The Interactive Fly
Genes involved in tissue and organ development
Formation of adult abdominal segments - gene expression in histoblasts
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Unlike the process in vertebrate development, the adult fly is not formed as a result of the continuous development of embryonic tissues, rather imaginal cells from which various adult structures eventually arise are set apart from embryonic tissues early in embryonic development. Imaginal precursor cells are established as discrete groups of cells localized to specific regions of the embryo. The precursors of the adult head structures, appendages and genitalia form from invaginations of the embryonic epithelium and make up the imaginal discs; these are groups of cells not directly associated with the larval integument. The precursors of the abdomen and the internal organs of the adult, such as the gut, salivary glands and brain, arise from nests or rings of cells intimately associated with larval structures. For example, the salivary gland imaginal rings are embedded in the larval salivary glands; the midgut imaginal histoblast nests arise in the larval midgut and the abdominal histoblast nests form among the cells of the larval abdomen (Curtiss, 1995).
Each adult abdominal segment forms from four pairs of histoblast nests: the anterior and posterior dorsal pairs (which produce the tergites); the ventral pair (which produce the sternites and pleurites), and the spiracular pair (which form the spiracle and the surrounding pleurite tissues). Each anterior dorsal and ventral histoblast nest is composed of approximately 16 cells; each posterior dorsal histoblast nest consists of approximately five cells, and each spiracle histoblast nest has approximately three cells. The abdominal histoblasts do not divide during the larval stages, but begin to divide within the first 3 hours after pupariation. They continue to divide until approximately 15 hours of pupal development without displacing the larval cells. At about 15 hours of pupal life, the abdominal histoblast cells begin to migrate and displace the larval cells, which are then histolyzed. Following proliferation and migration, cells of adjacent segments fuse at the dorsal/ventral and segmental borders. During the terminal stages of abdominal development the cells differentiate to produce epidermal tissues, including the microchaetae and macrochaetae, and to secrete the adult cuticle (Curtiss, 1995 and references).
In the Arrowhead mutant pharate adult, a single row of bristles develop in the anterior-most segment. No other development of the abdominal epithelium occurs, as evidence by the absence of bristles and cuticle. Nevertheless, when partial development of abdominal epithelium occurs in mutant pupae, the cuticle and bristles appear normal. It has been concluded that Awh does not affect differentiation of the cells, but does affect the establishment or proliferation of the precursors. Examination of escargot (a gene required for cell cycle regulation of imaginal tissue) expression in abdominal histoblasts and other imaginal precursors shows that Awh mutants have significantly fewer cells in each histoblast nest. This suggests that Awh is necessary to generate the proper number of abdominal histoblasts in the embryo (Curtiss, 1995).
Expression of Awh in histoblast and imaginal ring tissue, and the requirement for Awh for the proliferation of these tissues, points to a clear distinction between two types of imaginal tissues: (1) imaginal discs that give rise to adult structures such as wings, legs and gonads do not require Awh; this is in contrast to (2) histoblasts and imaginal ring tissue that do require Awh function for establishment or proliferation. Curtiss and Helwig (1995) define as incorporate those imaginal precursor cells, including the abdominal histoblasts and salivary gland imaginal rings, that are embedded in larval tissue. During metamorphosis, incorporate imaginal cells replace the cognate larval organ in which the precursor cells are located. excorporate imaginal precursor cells are defined as imaginal discs, which develop separately from larval larval tissue. During metamorphosis, excorporate imaginal cells elaborate structures unique to the adult.
