The Interactive Fly

Genes involved in tissue and organ development

Histoblast nests

What are histoblasts?

Formation of adult abdominal segments - gene expression in histoblasts

Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis

Cell rearrangements, cell divisions and cell death in a migrating epithelial sheet in the abdomen of Drosophila

miR-965 controls cell proliferation and migration during tissue morphogenesis in the abdomen

Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium

The Dachsous/Fat/Four-Jointed pathway directs the uniform axial orientation of epithelial cells in the Drosophila abdomen

Mechanical coordination is sufficient to promote tissue replacement during metamorphosis in Drosophila

Tissue mechanical properties modulate cell extrusion in the Drosophila abdominal epidermis

The Microtubule Minus-End Binding Protein Patronin Is Required for the Epithelial Remodeling in the Drosophila Abdomen

ECM degradation in the Drosophila abdominal epidermis initiates tissue growth that ceases with rapid cell-cycle exit

Genes expressed in histoblasts

What are histoblasts?

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).

Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis

The fusion of epithelial sheets is an essential morphogenetic event. The development of the abdomen of Drosophila was studied as a model of bounded epithelia expansion; a complex multistep process was uncovered for the generation of the adult epidermis from histoblasts, founder cells that replace the larval cells during metamorphosis. Histoblasts experience a biphasic cell cycle and emit apical projections that direct their invasive planar intercalation in between larval cells. Coordinately, the larval cells extrude from the epithelia by apical constriction of an actomyosin ring and as a consequence die by apoptosis and are removed by circulating haemocytes. The proliferation of histoblasts and the death of larval cells are triggered by two independent extrinsic Ecdysone hormonal pulses. Histoblast spreading and the death of larval cells depend on a mutual exchange of signals and are non-autonomous processes (Ninov, 2007).

Ecdysone acts as a significant temporal signal in Drosophila, triggering each of the major developmental transitions. Although most of the genetic elements involved in Ecdysone signal transmission are known, the difficulty in visualizing morphogenetic changes in vivo and interfering with signal reception in individual cells has become a major impediment in understanding of Ecdysone actions during metamorphosis (Ninov, 2007).

In vitro culture studies have shown that Ecdysone pulses are crucial for the morphogenesis of adult appendages. Other studies have uncovered the ecdysteroid dependence of multiple differentiative and maturational responses. Nonetheless, little is known about ecdysteroid control of cell proliferation. A revealing analysis in Manduca showed that proliferating cells of the optic lobe reversibly arrest in G2 whenever the concentration of ecdysteroid drops below a critical threshold. Furthermore, earlier studies had shown that the number of histoblasts is reduced in hypomorph mutants for EcR isoforms. Whether this was the result of lack of proliferation or cell death had not been defined. This study shows in vivo a direct role for Ecdysone in cell proliferation. The rapid cell cycles experienced by abdominal histoblasts at the end of the third larval instar halt if Ecdysone-signalling reception is cell-autonomously compromised. Histoblasts remain quiescent in G2 and competent to resume proliferation in response to late Ecdysone pulses. These observations suggest that Ecdysone signalling controls the cell cycle by regulating the expression of genes involved in the G2-to-M transition (Ninov, 2007).

The destruction of larval tissues in Drosophila also results from a major transcriptional switch triggered by Ecdysone. The anterior larval muscles and larval midgut and the head and thoracic LECs degenerate during the first half of prepupal development (prepupal Ecdysone peak), while the larval salivary glands, abdominal muscles and abdominal polyploid larval epidermal cells (LECs) histolyze after the second Ecdysone pulse (pupation). Given that the exposure to Ecdysone is systemic, the stage-specific cell death responses of different cell types to Ecdysone must be differentially regulated (Ninov, 2007).

The death of abdominal LECs shows apoptotic characters and proceeds in two steps: the basal extrusion of cells initiated by the contraction of an apical actomyosin ring, and their removal by haemocytes. The cell-autonomous inhibition of EcR activity in LECs led to abortive extrusion and cell survival. Thus, the death of LECs share with other obsolete larval cells a common priming hormonal (Ecdysone) input (Ninov, 2007).

It is still not clear how cell proliferation and cell death are differentially controlled by Ecdysone. The trigger of histoblast proliferation seems to be directly dependent on Ecdysone signalling. However, it is still not known how the onset of LEC death is set. In other words, how do LECs distinguish between the late larval and the pupal Ecdysone pulses? In a plausible scenario, to avoid detrimental epithelial gaps at the surface, signalling clues from 'matured' histoblasts (after their rapid proliferation in response to the initial prepupal Ecdysone pulse), could assist Ecdysone signalling to instruct LECs to die. Indeed, LECs do not die in response to the pupal Ecdysone pulse if histoblast proliferation (and hence, 'maturation') has been experimentally delayed. The identification and characterization of this putative signal awaits further genetic and molecular analysis. Thus, Ecdysone signalling is necessary, but not sufficient, for LEC death (Ninov, 2007). The developmental control of cell cycle dynamics and diversity represents a key regulatory mechanism that directs cell size, cell number and ultimately the organ size of adult individuals. Despite numerous elegant experiments, the details of how cell division is regulated and coupled to cell growth remain poorly understood (Ninov, 2007).

During abdominal morphogenesis, the trigger of cell proliferation occurs simultaneously in all histoblast nests within each segment. Cell counting reveals that up to eight cell divisions are required to build the complete adult hemitergite. The same proportions apply to the ventral and spiracular nest. The first three histoblast divisions during pupariation are synchronous and extremely fast, skip the G1 phase and resemble the early embryonic blastoderm divisions. In this early stage, histoblast cleave and progressively reduce their size. Ecdysone signalling was found to be involved in the initiation of the proliferation programme. But, how is the histoblast cell cycle regulated to achieve fast proliferation in the absence of cell growth? Does it rely on the storage of preexistent control molecules, as in early embryos, or is it linked to signals impeding their growth? While this issue remains to be unravelled, the extreme growth of histoblasts during previous larval stages makes plausible the accumulation of G1 regulators, which, upon Ecdysone signalling, could allow a fast transition through G1 phase. Indeed, it was found that Cyclin E concentration (which regulates entry into S phase) in histoblasts builds up during the larval period. The observed deceleration of histoblast proliferation could then be the consequence of the exhaustion of the entire stock of Cyclin E. Still, the implication of growth control mechanisms in the regulation of histoblast proliferation cannot be ruled out. Multiple cell types, such as the animal-cap blastomeres from Xenopus embryos, change their cell cycles from size-independent to size-dependent after they become smaller than a critical cell size. Histoblasts might sense size in an analogous way. Thus, pathways that regulate growth, such as insulin-mediated signalling, Myc and Ras oncoproteins and the products of the Tuberous sclerosis complex 1 and 2 genes (reviewed by 22-122">Jorgensen and Tyers, 2004), should be explored to evaluate their potential roles in the coupling mechanism linking growth and cell cycle progression (Ninov, 2007).

