Drosophila gene families: Germ band retraction

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Zygotically transcribed genes

Germ band retraction

  • Cellular processes associated with germ band retraction
  • Scarface, a secreted serine protease-like protein, regulates polarized localization of laminin A during germ-band retraction and dorsal closure
  • Cellular mechanics of germ band retraction in Drosophila
  • Elongated cells drive morphogenesis in a surface-wrapped finite-element model of germband retraction
  • Cytocortex-dependent dynamics of Drosophila Crumbs controls junctional stability and tension during germ band retraction
    Cellular processes associated with germ band retraction

    Large-scale movements of epithelial sheets are necessary for most embryonic and regenerative morphogenetic events. The cellular processes associated with germ band retraction (GBR) have been characterized in the Drosophila embryo. During GBR, the caudal end of the embryo retracts to its final posterior position. Using time-lapse recordings, it has been shown that, in contrast to germ band extension, cells within the lateral germ band do not intercalate. In addition, the germ band and amnioserosa move as one coherent sheet, and the amnioserosa strongly shortens along its dorsal-ventral axis. Furthermore, during GBR, the amnioserosa adheres to and migrates over the caudal end of the germ band via lamellipodia. Expression of both dominant-negative and constitutively active RhoA in the amnioserosa disrupts GBR. Since RhoA acts on both actomyosin contractility and cell-matrix adhesion, it suggests a role for such processes in the amnioserosa during GBR. The results establish the cellular movements and shape changes occurring during GBR and provide the basis for an analysis of the forces acting during GBR (Schock, 2002).

    GBR is completed during embryonic stage 12. This is a time of exceptional morphogenetic activity: the midgut fuses and encloses the yolk sac laterally; the tracheal pit extensions fuse to form the tracheal tree, and the segmental furrows form from anterior to posterior. At stage 12, the embryo consists of two major epithelia, the squamous extraembryonic amnioserosa and the ectodermal germ band epithelium, as well as a mesenchymal mass of mesodermal and central nervous system precursor cells. Also found in the embryo are the epithelia of the foregut, hindgut, and salivary and tracheal pit invaginations, which are of ectodermal origin, and the midgut epithelium, which forms at that stage by mesenchymal to epithelial transition. The syncytial yolk sac, which is enclosed by a yolk sac membrane, sits in the middle of the embryo at the beginning of GBR, but moves more dorsally, directly beneath the amnioserosa, by the end of GBR. Analysis was focused on the amnioserosa and the germ band, because they appear to be the most likely candidates of the above tissues to participate in GBR. Cells in the germ band do not intercalate. GBR is therefore not a reversal of germ band extension (Schock, 2002).

    Rather, reciprocal cell shape changes within the amnioserosa and the germ band are associated with the changes in embryo morphology at this stage. That is, the amnioserosa shortens along the DV axis, while the germ band elongates along the DV axis. This is possible because the amnioserosa and germ band are tightly attached to each other via adherens junctions, i.e., both epithelia move as one coherent sheet (Schock, 2002).

    The boundary between amnioserosa and germ band was investigated at high magnification to obtain an idea of whether the shape changes observed are of an active or a passive nature. It was assumed that contractile forces would be exerted along the plasma membranes, because the actin cytoskeleton is localized cortically in both germ band and amnioserosa cells. The row of leading edge germ band cells is pulled in where the amnioserosa membranes perpendicular to the leading edge are attached. This suggests that amnioserosa cells contract along their DV axis. Cells of the germ band would be expected to push into the larger amnioserosa cells in the case of an active DV extension of the germ band, thus resulting in a convex shape of the amnioserosa-germ band boundary (Schock, 2002).

    The presence of protrusions is demonstrated; these are formed predominantly at the posterior edge of the amnioserosa projecting toward the posterior. These protrusions exhibit high levels of dynamic actinGFP at the migration front, indicating actin polymerization. These protrusions have been classified as lamellipodia, because their appearance, behavior, and dynamic actin content are identical to lamellipodia in other motile cells. These lamellipodia migrate over the germ band instead of being passively dragged by the retracting germ band. The lamellipodia may migrate on an apical extracellular matrix secreted by germ band cells as a precursor to the larval cuticle. These observations indicate that the overlap of the amnioserosa over the caudal end of the germ band during GBR is maintained by lamellipodia-mediated migration. Furthermore, both constitutively active and dominant-negative RhoA disrupt GBR, when expressed in the amnioserosa. This suggests that actomyosin contractility or cell migration within the amnioserosa contribute to GBR, since these processes are affected by expression of rhoA mutants in tissue culture (Schock, 2002).