Very little information is available about gene expression during the larval period, a developmental interval critical to the formation of the adult. To what extent does gene expression during this period resemble that in the embryonic stages, and how does gene expression during the larval period contribute to segment polarity in the adult? In fact, all the genes expressed during embryonic segment polarity also play a similar role in the formation of the adult. Cells destined to form the cuticle of the adult abdomen are present as clusters of small, non-dividing diploid cells (the anterior dorsal, posterior dorsal and ventral histoblast nests) located at stereotyped postions in the larval epidermis. These cells, just as do their embryonic counterparts, express engrailed, hedgehog, wingless, patched, cubitus interruptus and sloppy paired in a stereotyped manner dependent on their positions within each segment. Each segment is subdivided into an anterior (A) and posterior (P) compartment, distinguished by activity of the selector gene engrailed (en) in P but not A compartment cells. The ventral epidermis of each abdominal segment forms a flexible cuticle, the pleura, with a small plate of sclerotised cuticle, the sternite, centered on the ventral midline. The pleura is covered with a uniform lawn of hairs, all pointed posteriorly, whereas the sternite contains a stereotyped pattern of bristles. Posterior compartments are to a large degree devoid of hairs and bristles, while the sternite cuticle of the A compartment consists of an anterior-to posterior progression of six types of cuticle distinguished by ornamentation and pigmentation. Just anterior to the posterior compartment, A6 is unpigmented, with hairs and none of the larger ornaments called bristles. A5 is darkly pigmented with hairs and bristles of large size. A4 and A3 are darkly and lightly pigmented respectively with moderately sized hairs and bristles. A2 is lightly pigmented with hairs, and A1, adjacent to the next more anteriorly located "posterior" compartment is unpigmented without hairs (Struhl, 1997).
Formation of adult abdominal segments - gene expression in histoblasts
The cuticle of the adult abdomen of Drosophila is produced by nests of imaginal histoblasts, which proliferate and migrate during metamorphosis to replace the polyploid larval epidermal cells. In this report, a detailed description is presented of the expression of four key patterning genes, engrailed (en), hedgehog (hh), patched (ptc), and optomotor-blind (omb), in abdominal histoblasts during the first 42 h after pupariation, a period in which the adult pattern is established. In addition, the expression is described of the homeotic genes Ultrabithorax, abdominal-A, and Abdominal-B, which specify the fates of adult abdominal segments. The results indicate that abdominal segments develop in isolation from one another during early pupal stages, and that some patterning events are independent of hh, wg, and dpp signaling. Pattern and polarity in a large anterior portion of the segment are specified without input from Hh, and evidence is presented that abdominal tergites possess an underlying symmetric pattern upon which patterning by Hh is superimposed. The signals responsible for this underlying symmetry remain to be identified (Kopp, 2002).
The dorsal cuticle of a typical abdominal segment contains a stereotyped sequence of pattern elements. At the anterior edge of each segment is the acrotergite, a narrow strip of naked sclerotized cuticle (a1). The remainder of the tergite is covered by trichomes, and can be subdivided into four regions. From anterior to posterior these regions are: a lightly pigmented region with no bristles (a2 fate); a lightly pigmented region that contains two to three rows of microchaetes (a3); a darkly pigmented region with one to two rows of microchaetes (a4); and a darkly pigmented region with a single row of macrochaetes (a5). The tergite is followed by the unpigmented posterior hairy zone (PHZ), which is composed of both anterior (a6) and posterior (p3) compartment cells. All trichomes and bristles in the segment are oriented uniformly from anterior to posterior. Finally, at the posterior edge of the segment is a zone of thin, naked intersegmental membrane (ISM), which can be subdivided into anterior smooth (p2) and posterior crinkled (p1) regions (Kopp, 2002).
The adult abdominal pattern is established in the first 2 days of pupal development, concurrent with the proliferation and migration of histoblasts and the destruction of the larval epidermal cells (LECs.) The spatial and temporal evolution of en, hh, ptc, and omb expression is followed during this critical period. The cuticle of each abdominal hemisegment is formed by three major histoblast nests. The anterior dorsal nest (aDHN) is composed of anterior compartment histoblasts and produces the tergite and part of the PHZ (a1-a6), whereas the posterior dorsal nest (pDHN) is composed of posterior compartment cells and produces the intersegmental membrane and the remainder of the PHZ (p1-p3). The ventral histoblast nest, which produces the sternite and pleura, contains both anterior and posterior compartment cells. en, hh, ptc, and omb are expressed in similar patterns in dorsal and ventral histoblasts, and the description is limited to the dorsal abdomen (Kopp, 2002).