Histoblast nest spreading initiates with the projection of leading comet-like protrusions, followed by apical cytoskeletal activity and active crawling over the underlying basal membrane, and terminates with the implementation of an apparent purse string, reminiscent of those described during dorsal closure, C. elegans ventral enclosure or wound healing (Ninov, 2007).

The comet-like protrusions of guiding histoblasts break through the LEC epithelial barrier, leading to planar intercalation of histoblast cell bodies. They account for the capacity of histoblasts to achieve migration within the bounded epithelial layer. Indeed, electron micrographs reveal that the advancing histoblasts form junctions with non-adjacent LECs before the adjacent LECs histolyze, thus insuring the continuity of the epidermis. Time-lapse observations suggest that these protrusions grow by sequential addition of actin molecules at their forward end. In this sense, they resemble, although being considerably slower, the actin tails employed by Listeria to propel through the cytoplasm of infected cells, or the actin-rich pseudopodia extended by neutrophils in response to chemoattractants. Proper actin cytoskeleton dynamics appear to be essential to build up these protrusions and the full repertoire of activities leading to the expansion of histoblast nests. The equilibrium between actin polymerization and depolymerization activities should be exquisitely regulated, and the forced polymerization of actin by Profilin overexpression not only blocks the cytoskeletal dynamics of single cells, but impedes the spreading of the whole histoblast nest. Potential roles for further actin dynamics regulators, the Arp2-Arp3 (Arp14D-Arp66B - FlyBase) complex, Dynamin (Shibire), membrane polyphosphoinositides, Cdc42, WASp-family proteins and other molecules in building up these projections remain to be explored. Further, although these protrusions appear to have a mechanical role, they also seem to be involved in the recognition of guidance cues, as they follow stereotyped paths. Indeed, gradients of cell affinity have been described for the patterning of the Drosophila abdomen, and it would be of major interest to understand how these cells interpret the larval landscape (Ninov, 2007).

The mechanisms involved in the death of LECs have been a matter of debate. While ultrastructural analysis suggests that LECs are phagocytosed, other studies suggested that LECs are histolyzed and die by autophagy. The current findings are conclusive in this respect. The death of LECs involves a caspase-mediated apoptotic process that implicates cytoskeletal remodelling and apical cellular constriction leading to delamination. The actomyosin mediated contractile force of dying LECs contributes in bringing together neighbouring histoblasts. Once the LECs initiate extrusion, they become immediate targets for circulating haemocytes, which extend membrane projections and engulf them. Finally, LECs are degraded inside haemocytes (Ninov, 2007).

Apical constriction is a process shared by multiple morphogenetic events, e.g., Drosophila mesodermal cells accumulate myosin and apically constrict during gastrulation under the control of the small GTPase Rho. Myosin activity is also sufficient to promote the apical constriction and elimination of photoreceptor cells in the Drosophila eye in response to the overexpression of an activated form of the Rok kinase. Indeed, this study found that the apical contractility of LECs depends on the level of phosphorylation of the MRLC and could be enhanced or abolished by modulating the counteracting kinase and phosphatase activities of Rok and MLCP. As a consequence, LEC delamination is either accelerated or delayed. How these regulatory activities are themselves regulated remains to be established. Yet, the LEC extrusion defects observed in weakened caspase cascade conditions after P35 overexpression strongly suggest that apoptotic signals could be involved in the trigger of actomyosin contractility in LECs. Apical contraction would thus be an early event in the LEC apoptotic process. Being particularly important to analyse the differences that modulate the activity of myosin during apical constriction of living cells and during extrusion of apoptotic cells, the replacement of LECs could become an exceptionally suitable model to unravel how myosin activity is regulated in apoptotic cells in vivo (Ninov, 2007).

The recruitment of haemocytes to dying LECs during abdominal cell replacement is extremely fast. The apical constriction of LECs takes about 2 hours, but the time that a haemocyte needs to fully engulf a LEC is less than 10 minutes. This entails a very reliable chemoattracting mechanism. In mammals, caspase 3-dependent lipid attraction signals, released by dying cells, induce the migration of phagocytes. Furthermore, several receptors are implicated in corpse recognition, including lectins, integrins, tyrosine kinases, the phosphatidylserine receptor (PSR) and scavenger receptors. In Drosophila, the elements involved in cell recognition by macrophages are mostly unknown. Haemocytes express Croquemort, a scavenger receptor homologue, which is required for the uptake of dead cells, and Pvr, a homologue of the vertebrate PDGF/VEGF receptor that seems to affect their motility. Still, the signals that haemocytes recognize in dying cells and the links between those signals and the apoptotic cascade are essentially unknown (Ninov, 2007).

As macrophages are responsible for much of the engulfment of dead cells in developing animals, an important role for macrophages in tissue morphogenesis has been suggested. However, this is not the case during abdominal morphogenesis, as the inhibition of haemocyte motility, which abrogates the removal of LECs, does not affect their replacement by histoblasts. These results are consistent with studies showing that macrophage removal of cell debris is not required for the regeneration of laser-induced wounds in Drosophila (Ninov, 2007).

