Functional domains are specified to single-cell resolution in a Drosophila epithelium

Tubules Are Divided into Multiple Domains.

Classically, the two anterior Malpighian tubules are described as comprising an initial segment (containing white luminal concretion bodies) and a main segment, joined by a narrow transitional segment; the two posterior tubules, in contrast, were thought to comprise solely a main segment (Fig. 1; refs. 15 and 29). Domains with similar relative sizes and positions are visible from enhancer analysis (Fig. 2), and we have thus adopted the nomenclature “initial,” “transitional,” and “main” segments to describe these genetically deduced domains. Surprisingly, enhancer trap lines that delineate the initial and transitional segments of anterior tubules (Fig. 2 D and E) reveal previously undescribed analogous domains in posterior tubules (Fig. 2 A–C). The latter are very small, however, and have probably been overlooked previously because they were too short to allow accumulation of concretions.

It is also possible to subdivide the main segments. Again, while the transitional–main segment boundary can be established in accordance with previous studies (Fig. 2 A–F), an additional domain is found marking the lower third of the tubule and the ureter (Fig. 2G). This region, in turn, can be resolved into three subregions: a lower tubule and an upper and lower ureter (Fig. 2 H and I).

Tubules Contain Multiple Cell Subtypes.

Such an analysis can be extended to cell subtypes. Previous studies have described just two tubule cell types: principal (type I) and secondary or “stellate” (type II; ref. 15). Both could be further subdivided. Principal cells, for example, comprise at least two distinct subpopulations (Fig. 3A); indeed, no line marked all the principal cells of the main segment. Interestingly, mutations at the white eye color locus also affect tubule color, and a similar mosaicism in principal cell color has been described in those white mutants that show position effect variegation of eye pigmentation (30, 31). Although position effect variegation is not the cause of our own observations, both lines of evidence demonstrate differences in otherwise indistinguishable cells with respect to transcriptional properties and thus presumably with respect to function. This heterogeneity may emerge as a general feature of epithelial structure.

Type II cells (Fig. 3B) are distributed evenly throughout the initial, transitional, and main segments of posterior tubules and within the main segment of anterior tubules. The two lines that mark stellate cells of the main segment also mark bar-shaped cells in the initial and transitional segments of the anterior tubule, which may thus be stellate cell counterparts (Fig. 3 C and D), although one line marks just the bar-shaped cells (Fig. 3E), implying that these are transcriptionally, and thus functionally, distinct. The transition between bar-shaped and stellate cells occurs at the transitional–main segment boundary (Fig. 3D). None of the above lines mark cells in the lower tubule or ureter (Fig. 4D), suggesting that the mechanism by which type II cells are specified respects the newly defined lower tubule boundary. Although type II cells are occasionally found to touch (data not shown), it is tempting to speculate that the overall evenness of their distribution in adults reflects spacing by lateral inhibition at some stage in their specification, reminiscent of the cuticular bristles of Drosophila (32).

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Figure 4

Counterstaining with ethidium bromide allows cell numbers to be counted. (A) Reporter gene expression was detected with fluorescein-coupled anti-β-galactosidase secondary antibody (green) in line c507 and counterstained with ethidium bromide. The boundary of β-galactosidase expression can be identified to single-cell resolution, and the number of principal cell nuclei to the tubule bifurcation can be counted. (B) Line c724 shows the relative abundance of principal and stellate cells in the main segment. In addition, the nuclei of the stellate cells are clearly smaller than those of principal cells. (C) Line c710 shows staining of the tiny cells of the ureter. Fine processes are visible in some cells (arrows). (D) Line c724, showing that stellate cells do not overlap with tiny cells in the lower tubule. A single stained stellate cell is visible at the junction of the lower tubule, and the tiny cell nuclei are visible in the lower tubule (cf. C). (E) Vital staining of tubule main segment principal cell nuclei with 10 μg/ml ethidium bromide for 5 min and viewed by epifluorescence. (F) Vital labeling of stellate cells with genetically modified (S65T) GFP. Flies carrying the P{UAS-GFP.S65T}T10 construct (Bloomington stock) were crossed to P{GAL4} line c724, and red-eyed adults were dissected in Drosophila saline and viewed without further treatment with mixed bright-field and fluorescein optics.