en-lacZ and hh-lacZ are expressed throughout the pDHN, but are not expressed in the aDHN. hh-lacZ is expressed in a gradient within the pDHN, with expression highest at the anterior edge. A similar gradient can be detected in understained preparations of en-lacZ. ptc-lacZ expression is present in only a few cells at the posterior edge of the aDHN. omb-GAL4 expression is seen in the posterior of the aDHN and the anterior of the pDHN. omb-GAL4 expression is highest near the compartment boundary and decreases symmetrically in both anterior and posterior directions. By 20-24 h APF, the aDHN and pDHN fuse to form a combined dorsal histoblast nest (DHN). The gradients of en-lacZ and hh-lacZ expression within the posterior compartment become more pronounced at this stage. ptc-lacZ is expressed in a narrow stripe in the middle of the DHN, which is presumably located just anterior to the compartment boundary. The posterior border of this stripe is sharply defined, whereas a short gradient forms in the anterior direction; no ptc-lacZ expression can be detected at the anterior edge of the DHN at this time. omb-GAL4 is expressed in a wide, double-sided gradient in the middle of the DHN. Double labeling for ß-galactosidase and En protein in omb-GAL42/UAS-lacZ pupae shows that omb-GAL4 is expressed in both compartments (Kopp, 2002).
At ~30 h APF, the DHN of consecutive segments begin to merge. Contact occurs as the border cells, a specialized row of LECs located at the posterior edge of each segment, are lost. At this stage, expression of en-lacZ and hh-lacZ is still highest at the compartment boundary, and lowest at the posterior edge of the segment. At high magnification, a clear gradient of En protein can be seen at this stage on a cell-by-cell basis. The ptc-lacZ stripe in the middle of the segment widens somewhat, but retains a sharp posterior limit. As the border cells are eliminated and histoblasts of consecutive segments come into contact, cells at the anterior edge of each segment activate ptc-lacZ. Activation occurs only where border cells have been lost; no expression of ptc-lacZ is detected posterior to persisting border cells. This pattern strongly suggests that the border cells insulate anterior histoblasts from the Hh protein secreted by the posterior compartment cells of the preceding segment. Consistent with such a role, the border cells do not express hh transcript, although they do express En. omb-GAL4 continues to be expressed in a symmetric, double-sided gradient at this stage (Kopp, 2002).
By 40-42 h APF, the border cells, which are the last LECs to be replaced by histoblasts, have been eliminated and segmental fusion has been completed. en-lacZ and hh-lacZ are upregulated at the posterior edge of the segment at this time, and soon the expression of both genes becomes uniform within the posterior compartment. For a short time, En levels are highest in cells at both edges of the posterior compartment, and lower in the middle cells, suggesting that en expression is upregulated by contact of anterior and posterior compartment histoblasts. In addition to the main ptc-lacZ stripe, a weak second stripe develops at the anterior edge of the segment. omb-GAL4 expression becomes asymmetric, with a well-defined posterior and graded anterior boundaries; based on the positions of muscle insertion points, most or all of omb-GAL4 expression at this stage is in the anterior compartment (Kopp, 2002).
Segment identities in the abdomen are specified by the Ubx, abd-A, and Abd-B genes of the bithorax complex (BX-C). More precisely, BX-C genes control the development of parasegments (ps), which are composed of the posterior compartment of one segment and the anterior compartment of the following segment. Ubx controls the identity of ps6, which includes the anterior compartment of the first abdominal segment (A1); abd-A functions primarily in ps7-ps9 (A2-A4), although it also contributes to the identities of ps10-ps12; and Abd-B is the main determinant of the identities of ps10-ps12. In the pupal abdomen, Abd-B is expressed strongly in ps12 (A7) (in females; the last abdominal segment is rudimentary in males), weaker in ps11 (A6), and at very low levels in ps10 (A5). This pattern is consistent with the view that different levels of Abd-B expression promote distinct segment identities in the posterior abdomen. abd-A is expressed in ps7 (A2) through ps12 (A7), at levels gradually increasing from the anterior to the posterior parasegments. Ubx is expressed only in the anterior compartment of A1 (ps6) in the abdominal epidermis. Double staining for Ubx and hh-lacZ shows that the posterior boundary of Ubx expression coincides precisely with the ps6/ps7 boundary. Thus, Ubx and abd-A are expressed in adjacent nonoverlapping domains, contrasting sharply with their overlapping expression in the embryo. Ubx expression is eliminated from A1 in the abd-A gain-of-function mutant Uab5, suggesting that abd-A represses Ubx during the pupal stage (Kopp, 2002).