Histoblast nest expansion is tightly coordinated with LEC removal. A naive view of the process of LEC extrusion suggests that their death is altruistic - it would promote the expansion of histoblasts. However, several results suggest that LECs do not execute this process autonomously. First, histoblast nests initiate their expansion in the absence of LEC death. Second, histoblast nests, during their spreading, grow, with no obvious planar orientation, by stochastic cell divisions not restricted to their edges. Finally, and most importantly, the inhibition of histoblast proliferation exerts non-autonomous effects on both extrusion and removal of LECs. A working model in which histoblast proliferation and LEC death are synchronized by a spatially and temporally controlled exchange of signals (secreted ligands or cell-to-cell communication modules) is thus strongly appealing. This potential mechanism for replacement of LECs by histoblasts somewhat resembles the elimination and death by anoikis of amnioserosa cells upon dorsal closure completion during Drosophila embryogenesis. Through this process, physical contacts and intracellular signalling among epithelial leading cells, the amnioserosa and the yolk sac coordinate the different behaviour of these cell types, which is essential for the accurate progress of both germ band retraction and dorsal closure. In this scenario, coordinated extrinsic and intrinsic events, hormonal inputs, cell contacts and cell signalling events will be responsible for the ordered proliferation and expansion of histoblasts and the extrusion and death of LECs (Ninov, 2007).

An alternative mechanism for the ordered cell substitution taking place during abdominal morphogenesis involving cell competition could also be proposed. Competition can be defined as an interaction between individuals brought about by a shared requirement leading to a reduction in the survivorship, growth and/or reproduction rates. Classical experiments in Drosophila imaginal discs have shown that cells heterozygous mutant for ribosomal protein genes (Minutes) placed beside wild-type cells are outcompeted and eliminated from the epithelium. More recent work has shown that imaginal wild-type cells are outcompeted by cells with growth advantage overexpressing the proto-oncogene Myc. Cell competition does not just apply to the fight for survival of cells with their 'fitness' experimentally altered, but also applies to the homeostasis of self-renewing cell pools such as lymphocytes or stem cells. The substitution of LECs by histoblasts closely resembles cell competition. Rapidly dividing and expanding histoblasts may become competent to displace the surrounding less-metabolically-active LECs. During normal development, having 'weaker' neighbours, histoblasts do not compete against each other, and cells from Minute clones in the abdomen are not eliminated in heterozygous animals. However, when confronted with death-resistant LECs, 'winner' histoblasts may become 'losers'. Histoblasts in an increasingly crowded environment will compete against each other, and the less fit individuals (less competent in signalling reception and transduction, or with slower proliferation rates) would eventually become more sensitive to 'killing' signals and would die (Ninov, 2007).

These findings demonstrate that the replacement of LECs by histoblasts, independently of being driven by cooperative mechanisms, cell competition or both, represents an extremely amenable morphogenetic model for the study of the dynamic control of the cell cycle and cell death, of the coordination of cytoskeleton activities and cell adhesion, and for the study of cell invasiveness (Ninov, 2007).

Cell rearrangements, cell divisions and cell death in a migrating epithelial sheet in the abdomen of Drosophila

During morphogenesis, cell movements, cell divisions and cell death work together to form complex patterns and to shape organs. These events are the outcome of decisions made by many individual cells, but how these decisions are controlled and coordinated is elusive. The adult abdominal epidermis of Drosophila is formed during metamorphosis by divisions and extensive cell migrations of the diploid histoblasts, which replace the polyploid larval cells. Using in vivo 4D microscopy, the behaviour of the histoblasts was examined and how they reach their final position and to what extent they rearrange during their spreading was analysed in detail. Tracking individual cells, it was shown that the cells migrate in two phases that differ in speed, direction and amount of cellular rearrangement. Cells of the anterior (A) and posterior (P) compartments differ in their behaviour. Cells near the A/P border are more likely to change their neighbours during migration. The mitoses do not show any preferential orientation. After mitosis, the sisters become preferentially aligned with the direction of movement. Thus, in the abdomen, it is the extensive cell migrations that appear to contribute most to morphogenesis. This contrasts with other developing epithelia, such as the wing imaginal disc and the embryonic germband in Drosophila, where oriented mitoses and local cell rearrangements appear to direct morphogenesis. Furthermore, the results suggest that an active force created by the histoblasts contributes to the formation of the adult epidermis. Finally, it was shown that histoblasts occasionally undergo apoptosis (Bischoff, 2009).

The formation of the abdominal epidermis differs from that of other epithelia, such as in the wing imaginal disc and during germband extension in the Drosophila embryo. In contrast to these more static epithelia, the formation of the abdominal epidermis is driven by extensive cell migrations. The final positioning of cells appears not to depend on the orientation of cell divisions, but particularly on cell movements, the speed and extent of which vary with the position along the AP axis. These movements also appear to lead to a rearrangement of sister cells in the direction of movement. During migration, cells only occasionally change their neighbours; the most extensive changes occur near the A/P boundary. These results explain why fluorescently marked wild-type clones tend to be elongated within the DV axis and do not split (Bischoff, 2009).

The movements and divisions of the histoblasts were tracked while they spread more than 300 µm in 24 hour. The cells at the tip of the moving cell mass were tracked, their positions were documented, and their divisions, traced their descendants documented and and their cell rearrangements studied. At 18 hours after puparium formation (APF), the anterior and posterior dorsal histoblast nests fuse shortly before histoblast migration begins. At 47 hours APF, the histoblasts have met at the dorsal midline and the whole abdomen is covered with adult cells. The segmental fold develops. Cells move in a more or less straight line towards the dorsal midline. Cells of segment 2 and 3 turn anteriorly when they approach the midline. Cells of segment 1 do not turn. The most anterior cell of the opposite hemisegment is positioned next to the most anterior cell of the abutting hemisegment, illustrating the matching of cells of the two hemisegments at the midline. To achieve this registration, the one cell, which is positioned more posteriorly during its dorsal migration, moves further in an anterior direction than the adjacent cell. An accompanying movie shows the entire migration of the histoblasts (18 to 47 hours APF) using a Histone::GFP marker (segments 2 and 3). At the beginning of the movie, the anterior and posterior dorsal histoblast nests fuse. Then the histoblasts move as a single epithelial layer towards the midline and replace the the polyploid larval epithelial cells (LECs). One row of LECs separates the histoblasts of neighbouring segments laterally from each other and is only subsequently removed (Bischoff, 2009).

One explanation for the differential movements of cells within the AP axis could be a gradient of cell affinities. This gradient might be manifest in a differential stickiness of cells along the AP axis, with posterior cells adhering less to each other, allowing their more extensive rearrangement (Bischoff, 2009).