Interestingly, several lines mark a “tiny” cell type found in lower tubules and posterior midgut (Fig. 3F) but never seen in the same genetic domain as stellate cells (Fig. 4D). These cells contain a reduced cytoplasm and a compact nucleus, are just 3–4 μm in diameter, and have thin processes (Fig. 4C). To our knowledge, such cells have not previously been described in D. melanogaster; possibly they are counterparts of the myoendocrine cells recently described in Formica (33). Our speculation is that tiny cells are neuroendocrine, monitoring the fluid collecting in the ureter and secreting neurohormones basally into the hemolymph to regulate muscle contractility or ion transport. Supporting this model, antiserum against horseradish peroxidase, which selectively marks the central and peripheral neurons of insects, including D. melanogaster (34, 35), labels just the tiny cells within the tubule (Fig. 5H; cf. Figs. 3F and 4C).

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Figure 5

Functional mapping of tubule regions. (A) Alkaline phosphatase staining of lower tubule. Tubules were fixed and stained for alkaline phosphatase. This shows a perfect overlap with line c507 (cf. Fig. 2G). (B and C) Short-term (10 min) labeling with 10 μg/ml rhodamine 123 identifies the main segment of both posterior (B) and anterior (C) tubules as the region responsible for transport of this organic molecule. (D) Initial and transitional segments express V-ATPase at a far lower level than the main segment, as detected by a reporter gene inserted into the vha55 gene in the P-element lethal line vha55^j2e5 (28). This corresponds to the boundary reported by lines 155Y, c709, and c776 in Fig. 2 A and B. (E) A boundary midway along the ureter is marked by a change in the size of nuclei labeled in line vha55^j2e5. This corresponds to the boundary reported by lines c649 and c601 in Fig. 2 H and I. (F) Within the main segment, only principal cell nuclei (cf. Fig. 4B) are labeled in line vha55^j2e5. (G) Within the main segment, only principal cell nuclei (cf. Fig. 4B) are labeled in P-element line l(2)k02508, a recessive lethal insertion within the gene encoding the body-enriched vha68–2 V-ATPase A subunit (36). (H) The tiny cells (cf. Figs. 3F and 4C) are labeled by antibodies to horseradish peroxidase, which recognize an epitope on a Drosophila nervous system-specific Na⁺,K⁺ ATPase β-subunit (37).

Genetic Domains Are Fixed to Single-Cell Precision.

Are genetically defined domains precisely specified with respect to cell number as well as cell type? GAL4-expressing tubules were counterstained with ethidium bromide to reveal cell nuclei. Distances between expression boundaries were measured by counting cell nuclei from either end of the tubule (Fig. 4A). It was also possible to distinguish nuclei of principal, stellate, and tiny cells on the basis of nuclear size (Figs. 4 B–D). This served as an objective method of demonstrating congruence between boundaries in different lines. For example, the number of ethidium bromide-stained principal cell nuclei in the upper domain of anterior tubules marked by bar-shaped stellate cells (line c649, Fig. 3E) is 40; this corresponds closely with the sum of such nuclei in the initial (22 ± 0.3) and transitional (22 ± 0.3) domains marked by line c825, or with the region unstained in line c568A, containing 44 ± 1.7 principal cells (Table 2).

Totals of principal cells (145 ± 1 and 111 ± 1 in the anterior and posterior tubules, respectively) are comparable with previous studies on larvae (38); but our data further show that, between the anterior and posterior tubules, there is no difference in cell numbers in the main or lower tubule segments, and that overall differences are entirely attributable to the relative sizes of the initial and transitional segments (Fig. 6).

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Figure 6

Summary of tubule regional architecture. Regions defined in this study are labeled (cf. Fig. 1); the numbers of principal and stellate cells in each region are shown. Numerical data are derived from Table 2; SEs are not shown, as they are <1 in each case.

Genetic Domains Can Be Established in Living Tubules.

Although our measurements derive from independent P{GAL4} lines that may well differ with respect to genetic background, the numbers of cells in the tubule overall, the positions of genetic boundaries, and the numbers of each cell type in domain were nearly invariant (Table 2). This implies that tubule development is robust and precise, and allows the possibility that it will be feasible to study the development, placement, morphology, and physiology of individual, identified cells. As we have found, unexpectedly, that ethidium labels many living Drosophila tissues, including the tubules (Fig. 4E), it is straightforward to establish cell coordinates in living tubules under fluorescent illumination by counting nuclei.

An added advantage of the GAL4-UAS system is that new reporters can be adopted for existing lines without having to perform a new screen. Since this work was undertaken, new vital reporters using genetically modified green fluorescent protein (GFP) have become available (39). GFP-marked domains are clearly visible in living tubules, in a pattern indistinguishable from that of the lacZ reporter in fixed preparations (Fig. 4F). Accordingly, it is now possible to perform experiments on identified cells in a living epithelium.

Physiological Properties of Tubules Respect Genetic Domains.