To test whether Hh signaling is required for ptc and omb expression, homozygous hhts2 individuals were grown at 29°C for 48 h prior to dissection. Under these conditions, ptc-lacZ expression was completely eliminated at all stages. However, the effect on omb-GAL4 expression was different, depending on the stage of development. In early pupae, the symmetric expression of omb-GAL4 about the compartment boundary was only slightly reduced, while expression in the LECs appeared normal. In contrast, the later asymmetric expression of omb-GAL4 in the anterior compartment was virtually eliminated. No change was seen in the expression of en-lacZ or En protein in hhts2 pupae raised at 29°C, suggesting that the gradients of en expression in the posterior compartment are established independently of Hh function (Kopp, 2002).
After replacement of the LECs by the histoblasts, the pupal abdomen consists of a chain of alternating anterior and posterior compartments. Therefore, at this stage each anterior compartment can be exposed to Hh protein diffusing across both its anterior and posterior edges. It is well documented that Hh diffusing from the posterior (across the compartment boundary) plays a key role in patterning the posterior tergite (a4-a6 fates). Hh diffusing from the anterior (across the segment border) appears to be less important, playing a direct role in specifying the acrotergite (a1), but not other anterior tergite (a2 and a3) fates (Kopp, 2002).
However, it has been suggested that Hh diffusing across the segment border may act indirectly through a secondary signal to specify polarity throughout the anterior tergite. To test this model, smo mutant clones located at the segment boundary were analyzed. Such clones should be unable to receive the Hh signal, and according to the model would be predicted to alter cell polarity in the anterior tergite. smo2 clones in the a1 region are transformed to a2 identity and secrete trichomes, making it possible to determine the polarity of each cell. Two types of clones were examined. The first type consists of large clones that abut the segment boundary and span the a1 and a2, and sometimes also the a3, regions. 33 clones of this type were examined, of which 16 could clearly be seen to contact the segment border along their entire width. All such clones had completely normal polarity both within the clone and in the surrounding wild-type cells, suggesting that no anterior Hh-responsive cells are required to polarize the a2 and a3 regions. Rather, these observations argue strongly that these regions are polarized independently of Hh (Koop, 2002).
The second type of clone consisted of small clones contained entirely within the a1 region, and separated from the a2 region by a strip of untransformed a1 cuticle. Of 13 such clones examined, 11 had completely normal polarity throughout, and 2 showed altered polarity in 1 or 2 cells along the posterior edge of the clone. It is suggested that these polarity reversals, which are the exception rather than the rule and extend for only one cell diameter, are a strictly local effect of a2 cells coming into contact with a1 cells improperly located to their posterior (Koop, 2002).
Several genotypes have been described in which abdominal tergites show mirror-symmetric patterning. A series of experiments was conduced to test whether this mirror symmetry is the result of Hh signaling. The results are uniformly negative, suggesting that abdominal tergites possess an underlying mirror-symmetric pattern that is specified independently of hh (Koop, 2002).
Ubiquitous expression of omb causes double-posterior patterning of the tergite (a6-a5-a4-a4-a5-a6), whereas loss of omb function can cause reciprocal, double-anterior patterning (a2-a3-a3-a2). Ubiquitous expression of omb driven by the gain-of-function allele QdFab has no effect on expression of en-lacZ, hh-lacZ, hh transcript, or the omb-GAL42 enhancer trap. Moreover, pupae hemizygous for the null allele omb282 show normal expression of hh-lacZ, en-lacZ, and En protein (Koop, 2002).
These observations indicate that omb does not regulate the expression of hh , en, or omb. ptc-lacZ expression is also unaffected in omb282 pupae, indicating that omb is not required for Hh signaling. However, Omb may potentiate Hh signaling: in QdFab, the level of ptc-lacZ expression is increased relative to that of wild-type at both edges of the anterior compartment, although the timing of ptc-lacZ activation is not affected (Koop, 2002).