Interestingly, the behaviour of the cells in the moving epithelial sheet appears to be influenced by the presence of the A/P boundary. The A/P boundary, with its differential adhesive properties, seems to act like an expansion joint, allowing cells to move more freely along each other. Thus, the A/P boundary is not only important for the patterning of the A and P compartments, but also appears to influence the positioning of the histoblasts (Bischoff, 2009).

In addition, it was found that the A and the P compartment cells behave differently, with cells of the P compartment rearranging more extensively and also being more likely to undergo cell death. These findings highlight the differences between the A and P compartments, which may act as two independent fields, and provide insights into differences in cellular behaviours (Bischoff, 2009).

In many developmental contexts, cells need to coordinate their behaviour; for example, in order to move as a group. This work complements the study of Ninov (2007), who focussed their analysis on the interactions of histoblasts and LECs, and highlights the behaviour of individual cells, the sum of which is responsible for morphogenesis. A combination of cell tracking using 4D microscopy and clonal analysis should help tackle questions such as what mechanisms guide the cells to their final position and what positions them relative to each other. Addressing these questions is important in order to understand the morphogenesis of all epithelia, including in gastrulation and neurulation (Bischoff, 2009).

miR-965 controls cell proliferation and migration during tissue morphogenesis in the abdomen

Formation of the Drosophila adult abdomen involves a process of tissue replacement in which larval epidermal cells are replaced by adult cells. The progenitors of the adult epidermis are specified during embryogenesis and, unlike the imaginal discs that make up the thoracic and head segments, they remain quiescent during larval development. During pupal development, the abdominal histoblast cells proliferate and migrate to replace the larval epidermis. This study provides evidence that the microRNA, miR-965, acts via string and wingless to control histoblast proliferation and migration. Ecdysone signaling downregulates miR-965 at the onset of pupariation, linking activation of the histoblast nests to the hormonal control of metamorphosis. Replacement of the larval epidermis by adult epidermal progenitors involves regulation of both cell-intrinsic events and cell communication. By regulating both cell proliferation and cell migration, miR-965 contributes to the robustness of this morphogenetic system (Verma, 2015).

The findings of this study link regulation of the miR-965 microRNA to the onset of histoblast proliferation at the larval to pupal transition. Previous reports have provided evidence that Ecdysone signaling activates string expression to trigger the onset of histoblast proliferation at the beginning of pupal development (Ninov, 2009). The current findings provide evidence that Ecdysone signaling works though regulation of miR-965, which in turn regulates string. Interestingly, evidence was also found for negative feedback regulation of miR-965 on EcR. Mutual repression circuitry of this type can contribute a switch-like function: EcR activity lowers miR-965 activity, which allows greater EcR expression/activity by alleviating miR-965 mediated repression. In a circuit of this design, there will be a delay between reduced transcription of the miRNA primary transcript and the decay of the mature miRNA product. Hence sustained EcR activity is needed to throw the switch (Verma, 2015).

EcR shows positive transcriptional autoregulation and this is buffered by miR-14 in a mutual repression circuit (Varghese, 2007). Positive feedback allows for a sharp switch-like response, but also makes the system very sensitive to stochastic fluctuation in EcR activity. Coupling EcR positive auto-feedback to miRNA-mediated repression allows a robust switch function upon Ecdysone stimulation, while protecting the system from the effects of biological noise. This study provides evidence that miR-965 plays an analogous role in regulating EcR response and suggests that miR-965 confers robustness to the EcR response in the histoblasts (Verma, 2015).

Upregulation of string in the miR-965 mutant contributes to the defects in histoblast proliferation. How misregulation of string might contribute to the migration defects is less immediately obvious. Previous work has shown that cell cycle progression in the histoblast population is required to trigger programmed cell death in the surrounding larval epidermal cells (LECs). Evidence has been provided that cell growth and the expansion of the histoblast nests may be required to elicit LEC apoptosis. Although the mechanism by which expansion of the histoblasts triggers LEC death is not clear, elevated string expression in the miR-965 mutant is likely to be responsible for the cell cycle progression defects during this phase, hindering normal LEC removal and histoblast migration (Verma, 2015).

Persistence of the LECs might also be a consequence of the increased expression of Wg protein in the mutant histoblast nests. Wg acts in combination with EGFR and Dpp signals to control abdominal segment patterning. These signals are thought to control differential cell adhesion, which may be important for elimination of the LECs as well as for proper segmental fusion of the histoblast nests. Elevated expression of Wg protein may lead to an expanded range of action, perhaps resulting in ectopic Wg activity in the LECs (Verma, 2015).

Each adult abdominal segment has a well-defined anterior-posterior polarity. Wg is required from 15–20 hr APF for bristle formation and from 18–28 hr APF for tergite differentiation and pigmentation. Overexpression of wg has been shown to cause ectopic bristle formation, and shaggy mutant clones, which constitutively activate wg signaling, can cause polarity reversal in abdominal bristles, while EGFR, FGF, dpp and Notch signaling have no effect on the polarity of bristles in adult epidermis. Wg levels are normally higher in the posterior region of the anterior histoblast nests and lower more anteriorly. The current finding that Wg levels were elevated and that the distribution of Wg was broader than normal suggests ectopic Wg activity throughout the histoblast nest, including cells that normally experience low Wg levels. Ectopic spread of Wg could be responsible for the formation of ectopic bristles and for the occasional instances of polarity reversal observed in the anterior part of tergites in the miR-965 mutants (Verma, 2015).

Replacement of the larval epidermis during metamorphosis involves regulation of both cell-intrinsic events in the abdominal histoblasts and communication between histoblasts and the larval cells they will replace. miR-965 acts on at least two separate processes required during histoblast morphogenesis. A miRNA with multiple targets can add a layer of regulation, acting across different pathways to integrate their activities. In doing so, the miR-965 miRNA appears to contribute to the robustness of this complex morphogenetic system (Verma, 2015).

Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium

Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring non-dying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, the contraction of actomyosin cables formed in apoptotic and neighboring cells drive cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows remodeling of adherens junctions (AJs) between apoptotic and non-dying cells, with a reduction in the levels of AJ components, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. A model is proposed in which modulation of tissue tension represents a mechanism of apoptotic cell extrusion, and would further influence biochemical signals of neighboring non-apoptotic cells (Teng, 2017).