    The amnioserosa is required for GBR because embryos that lack this tissue fail to undergo GBR. It has been proposed that this requirement for the amnioserosa may involve signaling from the amnioserosa to the germ band. The cell shape changes and motility observed within the amnioserosa and the overexpression experiments suggest that the amnioserosa additionally contributes to GBR in other ways than signaling (Schock, 2002).

    The processes observed in this study allow several mechanisms, which are not mutually exclusive, to participate in GBR. (1) Segment furrow formation within the germ band may facilitate GBR by causing AP shortening of the germ band. (2) Active DV shortening of the amnioserosa may contribute to GBR by pulling in the lateral sides of the anterior germ band, thereby resulting in retraction of the germ band behind the bend of the U-shaped germ band-extended embryo. (3) The pulling force that appears to be exerted by DV shortening of the amnioserosa may be assisted by active DV extension of the germ band cells. (4) The overlap of the amnioserosa over the germ band may allow proper deployment of forces occurring within the amnioserosa (Schock, 2002).

    Scarface, a secreted serine protease-like protein, regulates polarized localization of laminin A during germ-band retraction and dorsal closure

    Cell-matrix interactions brought about by the activity of integrins and laminins maintain the polarized architecture of epithelia and mediate morphogenetic interactions between apposing tissues. Although the polarized localization of laminins at the basement membrane is a crucial step in these processes, little is known about how this polarized distribution is achieved. This study analysed the role of the secreted serine protease-like protein Scarface in germ-band retraction and dorsal closure -- morphogenetic processes that rely on the activity of integrins and laminins. Evidence that scarface is regulated by c-Jun amino-terminal kinase and that scarface mutant embryos show defects in these morphogenetic processes. Anomalous accumulation of laminin A on the apical surface of epithelial cells was observed in these embryos before a loss of epithelial polarity was induced. It is proposed that Scarface has a key role in regulating the polarized localization of laminin A in this developmental context (Sorrosal, 2010).

    During germ band retraction (GBR), the tail end of the germ band, or embryo proper, interacts with the amnioserosa (AS), an epithelium of large flat cells that does not contribute to the larva, and moves to its final posterior position. After GBR, the ectoderm has a gap on its dorsal side that is occupied by the amnioserosa (AS). The dorsal-most cells of the ectoderm, on both sides of the embryo, are in contact with the AS and are called leading edge (LE) cells. During DC, LE cells direct the movement of the epidermal sheets migrating dorsally over the apical side of the AS until they meet and fuse at the dorsal midline. Attention of this study centered on scarf on the basis of its embryonic expression pattern. Two piggyBac insertions, scarf PBss(GFP) and scarf M13.M2(lacZ), were expressed at a high level in LE cells during GBR and DC and at a low level in AS cells. The expression of scarf was confirmed by in situ hybridization. It was also expressed in the head and tail regions, and in the ventral ectoderm in a segmental pattern (Sorrosal, 2010).

    The JNK pathway is activated in LE cells and leads to the expression of the signalling molecule Dpp and the phosphatase Puckered (Puc). The scarf gene was expressed in the same cells as puc and dpp at the LE. Reduced JNK - Basket (Bsk) in Drosophila - or expression of a dominant-negative version of Bsk or Puc, which mediates a feedback loop repressing JNK activity, led to the loss of scarf expression in LE cells. Loss of puc activity, which leads to increased levels of JNK activity, or expression of an activated version of JNK -activating kinase (Hemipterous in Drosophila), led to the expansion of the scarf expression domain throughout the lateral ectoderm. Dorsal cells had a stronger response to increased JNK, suggesting that JNK requires the activity of another factor expressed in this region to induce scarf. As stated, the JNK cascade drives the expression of the secreted molecule Dpp. In embryos mutant for the Dpp receptor Thickveins, scarf expression at the LE was not lost and ectopic activation of the Dpp pathway did not induce the ectopic expression of scarf. These results indicate that scarf expression is induced by JNK in LE cells (Sorrosal, 2010).