In an earlier report, it was found that the phenotype of QdFab is not suppressed in QdFab;hhts2 double mutants raised at the restrictive temperature, suggesting that the mirror-symmetric phenotype of QdFab is independent of Hh function. However, the new observation that ptc-lacZ expression is upregulated in QdFab prompted a reexamination of these double mutants. A large number of QdFab/FM6; hhts2/hhts2 animals shifted to 31o, C at pupariation were compared to their identically treated QdFab/FM6; hhts2/In(3LR)Cx, Sb siblings. In agreement with earlier results, no suppression is seen of the QdFab phenotype by hhts2. In a reciprocal experiment, it was asked whether cell fates or polarity in QdFab could be altered by ectopic hh expression. Flip-out hh-expressing clones were generated. These clones were not associated with any changes in cell fate or polarity. Taken together, these results argue strongly that mirror-symmetric patterning in omb mutants is established independently of hh (Koop, 2002).
Ectopic expression of en causes transformation of anterior compartment structures to posterior compartment identity, and produces a mirror-symmetric double-posterior pattern (p1-p2-p3-p3-p2-p1). This phenotype is seen in the en gain-of-function en mutant, which causes near-ubiquitous expression of en in the pupal abdomen and in T155-GAL4/UAS-en heterozygotes. Examination of En-expressing clones in otherwise wild-type flies reveals that the line of symmetry lies within the anterior compartment. En-expressing cells located posterior to this line orient to the posterior, whereas En-expressing cells located anterior to it orient to the anterior. This effect of En on cell fate and polarity is strictly cell autonomous. Whether Hh signaling plays a role in the symmetric polarization of en-expressing cells has been tested. No activation of en-lacZ is seen in the anterior compartment of gain of function en heterozygotes, although sporadic activation of hh-lacZ and hh transcript is observed. However, it is difficult to see how such variable activation of hh could be responsible for the highly regular mirror-symmetric cuticular pattern produced. ptc-lacZ expression is reduced at both edges of the anterior compartment in gain of function en, consistent with repression of ptc by En. omb-GAL4 expression appears unchanged relative to wild type (Koop, 2002).
To ask directly whether en-expressing cells in gain of function en flies are patterned by Hh, smo3 clones were generated in gain of function en heterozygotes. These clones had no effect on cell fate or polarity: smo mutant cells located posterior to the line of symmetry retained posterior orientation, whereas cells located anterior to this line retained anterior orientation. The affinity of smo mutant cells in a gain of function en background also appeared unchanged, since all clones interdigitated freely with surrounding cells (Koop, 2002).
In a reciprocal experiment, it was asked whether the patterning of en-expressing cells is affected by ectopic Hh expression. Flip-out Hh-expressing clones were generated in en gain of function heterozygotes. Hh-expressing clones had no effect on cell fate or polarity. Thus, Hh signaling does not appear to play a role in the mirror-symmetric polarization of en-expressing tergites (Koop, 2002).
Mirror-symmetric patterning is also caused by ectopic expression of Hh itself. Ubiquitous Hh expression driven by hs-hh or UAS-hh;T155-GAL4 results in a mirror-symmetric double-posterior pattern (p2-p3-a6-a5-a5-a6-p3-p2). Interpretation of this phenotype has been complicated by the observation that ectopic Hh induces localized expression of en-lacZ in the anterior compartment. This induction leaves open the possibility that the mirror-symmetric patterning may be mediated by changes in endogenous hh expression (Koop, 2002).
To test this possibility, the expression of en-lacZ, hh-lacZ, and ptc-lacZ was examined in the abdomens of UAS-hh;T155-GAL4 pupae that were shifted from 17°C to 29°C at pupariation to enhance GAL4-induced ectopic expression. During the early pupal stages, ptc-lacZ expression was strongly and evenly expanded to the anterior, while the expression of hh-lacZ, en-lacZ, and En protein was unchanged. However, by 40-42 h APF some pupae showed weak ectopic expression of en-lacZ and hh-lacZ in a narrow stripe in the middle of the anterior compartment. ptc-lacZ expression was upregulated to each side of this stripe as well as at both edges of the anterior compartment (Koop, 2002).