This study reports the temporal sequence of events during apoptotic cell extrusion, with a focus on the remodeling of AJs, the cytoskeleton, and mechanical tension. After caspase-3 starts to be activated in the polyploid larval epithelial cells (LECs), those undergoing apoptosis initiate apical constriction. It was reasoned that the initiation of this constriction could be due to a combination of actomyosin cable formation in the dying cell and the activity of caspase-3, which assists in the upregulation of actomyosin contractility. Indeed, it has been shown in tissue culture that the cleavage of Rho associated kinase by caspase- 3 is involved in phosphorylation and activation of myosin light chain, which regulates actomyosin contractility. It is proposed that the actomyosin cable that forms in apoptotic LECs is responsible for the early stages of apoptotic cell extrusion. During apical constriction, the level of AJ components including E-cad strongly reduced in a caspase-3-dependent manner. In the neighboring non- dying cells, this reduction is found only at the interface between the apoptotic cell and its neighbors. Since caspase-3 is not activated in the neighboring cells, it is speculated that the reduction of E-cad is a consequence of a loss of trans-interactions between E-cad of the neighboring cell, and E-cad of the apoptotic cell, which undergoes caspase-3-dependent cleavage. This often, but not always, leads to plasma membrane separation, which is suggestive of a loosening of AJ-dependent adhesion. It has been reported that anillin organizes and stabilizes actomyosin contractile rings at AJs and its knock-down is associated with a reduction of E-cad and β-Catenin levels at AJs, leading to AJ disengagement. A gradual decrease in the level of E-cad, and a gradual increase in MyoII accumulation in apoptotic cells was observed prior to the strong reduction of E-cad levels. This lead to the hypothesis that mechanical tension exerted on the cell interface between apoptotic LECs and neighboring cells by the contraction of the actomyosin cable, which forms in the apoptotic cell, is large enough to rupture the weakened contacts between plasma membranes at AJs upon the strong reduction of E-cad levels (Teng, 2017).

Interestingly, and by contrast, there are cases when AJs are not disengaged even after the level of E-cad is reduced. In these cases the cells exhibit a separation of actomyosin cables from the membrane. It is speculated that the state of cell-cell contacts at AJs, i.e., whether they will disengage or remain engaged during apoptosis, is dependent on which of the following links is weaker: The link between two plasma membranes, or the link between the plasma membrane and the actomyosin cable. Both of these links would be weakened by a strong, albeit incomplete, reduction of E-cad levels. When the former is weaker than the latter, the two plasma membranes could be detached. When the former is stronger than the latter, the two plasma membranes could remain in contact, and the actomyosin cable could be detached from the plasma membrane (Teng, 2017).

In parallel with the reduction of E-cad levels and the associated release of tension, a supra-cellular actomyosin cable begins to form in neighboring cells. These observations prompted a speculation that the release of tissue tension triggers MyoII accumulation in neighboring cells. Subsequent contraction of this outer ring helps to reshape tissue tension, which is transiently released when E-cad is reduced. As a consequence, the neighboring cells are stretched. Upon completion of apical constriction, neighboring non-apoptotic cells form de novo AJs and the stretched cells undergo cell division and/or cell-cell contact rearrangement. These processes allow a relaxation of the high tension associated with the stretching of cells. Finally, measurements of caspase-3 activity, and the observations from caspase inhibition experiments, lead to a conclusion that the characteristics associated with apoptotic cell extrusion reported in this study are the consequences of the apoptotic process, rather than the cause (Teng, 2017).

In addition to the progressive remodeling of AJs and modulation of tissue tension during apoptosis, the mechanical role was examined of apoptosis 'apoptotic force' in tissue morphogenesis, which has been proposed, demonstrated, and discussed. It was shown that the mechanical force generated by the contraction of actomyosin cables formed when LECs undergo apoptosis, especially boundary LECs, promotes tissue expansion, along with histoblast proliferation and migration. Nonetheless, it cannot be ruled out that this apical contraction is in part driven by a decrease in cell volume, which can be triggered by caspase activation. Intriguingly, it was found that apoptosis of non-boundary LECs did not affect tissue expansion. This raised the possibility that the mechanical influence of apoptosis in neighboring tissues is dependent not only on the physical connections between cells, but also on the mechanical properties of cells, including cell compliance. If a tissue is soft, for instance, the tensile forces generated by apoptotic process could be absorbed by nearest-neighbor cells and would not propagate to cells further than a single cell away. It is speculated that the apoptotic process could mechanically contribute to cell death-related morphogenesis, only when apoptosis takes place at optimal mechanical properties of a tissue (Teng, 2017).

This study presents a framework for understanding how cell adhesions and tissue tension are progressively modulated during apoptosis in a developing epithelium. It is concluded that tissue tension reshaping, including the transient release of tension upon a reduction in the levels of AJ components, represents a mechanism of apoptotic cell extrusion. It would be important to explore how this transient modulation in mechanical tension would further influence the biochemical nature of neighboring non-apoptotic cells (Teng, 2017).

The Dachsous/Fat/Four-Jointed pathway directs the uniform axial orientation of epithelial cells in the Drosophila abdomen

The achievement of the final form of an individual requires not only the control of cell size and differentiation but also integrative directional cues to instruct cell movements, positions, and orientations. In Drosophila, the adult epidermis of the abdomen is created de novo by histoblasts. As these expand and fuse, they uniformly orient along the anteroposterior axis. The Dachsous/Fat/Four-jointed (Ds/Ft/Fj) pathway is key for their alignment. The refinement of the tissue-wide expression of the atypical cadherins Ds and Ft result in their polarization and directional adhesiveness. Mechanistically, the axially oriented changes in histoblasts respond to the redesign of the epithelial field. It is suggested that the role of Ds/Ft/Fj in long-range oriented cell alignment is a general function and that the regulation of the expression of its components will be crucial in other morphogenetic models or during tissue repair (Mangione, 2018).

