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Genes involved in tissue and organ development
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Embryonic origin, functional domains and physiology of Malpighian tubules
The tubules arise during embryogenesis as four protuberances extending from the proctodeum. As this tissue is considered ectodermal, Malpighian tubules are classified as an ectodermally derived secretory tissue. The protuberances grow, first by cell proliferation and then by extensive rearrangement of the cells, to produce elongated blind end tubes composed of a single-cell-layered epithelium (Hoch, 1994).
The development of Malpighian tubules reveals an essential role for the gene Krüppel. In each Malpighian tubule, one cell is singled out, the tip cell, whose function during embryogenesis is to promote cell division in its neighbours. The tip cell arises by division of a tip mother cell, which is selected from a cluster of equivalent cells, each expressing Krüppel in each tubule primordium. Each cluster is delineated by the expression of proneural genes; the selection of a single cell from each group involves lateral inhibition, mediated by the neurogenic genes. achaete is responsible for tip cell allocation, but Kr acts as the selector gene, responsible for tip cell fate. The tip cell directs the growth of the Malpighian tubules and organizes the mitotic response and migration of the other cells forming each tubule (Hoch, 1994). Therefore Krüppel is responsible for cell fate in the Malpighian tubules, a function quite distinct from Krüppel's role as a gap gene.
Drosophila possesses two pair of Malpighian tubules. The right pair of tubules project forward from their point of insertion within the hindgut and lie at the anterior end of the abdomen, and the left pair extend backwards so that their tips become attached to the posterior part of the hindgut. Each tubule pair unites to form a common ureter, which enters the intestine between the midgut and hindgut. The two anterior Malpighian tubules are classically described as comprising a distal initial segment and a proximal main segment, joined by a narrow transitional segment; the two posterior tubules, in contrast, were thought to consist solely of a main segment. Contemporary studies, using enhancer trap lines, which place reporter genes under the control of tissue specific enhancers, confirm this viewpoint and thus the nomenclature "initial," "transitional," and "main" segments has been adopted to described these genetically deduced domains (Sözen, 1997).
Enhancer trap studies reveal an unexpected complexity in Malpighian tubules in terms of both regions and cell types. Enhancer trap lines that delineate the initial and transitional segments of anterior tubules, reveal previously undescribed analogous domains in posterior tubules. It is also possible to subdivide the main segments. While the transitional-main segment boundary has been established in accordance with classical studies, an additional domain is found marking the lower third of the tubule and the ureter. This latter region, in turn, can be resolved into three subregions: a lower tubule and an upper and lower ureter (Sözen, 1997).
Previous studies have described just two tubule cell types: principal (type I) and secondary or stellate (type II). Both can be further subdivided. Principal cells, for example, comprise at least two distinct subpopulations. Thus there appear to be differences in otherwise indistinguishable cells with respect to enhancer trap expression patterns and presumably with respect to function as well. Type II cells are distributed evenly throughout the initial, transitional and main segments of posterior tubules and within the main segment of anterior tubules. None of the enhancer trap markers mark cells in the lower tubule or ureter, suggesting that the mechanism by which type II cells are specified respects the newly defined lower tubule boundary. Several lines mark a "tiny" cell type found in lower tubules and posterior midgut but do not mark the same genetic domain as stellate cells. Possibly these previously undescribed cells are counterparts of the myoendocrine cells recently described in Formica. These cells may monitor the fluid collected in the ureter and secrete neurohormones basally into the hemolymph to regulate muscle contractility or ion transport (Sözen, 1997).
Do discrete physiological properties map to the genetic domains that have been identified? With respect to a number of different transport processes, this is indeed the case. The obvious functional property of the tubule is to secrete urine. It has been reported that the initial segment of Drosophila anterior tubule does not secrete detectable fluid, that the lower third of the tubule is reabsorptive, and that only the main segment is responsible for fluid production. High levels of proton-pumping V-ATPases energize apical plasm membranes of several epithelia, including Malpighian tubules. The B-subunit of Drosophila V-ATPase is expressed in the initial and transitional segments and is much weaker in the reabsorptive main segment. The main segment consists entirely of the large, principal cell. This provides the first evidence that cation transport into the lumen of Malpighian tubules may be a unique property of principal, rather than type II, cells. A putative aquaporin has been cloned in Drosophila: this channel is found in stellate cell basolateral membranes. Given that stellate cells are found in secretory but not reabsorptive tubule regions, they may well play an essential role in fluid secretion. Another function ascribed to Malpighian tubules is the secretion of organic metabolites. This function is confined to the main segment. It is clear that the staining pattern for alkaline phosphatase precisely matches the lower tubule boundary, and is associated with the reabsorptive, rather than the secretory, domain of the tubule (Sözen, 1997).