Do discrete physiological properties map to the genetic domains we have 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 previously reported that the initial segment of Drosophila anterior tubule does not secrete detectable fluid (10), that the lower third of the tubule is reabsorptive, and that only the main segment is responsible for fluid production (40). This presages the main/lower tubule distinction described here.

High levels of proton-pumping V-ATPases energize apical plasma membranes of several epithelia (41, 42), including Malpighian tubules (43), which are known to be exquisitely sensitive to the V-ATPase inhibitor, bafilomycin (10, 25). V-ATPase expression might thus offer an informative physiological indicator. The gene encoding the B-subunit of the Drosophila V-ATPase has been inactivated by insertion of a P[lacZ] enhancer trap element (28). lacZ expression in the initial and transitional segments is much weaker than in the main segment (Fig. 5D). Moreover, a clear subdivision between proximal and distal ureter (Fig. 5E) respects the boundary defined by Fig. 2 H and I. Nuclei labeled within the main segment (Fig. 5F) are entirely the larger, principal cell nuclei; stellate cell nuclei do not stain (cf. Fig. 4B). Although a single reporter element may not provide a definitive view of V-ATPase distribution, we have similarly cloned and inactivated a further three subunits of the Drosophila V-ATPase, and each P-element line reports the same expression pattern in Malpighian tubules (Fig. 5G and ref. 44). This provides the first evidence that cation transport in this tissue may be a unique property of principal, rather than type II, cells.

These data provide not only clear supporting evidence for each of the regional divisions, but also for a proposed functional difference between principal and secondary cells (14). We have previously cloned a putative aquaporin by reverse transcriptase-PCR from tubule mRNA and have shown specific labeling of stellate cell basolateral membranes (12) with antibodies against a vertebrate kidney aquaporin (45). Given that stellate cells are found in secretory (Fig. 3B), but not in reabsorptive (Fig. 4D), tubule regions, they may well play an essential role in fluid secretion (46).

Another function ascribed to insect tubules is the excretion of organic metabolites, and several dyes are actively transported by tubules (21). When the cationic dyes rhodamine 123 (22) or ethidium bromide are added to tubules, they appear initially only in the lumen of the main segment (Fig. 5 B and C; cf. Fig. 2 B and F). Both the initial and transitional segments of anterior tubules and the miniature analogues in posterior tubules fail to transport dye, confirming that they are functionally, as well as genetically, similar. This functional domain aligns precisely with a genetic boundary; by performing an ethidium transport experiment on c724 tubules with stellate cell-directed GFP, it was found that the number of type II cells in the distal domain of anterior tubules that did not transport ethidium did not differ significantly from the initial and transitional segments identified by line c649 (Fig. 7).

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Figure 7

Numerical reconciliation of genetic and functional domains. (Top) Line c724 drives GFP expression in 33 type II cells of initial, transitional, and main segments. (Middle) Line c649 drives expression in 13 bar-shaped cells, diagnostic of initial and transitional segments (Table 2). (Bottom) When ethidium bromide is added to c724, it is transported into the cells, obscuring the view of all but the most distal 13 cells. This shows that the organic cation transport system operates in a compartment statistically indistinguishable from main segment.

The genomic DNA flanking several of these P-element insertions has been characterized. In the case of two independent lines that mark the lower tubule (Figs. 2G and 4A), a neighboring transcription unit encodes an alkaline phosphatase (M.Y., unpublished work). The staining pattern for alkaline phosphatase (Fig. 5A) clearly matches precisely the lower tubule boundary (Fig. 2G). Although widely used as a histochemical marker, the functions of alkaline phosphatase are not clear. However, alignment with the genetically and functionally derived maps show that it is associated with the reabsorptive, rather than the secretory, domain of the tubule.

Taken together, our data provide a unique insight into the functional organization of an epithelium. Even in this extremely simple epithelium enhancer trapping reveals unexpected complexity in both regions and cell types. Additionally, the use of cell counting to reconcile boundaries allows enhancer trap analyses to extend beyond the anecdotal and reveals robust genetic boundaries to be both precisely placed and functionally significant. To our knowledge, this is the first time that enhancer trap analysis has been used in this way to establish cell numbers for domains and to reconcile apparently similar domains numerically. However, this methodology will be, in principle, applicable both in Drosophila and in other organisms, as the genetic tools available for the study of vertebrates develop. Its utility in allowing a reconciliation with functional domains should be obvious; and as the Drosophila tubule is unlikely to be unique, these data imply that epithelia in general may be organized to a level of cellular sophistication that work to date has failed to reveal.