patched

DEVELOPMENTAL BIOLOGY

Embryonic

Patched RNA is first detected during cellularization throughout the cortex except for a dorsal anterior region and around the posterior pole, including pole cells. By stage 8, PatchedmRNA is expressed in broad stripes of segmental periodicity in the posterior part of the parasegment compartment. These later split into two stripes per segment primordium (Hooper, 1989). Additional regions of expression include ectodermally derived hindgut and the labrum of the head. Late embryos express ptc in internal structures like esophagus, foregut, visceral mesoderm and hindgut. In the head region, parts of the mandibular, maxillary and labial buds [Image] are stained, as well as the hyperpharyngeal lobe and pharynx (Capdevila, 1994a).

During neurogenesis, the transmembrane protein Patched promotes a wingless-mediated specification of a neuronal precursor cell, NB4-2. Wg, secreted by row 5 cells promotes wingless expression in adjacent row 4 cells; Wg in turn represses gooseberry. Novel interactions of these genes with engrailed and invected during neurogenesis have been uncovered. While in row 4 cells Ptc represses gsb and wg, in row 5 cells en/inv relieve Ptc repression of gsb by a non-autonomous mechanism that does not involve hedgehog. The non-autonomous mechanism originates in Row 6/7 cells where en/inv engender hedgehog and another unknown secreted signal which acts in turn on adjacent row 5 cells to heighten wingless, and consequently, the expression of gooseberry. This differential regulation of gsb leads to the specification of NB5-3 and NB4-2 identities to two distinct neuroblasts. The row 5, NB5-3, neuroblasts are specified by high levels of gsb, expressed autonomously in row 5. The fate of row 4, NB4.2, requires an absence of gooseberry, assured by Patched repression and Wingless signaling from adjacent row 5 cells. The uncoupling of the ptc-gsb regulatory circuit by hedgehog and the unknown secreted signal from row 6/7 cells enables gsb to promote Wg expression in row 5 cells. These results suggest that the en/inv->ptc->gsb->wg pathway uncovered here and the hh->wg are distinct pathways that function to maintain the wild-type level of Wg. These results also indicate that Hh is not the only ligand for Ptc and similarly, that Ptc is not the only receptor for Hh (Bhat, 1997).

The genital disc consists of three primordia: moving from anterior to posterior they are the female genital primordium, the male genital primordium and the anal primordia. Only one of the two genital primordia develops, depending on the individual's sex, whereas the anal primordium develops in both sexes. It is proposed here that the genital disc, which is of ventral origin, is organized in a manner similar to the antennal and leg discs: the expression domains of decapentaplegic and wingless are mostly complementary and abut engrailed expression. An analysis was made of the roles of the genes hedgehog, patched, dpp and wg in the development of the three primordia that form the genital disc. The morphogenetic alterations produced by ectopic expression of hh mimic a lack of ptc function. Both genetic conditions cause derepression of dpp and wg. Ectopic expression of either of these genes causes non-autonomous duplications and/or reductions of genital and anal structures. Some of these alterations are explained by the mutual repression of wg and dpp. In the development of the genital disc, the functional relationships between these genes seem to be analogous to those described for leg and antennal discs: dpp and wg are induced in the anterior compartment by Hh protein blocking the repressive effect of Ptc, and the mutual repression of dpp and wg restrict one another to their respective domains. It may be concluded that dpp and wg act as general organizers for development of the genital disc (S·nchez, 1997).

The development of the genital primordia is based on two processes: cell proliferation and sexual differentiation. Cell proliferation refers to the capacity of each genital primordium to grow or to be kept in the repressed state. Sexual differentiation refers to the type of adult structure formed by each genital primordium. It is proposed that the control of cell proliferation in the male and female genitalia requires the concerted action of Abd-B and doublesex, either directly or indirectly, through the expression of the genes dpp and wingless. Thus, in female genital discs, the repressed male primordium does not express dpp whereas the repressed female primordium of the male genital discs expresses a reduced level of dpp. This reduced level seems to be insufficient to stimulate cell growth. In contrast, when strong dpp levels are obtained in the repressed female primordium of male discs, repressed female primordia overproliferate in mutants for patched or costal-2, as well as in the discs where uniform ectopic expreession of hedgehog is produced. The genes dpp and wg, however, do not participate in the sexual differentiation process, which depends on sexual cytodifferentiation genes. Thus the growth of repressed female primordia of the patched mutant male discs would give rise to no adult female genital structures since the genetic sex is male (S·nchez, 1997 and references).

Larval

Patched is involved in the formation of the boundary between anterior and posterior compartments of the wing disc (Johnson, 1995).

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, 1997a).

Hedgehog (Hh), a protein secreted by engrailed expressing P compartment cells, spreads into each A compartment across the anterior and the posterior boundaries to form opposing concentration gradients that organize cell pattern and polarity. Anteriorly and posteriorly situated cells within the A compartment respond in distinct ways to Hh: they express different combinations of genes and form different cell types. patched is expressed at both boundaries. patched is expressed in a graded fashion within each stripe, just anterior to each P compartment. ci peaks at high level in those cells abutting Hh- secreting cells of the P compartment and declines progressively in cells further away. wingless is also expressed in this domain and sloppy paired is expressed in the same region as wingless. decapentaplegic is expressed only in the ventral pleura in those A compartment cells neighboring P compartment cells within the same segment. dpp is not expressed immediately behind posterior compartments (Struhl, 1997).

Pupal

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).

To test whether Hh signaling is required for ptc and omb expression, homozygous hh^ts2 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 hh^ts2 pupae raised at 29°C, suggesting that the gradients of en expression in the posterior compartment are established independently of Hh function (Kopp, 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).

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