PCP pathways act as key coordinators in the makeover of planar epithelial tissues during development by modulating adhesive interactions and mechanical forces. However, the regulatory means that these pathways use to direct the topographical organization of epithelial cells are far from being clear. By applying in vivo analyses to the morphogenesis of the adult abdominal epidermis of Drosophila, this study has characterized a new mechanism for the stepwise long-range cobblestone organization of the tissue. The organization of the abdominal epithelial landscape was found to be the result of an axially oriented adhesiveness mediated by the Ds/Ft/Fj pathway. The directional cues dictated by this pathway put the epithelial cells on the right track, orienting their otherwise changing shapes along the A/P axis (Mangione, 2018).

Global tissue changes may involve many different activities: coordinated cell-cell rearrangements triggering tissue reorientation or convergent extension,or spatially controlled proliferation and growth, division orientation, and death. The uniform axially oriented alignment of histoblasts emerges in a precise spatiotemporal manner through coordinated changes in cell shape orientation. Histoblasts constitute a highly proliferative tissue with much room for expansion. In this scenario, which is very different from that of epithelial tissues constrained in their dimensions like the fly notum, to reach uniformity in cell alignment orientations, changes in cell shape and area would be preferred over cell intercalations. When the expressions of ds, ft, or fj are affected, the relative orientations of cell alignments are severely disturbed without alterations in tissue differentiation (Mangione, 2018).

It is not known which positional cues the Ds/Ft/Fj pathway interprets to dictate the stereotyped uniform anisotropic polarization of the abdominal epidermis along the A/P axis. Some tips may come from analyses on the regulation of the components of the core Fz-PCP system. This system is a required partner of the Ds/Ft/Fj pathway for proper planar polarity acquisition. Both pathways rely on primary long-range global cues to coordinate short-range cell polarity. During the development of the eye and wing in Drosophila, the secreted factor Wingless (Wg/Wnt1) modulates intercellular interactions in both systems. In the Ds/Ft/Fj pathway, Ds and Ft form heterodimers, whereas in the Fz-PCP system, there are Fz-Vang heterodimers and Stan-Stan homodimers. In the eye and the wing discs, Wg binds to Fz, which affects Fz-Vang interactions and the activity of the Fz-PCP system. As a result, cells orient toward the source of Wnt expression (Wg and Wnt4) at the compartment margins. Wg/Wnt4 could be playing an equivalent role during the axial uniform alignment of oriented cells in the abdomen. While Wg specifies the tergite and sternite cell fates, how it could regulate the graded expression of ds, ft, or fj or influence the uniform axial orientation of histoblasts remains poorly explored (Mangione, 2018).

It is known that differential adhesive properties between neighboring cells prevent intermingling, as they tend to minimize their contacts. In clones, this leads to smooth borders. Major differences were found in roughness, perimeter, and, to a lesser extent, roundness in mutant clones for members of the Ds/Ft/Fj pathway. These differences strongly support a role for the Ds/Ft/Fj pathway and, in particular, the opposing graded expressions of the atypical cadherins Fat and Ds, in generating directional information at cell contacts. Mutant clones generate planar conflicts between cells with different adhesive properties and smooth borders at specific edges, and the directional information is lost (Mangione, 2018).

The evolving functional pattern delineated by the Ds/Ft/Fj pathway arises as an elegant and efficient way to dictate directional order across developmental fields. Several pieces of evidence point to it as a key element modulating similar processes in different organisms. Mitral valve prolapse (MVP) is a common cardiac valve disease, the genetic etiology of which has remained elusive. Recently, it has been shown that MVP could be traced back, both in mice and fish models, to developmental errors in valve morphogenesis. The epicardial-derived cells fail to uniformly align into the posterior leaflet, and this failure correlates to a missense mutation in the DCHS1 gene, the human homolog of ds. Similarly, the establishment of polarized arrays of aligned chondrocytes in the zebrafish developing pharyngeal arch requires Fat3 and Dchs2 cadherins. During postnatal stages in mammals, hair follicles progressively get in line, both locally and globally, precisely with the A/P axis. This process of postnatal refinement has been suggested, although not proved, to be the result of instructive functions originating from the Ds/Ft/Fj pathway. Whether vertebrate Ds/Ft/Fj signaling is essential to propagate polarity at a distance, is linked to molecular gradients, or may interact with other polarizing signals remains unknown (Mangione, 2018).

The subcellular polarization of the atypical myosin Dachs (D) in response to the Ds/Ft/Fj pathway appears to be uncoupled from the uniform orientation of cell alignments; D polarity is reoriented but sustained in ds, and its loss did not affect cell alignments uniformity. These data led to a hypothesis that a bias in contractility mediated by D at the cell cortex may be not critical for uniform cell orientation. Moreover, contractile anisotropy as a factor directing the axial uniformity of histoblasts is unsupported by the observed isotropic distribution of vinculin at cell vertices. In this scenario, asymmetric adhesiveness through heterodimeric interactions between Ds and Ft appears to be a more plausible element directing the uniform orientation of histoblasts (Mangione, 2018).

Assuming that cells and tissues tend to minimize their surface free energy, contacts through adhesion molecules and contractile activities at the cell cortex would be key determinants of cell and tissue shape. Adhesiveness will promote cells to spread their shared surface, while contractility will counterbalance the adhesive forces. Differential adhesive properties within histoblasts would introduce anisotropic tension affecting cell-cell contacts and the capacity to coordinately orient shape changes in the tissue plane. This anisotropic tension, which is revealed by laser microsurgery, is lost in ds mutants. Anisotropic tension is also uncovered in clones, in which contact angles and lengths between histoblasts adjust to their conflicting genotypes and their relative location. Differential adhesiveness at cell junctions would have direct input into surface tension. Tensile patterns rather than cell positions would therefore play instructive roles in the acquisition of uniform order. At any given point in time, they will reflect the recent developmental history of the tissue. Along this line, during the expansion and remodeling of the histoblasts, the expression pattern of Ft modulated by Ds and fj evolves into an A/P gradient spanning whole compartments. This expression refinement will result in the spreading throughout the epithelium of a counterbalanced adhesion share between Ft and Ds that will delineate the axially oriented surface tension landscape that will instruct uniform cell alignments (Mangione, 2018).

