in 5' UTR - 216

Exons - 3

Bases in 3' UTR - 213

PROTEIN STRUCTURE

Amino Acids - 770

Structural Domains

Like most FGFs, the BNL protein contains a signal peptide at the N-terminus, suggesting that it is secreted. BNL is unusual in that it contains large domains flanking the FGF domain that are not found in the mammalian proteins (Sutherland, 1996).

A 99 residue segment of BNL (residues 260-358) is 30-40% identical to human FGFs 1, 2, and 9 as well as other vertebrate FGFs. Within this region, 17 of the 23 residues that are highly conserved among vertebrate FGFs are also conserved with BNL. Two introns in the BNL FGF domain are in the same positions as the introns in mammalian FGF genes (Sutherland, 1996).

The three-dimensional structures of two members of the FGF family, bovine acidic FGF and human basic FGF, have been crystallographically determined. These structures contain 12 antiparallel beta strands organized into a folding pattern with approximate threefold internal symmetry. Topologically equivalent folds have been previously observed for soybean trypsin inhibitor and interleukins-1 beta and -1 alpha. Beta-sheet strand 10, implicated in receptor and heparin binding by FGF, is adjacent to the site of an extended sequence insertion in several oncogene proteins of the FGF family (Xhu, 1991).

date revised: 2 Jan 97  

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