The mirror-symmetric posterior tergite in UAS-hh;T155-GAL4 animals (a6-a5-a5-a6) develops between the ectopic en stripe and the normal posterior compartment. This region is flanked by hh-expressing cells and has peaks of ptc-lacZ expression at both its anterior and its posterior edges. Therefore, the symmetric patterning of the tergite could be caused by symmetric expression of the endogenous hh gene, rather than by ubiquitous expression of UAS-hh. To test this possibility, the hhts2 mutation was used to block endogenous Hh activity. UAS-hh;T155-GAL4 hhts2/hhts2 animals were shifted to 31°C at pupariation. Endogenous Hh signaling, as detected by ptc-lacZ expression, is eliminated under these conditions. In the pharate adults that developed, the mirror-image patterning of posterior tergite and PHZ structures was unaffected relative to that of identically treated UAS-hh;T155-GAL4 hhts2/TM6 siblings, although the transformation of anterior tergite to intersegmental membrane was partly suppressed (Koop, 2002).
To confirm the inactivation of endogenous Hh, en-lacZ and ptc-lacZ expression was examined in UAS-hh;T155-GAL4 hhts2/hhts2 pupae raised at 29o C. In this genotype, en-lacZ was activated in a stripe in the middle of the anterior compartment, as it was in UAS-hh;T155-GAL4 pupae. However, no separate peaks of ptc-lacZ expression were detected. Instead, ptc-lacZ was activated uniformly in the posterior half of the anterior compartment. Curiously, little or no expression of ptc-lacZ was seen in the anterior half (Koop, 2002).
Taken together, these observations suggest that localized activation of the endogenous hh gene is not responsible for the mirror-symmetric pattern caused by ubiquitous expression of exogenous Hh. However, in this case the results are not conclusive, as the hhts2 allele may allow residual Hh function at the restrictive temperature (Koop, 2002).
In conclusion, abdominal tergites display mirror-symmetric patterning in several different genotypes. These genotypes include loss-of-function mutants of omb or hh, and genotypes in which omb, en, or hh are expressed ubiquitously. It is thought that these cases reveal an underlying symmetric patterning of the tergite. However, after the loss of the border cells, anterior compartments are exposed to Hh from both anterior and posterior edges, raising the possibility that these mirror-symmetric phenotypes result from symmetric Hh signaling. Indeed, it has been suggested that a U-shaped gradient of Hh produced by diffusion across the compartment and segment boundaries specifies polarity throughout the tergite. This report, tested the role of Hh in three separate cases of mirror-symmetric patterning. The results are uniformly negative, and provide compelling evidence that abdominal tergites possess an underlying mirror-symmetric patterning that is specified independently of Hh (Koop, 2002).
There are two main conclusions which may be drawn from the work to define Hh requirements in abdominal patterning: (1) Hh signaling is not required to specify pattern or polarity in the a2 and a3 regions, which comprise most of the anterior tergite; (2) abdominal tergites possess an underlying mirror-symmetric patterning that is specified independently of Hh. The phenotypes of hhts2 and omb2 mutants, in which the a2 and a3 regions are often duplicated in mirror image, imply that a single patterning system is responsible for specifying both the a2 and a3 regions and the underlying mirror symmetry of the tergite. The identity of this system remains to be determined (Koop, 2002).
Several observations suggest that posterior compartments in the abdomen are organized in much the same way as anterior compartments. Ectopic expression of omb transforms the entire posterior compartment to PHZ (p3 fate) that has clear mirror-symmetry: trichomes in the anterior region orient toward the posterior, while those in the posterior region orient toward the anterior. Thus, anterior and posterior compartments in the abdomen may be organized in a similar fashion and patterned by similar mechanisms (Koop, 2002).
References
Curtiss, J. and Heilig, J. S. (1995). Establishment of Drosophila imaginal precursor cells is controlled by the Arrowhead gene. Development 121(11): 3819-3828
Kopp, A., and Duncan, I. (2002). Anteroposterior patterning in adult abdominal segments of Drosophila. Dev. Bio. 242: 15-30
Struhl, G., Barbash, D. A. and Lawrence, P. A. (1997). Hedgehog organizes the pattern and polarity of epidermal cells in the Drosophila abdomen. Development 124 (11): 2143-2154
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