Will the final arrangement of the cells be physically stable (minimal energy) upon completion or will a secondary event be necessary to stabilize it? In Caenorhabditis elegans, the epidermal cells elongate during development and subsequently attach to the cuticle to fix their shape. Thus, the collagenous exoskeleton secreted by the apical surface of the epidermis seems to be indispensable. The histoblasts use their apical surface to attach to the overlaying pupal cuticle very early on. These contacts are very dynamic during the period of expansion and become stabilized by the end of tissue remodeling. Whether they fulfill a hardening role on the tissue landscape is an open question (Mangione, 2018).

The role this study has uncovered for the Ds/Ft/Fj pathway implementing the uniform orientation of the alignment of histoblasts does not relate to any developmental function in patterning, cell specification, and/or differentiation. The directional evolution of the expression of the different Ds/Ft/Fj pathway elements sets a spatially and temporally controlled directional adhesive partnership between Ds and Ft. This provides the basis for the establishment of a dynamic adhesiveness pattern unfolding over time that directs cell shape changes, ultimately guiding the uniform alignment of the epithelial cells across the tissue (Mangione, 2018).

Mechanical coordination is sufficient to promote tissue replacement during metamorphosis in Drosophila
During development, cells coordinate to organize in coherent structures. Although it is now well established that physical forces are essential for implementing this coordination, the instructive roles of mechanical inputs are not clear. This study shows that the replacement of the larval epithelia by the adult one in Drosophila demands the coordinated exchange of mechanical signals between two cell types, the histoblasts (adult precursors) organized in nests and the surrounding larval epidermal cells (LECs). An increasing stress gradient develops from the center of the nests toward the LECs as a result of the forces generated by histoblasts as they proliferate and by the LECs as they delaminate (push/pull coordination). This asymmetric radial coordination of expansive and contractile activities contributes to epithelial replacement. These analyses support a model in which cell-cell mechanical communication is sufficient for the rearrangements that implement epithelial morphogenesis (Prat-Rojo, 2019).

Tissue mechanical properties modulate cell extrusion in the Drosophila abdominal epidermis

The replacement of cells is a common strategy during animal development. In the Drosophila pupal abdomen, larval epidermal cells (LECs) are replaced by adult progenitor cells (histoblasts). Previous work showed that interactions between histoblasts and LECs result in apoptotic extrusion of LECs during early pupal development. Extrusion of cells is closely preceded by caspase activation and is executed by contraction of a cortical actomyosin cable. This study identified a population of LECs that extrudes independently of the presence of histoblasts during late pupal development. Extrusion of these LECs is not closely preceded by caspase activation, involves a pulsatile medial actomyosin network, and correlates with a developmental time period when mechanical tension and E-cadherin turnover at adherens junctions is particularly high. This work reveals a developmental switch in the cell extrusion mechanism that correlates with changes in tissue mechanical properties (Michel, 2020).

This study has analyzed the replacement of LECs by adult histoblasts in the pupal abdominal epidermis in Drosophila. Previous work has shown that the interaction between the growing nest of histoblasts and LECs is required for LEC extrusion. LEC extrusion is closely preceded by caspase activation and involves contraction of a supracellular actomyosin cable. This study has identified a second hotspot of cell extrusion at the dorsal midline of the pupal abdomen. Cell extrusion at the dorsal midline occurs mainly late during development and is independent of histoblasts, but depends on the presence of neighboring LECs. LEC extrusion is preceded by a uniform rise in caspase activity throughout the larval tissue and involves pulsatile contractions of a medial actomyosin network. Mechanical tension on adherens junctions of LECs increases during pupal development. High mechanical tension during late pupal development depends in part on caspase activity and correlates with a high turnover of E-cadherin. E-cadherin turnover depends on Dynamin-dependent endocytosis and blocking Dynamin severely affects LEC extrusion. This work reveals a novel, histoblast-independent mechanism by which LECs extrude during late pupal development (Michel, 2020).

Extrusion of LECs located at the border of histoblast nests during early pupal development involves the remodeling and weakening of adherens junctions between the extruding LEC and its neighboring cells, and the accumulation of Myosin II at the cortices of both the extruding LEC and its neighboring cells. Cortical Myosin II accumulation in neighboring cells results in the formation of a supracellular actomyosin cable. Contraction of this cable facilitates the extrusion of the LEC (Teng, 2017). The data indicate that extrusion of LECs at the dorsal midline during late pupal development involves a distinct mechanism. In addition to a cortical pool of Myosin II and F-actin, Myosin II and F-actin was found also in an apical medial region of the extruding LECs. Similar accumulations of Myosin II and F-actin have been reported in several other epithelia, including in the Drosophila embryo in ventral furrow cells of the epidermis and in amnioserosa cells. The medial actomyosin network is pulsatile in these cells resulting in concomitant fluctuations in apical cell area. Similarly, it was found that in extruding LECs located in the vicinity of the dorsal midline, apical cell area fluctuates and apical constriction correlates with increased medial Myosin II intensity. Over longer time periods, apical cell area shrinks, indicating a ratchet-like mechanism. A similar ratchet-like contraction mechanism is involved in the Drosophila embryo during neuroblast delamination (Simoes, 2017) and the extrusion of amnioserosa cells. The reason for these different extrusion mechanisms of LECs during early and late pupal development remains unclear. It is noted, however, that mechanical tension on LECs increases approximately 5- to 10-fold between early (20 h APF) and late (27 h APF) pupal development, suggesting that overall tissue mechanical tension influences the mechanism by which cells extrude (Michel, 2020).

How is the extrusion of LECs at the dorsal midline initiated? Several mechanisms have been described that drive cell extrusion, including cell competition and tissue crowding. These two mechanisms, however, appear not to play a major role in the extrusion of LECs located at the dorsal midline of the pupal abdominal epidermis. Tissue crowding induces, for example, caspase-dependent cell extrusion at the dorsal midline in the pupal notum. Experimental induction of clones expressing an activated form of Ras, RasV12, leads to the crowding of neighboring wild-type cells and to their extrusion. In the abdominal epidermis, the growth or migration of histoblasts could result in crowding of the LECs. During the analyzed developmental time period, the average area of non-extruding LECs located at the dorsal midline, however, remains nearly constant, indicating that these LECs do not become crowded. Moreover, LECs located at the dorsal midline still extrude when histoblast division is halted or histoblasts are genetically ablated, providing further evidence that tissue crowding is not involved in the extrusion of these cells. Finally, cell competition refers to the out competition of slow-growing cells by fast-growing cell. Again, the finding that LEC extrusion at the dorsal midline is independent of histoblast division, strongly argues that cell competition does not have a major influence on the extrusion of these LECs (Michel, 2020).