Secretion by Malpighian tubules is under hormonal control. CAP2b, a cardioacceleratory peptide, is present in Drosophila and stimulates Malpighian tubule fluid secretion via cGMP, which in turn stimulates the nitric oxide signaling pathway. Liquid chromatography analysis of adult Drosophila reveals the presence of a CAP2b-like peptide, that coelutes with Manduca sexta CAP2b and synthetic CAP2b and that has CAP2b-like effects on the M. sexta heart. CAP2b stimulation elevates tubule cGMP levels but not those of cAMP. Both CAP2b and cGMP increase the transepithelial potential difference, suggesting that stimulation of vacuolar ATP action underlies the corresponding increases in fluid secretion (Davies, 1995).
Calcium mobilization in identified cell types within an intact renal epithelium (the Drosophila Malpighian tubule) was studied by GAL4-directed expression of an aequorin transgene. Aequorin is a Ca2+ sensitive liminescent protein isolated from the coelenterate Aequorea victoria. It is a complex of apoaequorin, a 21 kDA polypeptide, and coelenterazine, a hydrophobic luminophore. Aequorin is used for monitoring Ca2+ changes. CAP2b, causes a rapid, dose-dependent rise in cytosolic calcium in only a single, genetically-defined, set of 77 principal cells in the main (secretory) segment of the tubule. In the absence of external calcium, the CAP2b-induced calcium response is abolished. In Ca2+-free medium, the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, elevates [Ca2+]i only in the smaller stellate cells, suggesting that principal cells do not contain a thapsigargin-sensitive intracellular pool. Assays for epithelial function confirm that calcium entry is essential for CAP2b to induce a physiological response in the whole organ. The data suggest a role for calcium signaling in the modulation of the nitric oxide signaling pathway in this epithelium. CAP2b must act to increase fluid secretion rates solely by an initial rise of [CA2+]i in principal cells. CAP2b stimulates tubule Nitric oxide synthase activity. It is probable that the CAP2b induced rise in [CA2+]i is sufficient to trigger the activation of Drosophila calcium sensitive Nitric oxide synthase. The maximal CAP2b concentrations employed elevate principal cell calcium levels from 87 to 255 nM, a value close to the EC50 of Drosophila NOS. This implies that Drosophila Nos is responsive over the range of the CAP2b concentrations employed. This may account for the observation that thapsigargin treatment results in increased basal cGMP levels that are not further increased on CAP2b stimulation. Thus the data provide strong evidence for a calcium-mediated link between CAP2b and NOS/cGMP activation of fluid secretion. The GAL4-targeting system allows general application to studies of cell-signaling and pharmacology that does not rely on invasive or cytotoxic techniques (Rosay, 1997).
Other hormones likely to be involved in Malpighian tubule function are the leucokinins. Leucokinins are a group of widespread insect hormones. In tubules, their major action is to raise chloride permeability through stellate cells by binding to receptors on the basolateral membrane, and so ultimately to enhance fluid secretion (Julian Dow, personal communication).
For more information about Malpighian tubule function, see The Drosophila melanogaster Malpighian tubule WWW page, maintained by Julian Dow at the University of Glasgow. In addition, the Dow laboratory maintains a sensitive map illustrating the physiology of Malpighian tubule secretory cells. Functioning of the V-ATPase is described at Julian Dow's V-ATPase site.
References
Davies, S. A., et al. (1995). CAP2b, a cardioacceleratory peptide, is present in Drosophila and stimulates tubule fluid secretion via cGMP. Am. J. Physiol. 269: R1321-1326
Hoch, M., Broadie, K., Jackle, H. and Skaer, H. (1994). Sequential fates in a single cell are established by the neurogenic cascade in the Malpighian tubules of Drosophila. Development 120: 3439-3450
Rosay, P., et al. (1997). Cell-type specific calcium signalling in a Drosophila epithelium. J. Cell Sci. 110:1683-1692. Medline abstract: 97407707
Sözen, M. A., et al. (1997). Functional domains are specified to single-cell resolution in a Drosophila epithelium. Proc. Natl. Acad. Sci. 94: 5207-5212. Medline abstract: 97289745
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