During early pupal development, local interactions between histoblasts and LECs trigger caspase activation in LECs. Caspase activation then closely precedes extrusion of LECs, cell by cell, at the border of histoblast nests, indicating that caspase activation triggers extrusion (Teng, 2017). In LECs at the dorsal midline, by contrast, caspase activity, as measured by Apoliner cleavage, shows a population-wide fluctuation during pupal development. Caspase activity peaks during the observed developmental time period twice, around 20 h APF and around 26-29 h APF. Caspase activation in these cells does not appear to trigger their extrusion, because the first peak of caspase activity at around 20 h APF takes place several hours before LECs at the dorsal midline start to extrude. Interestingly, the second rise of caspase activity between 24 h and 26 h APF precedes the extrusion of LECs at the dorsal midline. Taken together with the observation that LEC extrusion at the dorsal midline requires caspase activity, these results indicate that the second rise of caspase activity is a prerequisite for LECs to extrude at the dorsal midline. How the population-wide fluctuations of caspase activity in LECs at the dorsal midline arise will be interesting to explore (Michel, 2020).

This study found that mechanical tension acting on LEC adherens junctions increases during pupal development. Increased mechanical tension is independent of the proliferation of histoblasts, but requires caspase activity, which in turn is necessary for LEC extrusion. It is speculated that LEC extrusion during late pupal development may, in part, facilitate the increased mechanical tension on adherens junctions of the remaining LECs. Mechanical tension may influence cell extrusion in different ways and by different mechanisms. In the pupal notum, for example, a similar developmental increase in mechanical tension has been observed, but, unlike the situation in the pupal abdomen, increased mechanical tension correlates with fewer cell extrusions. This different behavior may result from the different characteristics of the two epithelia. In the notum, cell extrusion occurs within a diploid, proliferating tissue with columnar cells. On the other hand, LECs of the pupal abdomen are polyploid, non-proliferating and squamous in shape. In these respects, the abdominal LECs resemble more the amnioserosa cells of the embryo (Michel, 2020).

How could an increase in mechanical tension on LEC adherens junctions facilitate extrusion? LEC extrusion involves the remodeling of adherens junctions of the extruding cell (Teng, 2017). Adherens junction remodeling depends on E-cadherin. The turnover of E-cadherin was found to have increased at 27 h APF compared with 20 h APF, thus positively correlating with increased mechanical tension. The turnover of E-cadherin depends on lateral diffusion on the plasma membrane and on trafficking between a cytosolic, presumably vesicular, pool of E-cadherin and a junctional pool of E-cadherin. The exchange between these two pools requires Dynamin-dependent endocytosis. Consistent with this notion, E-cadherin turnover was reduced in LECs of flies mutant for shibire1, which encodes Dynamin. Interestingly, extrusion of LECs in the lateral region of the abdominal segments is nearly blocked in shibire1 mutants. Although the possibility cannot be excluded that Dynamin-dependent endocytosis of proteins other than E-cadherin are important for LEC extrusion, these data lend support for the idea that high E-cadherin turnover is required for extrusion of these LECs. Surprisingly, a subset of LECs, those located in medial regions of the segment, do extrude in shibire1 mutants. These findings reveal an unexpected complexity of LEC extrusion that will require future work to be resolved (Michel, 2020).

The following model is proposed of how LECs in the pupal abdominal epidermis extrude. During early pupal development (20 h APF) mechanical tension on LEC junctions is low and E-cadherin turnover is low. LECs extrude only if they receive a specific 'extrusion signal' from the histoblasts. Extrusion is mediated by cortical actomyosin, which forms a contractile supracellular cable driving apical cell constriction (Teng, 2017). Later during pupal development (27 h APF), mechanical tension on LEC junctions, E-cadherin turnover and caspase activity are high. Stochastic extrusions of LECs commence, which are mediated by a pulsatile medial actomyosin network that leads to ratchet-like apical cell contractions (Michel, 2020).

The Microtubule Minus-End Binding Protein Patronin Is Required for the Epithelial Remodeling in the Drosophila Abdomen

In the developing Drosophila abdomen, the epithelial tissue displays extensive cytoskeletal remodeling. In stark contrast to the spatio-temporal control of the actin cytoskeleton, the regulation of microtubule architecture during epithelial morphogenesis has remained opaque. In particular, its role in cell motility remains unclear. This study shows that minus-end binding protein Patronin is required for organizing microtubule arrays in histoblast cells that form the Drosophila abdomen. Loss of Patronin results in a dorsal cleft, indicating the compromised function of histoblasts. It was further shown that Patronin is polarized in these cells and is required for the formation of highly dynamic non-centrosomal microtubules in the migrating histoblasts. Thus, this study demonstrates that regulation of microtubule cytoskeleton through Patronin mediates epithelium remodeling (Panzade, 2021).

ECM degradation in the Drosophila abdominal epidermis initiates tissue growth that ceases with rapid cell-cycle exit
During development, multicellular organisms undergo stereotypical patterns of tissue growth in space and time. How developmental growth is orchestrated remains unclear, largely due to the difficulty of observing and quantitating this process in a living organism. Drosophila histoblast nests are small clusters of progenitor epithelial cells that undergo extensive growth to give rise to the adult abdominal epidermis and are amenable to live imaging. Our quantitative analysis of histoblast proliferation and tissue mechanics reveals that tissue growth is driven by cell divisions initiated through basal extracellular matrix degradation by matrix metalloproteases secreted by the neighboring larval epidermal cells. Laser ablations and computational simulations show that tissue mechanical tension does not decrease as the histoblasts fill the abdominal epidermal surface. During tissue growth, the histoblasts display oscillatory cell division rates until growth termination occurs through the rapid emergence of G0/G1 arrested cells, rather than a gradual increase in cell-cycle time as observed in other systems such as the Drosophila wing and mouse postnatal epidermis. Different developing tissues can therefore achieve their final size using distinct growth termination strategies. Thus, adult abdominal epidermal development is characterized by changes in the tissue microenvironment and a rapid exit from the cell cycle (Davis, 2022).


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