    Homozygosis or trans-heterozygosis for the piggyBac insertions scarf PBss and scarf M13.M2 caused semi-lethality and survivors had scars on the head, phenotypes that were reverted by precise excision of the transposon elements. As embryos showed no obvious cuticle phenotype, stronger alleles of scarf were generated by imprecise excision of a P element located in the third intron of scarf (scarf KG05129). One allele (scarf Δ1.5) was isolated that led to the loss of scarf messenger RNA expression. This is an embryonic lethal allele and trans-heterozygous combinations of scarf Δ1.5 or Df(2R)nap14, a deficiency that uncovers the scarf locus, with scarf PBss or scarf M13.M2 were semi-lethal and resulted in scars on the head. Homozygous animals for scarf Δ1.5 or Df(2R)nap14 showed similar phenotypes. Their larval cuticles were either dorsally wrinkled or presented a dorsal hole or a U-shaped phenotype, which is suggestive of failures in GBR and DC. A high proportion of embryos showed undifferentiated cuticles. Ubiquitous expression of scarf largely rescued the scarf Δ1.5 phenotype (Sorrosal, 2010).

    Time-lapse recordings indicated that these embryos were delayed in development and showed a failure in GBR. Frequently, the attachment of the AS to the tail end of the germ band was compromised and the germ band did not retract. In embryos that were able to retract, DC started and LE cells began to direct the movement of the epidermal sheets migrating dorsally over the apical side of the AS. However, either the AS detached from the neighbouring epidermal sheet or the epidermal sheets met and fused at the dorsal midline but the dorsal epidermis reopened. The requirement of scarf in DC was analyzed in greater detail in fixed staged embryos. During the initial steps of DC, lateral ectodermal cells start to elongate dorsally and an actin cable at the LE is formed, which helps to generate a linear fence at the interface between LE and AS cells. In scarf Δ1.5 embryos, elongation of the lateral ectoderm was compromised frequently, the actin cable was not formed properly, the interface between LE and AS cells became irregular, and eventually rips appeared at the AS-LE interface. The elongation of lateral ectodermal cells and adhesion between AS and LE cells are mediated by the activity of Dpp and JNK activity controls the polymerization of actin into a cable at the LE. In embryos lacking scarf, the activity of the Dpp and JNK signalling pathways was not affected. Thus, Scarf has a role in DC without affecting the activity levels of these pathways (Sorrosal, 2010).

    Integrins and laminins have a fundamental role in GBR and DC by mediating interactions between AS cells and neighbouring cell populations. As defects were observed in these processes on scarf depletion and a reduction in scarf levels increased the frequency of cuticle phenotypes caused by depletion of the β-integrin position-specific (βPS) subunit, the contribution of Scarf to the regulation of the expression levels and subcellular localization of these proteins was examined. Cross-sectional views of properly staged wild-type and scarf Δ1.5 embryos stained for βPS integrin revealed similar levels and localization of integrins. The JNK signalling regulates the expression of the βPS integrin subunit during DC. Although ectopic activation of JNK induced increased expression of βPS integrin, ectopic expression of scarf did not exert this effect. The expression of LanA, one of the two existing fly α-laminins. In wild-type embryos, high levels of LanA were localized at the BM of AS cells. Interestingly, scarf embryos revealed strong defects in the polarized distribution of LanA. It was localized on the apical side of the AS epithelium and its protein levels were largely reduced in the BM. Ubiquitous expression of scarf largely rescued the levels of LanA at the BM. Similar defects were observed in the polarized distribution of LanA in the lateral ectoderm of scarf mutant embryos, and these defects were also rescued largely by the ubiquitous expression of scarf. Apico-basal polarity of AS cells, visualized by the basal localization of βPS integrin and the localization of E-cadherin (E-cad) at the adherens junctions, was not affected in hypomorphic scarf PBss and scarf M13.M2 embryos and in some scarf Δ1.5 embryos even though LanA was localized on the apical side. However, scarf depletion was frequently found to cause defects in the apico-basal polarity of AS cells and this was accompanied by multilayering of the AS epithelium. These observations suggest that before inducing a loss of epithelial integrity, the absence of Scarf caused aberrant localization of LanA on the apical side of the AS epithelium and reduced LanA in the BM, without affecting the distribution of apical and baso-lateral proteins. As integrins are involved in maintaining epithelial polarity, these results suggest that the observed loss of cell polarity is a consequence of reduced integrin-mediated adhesion to the BM. Consistent with this view, defects in the attachment of the AS cells to the underlying yolk cell were observed frequently (Sorrosal, 2010).

    Although the defects observed in scarf Δ1.5 embryos resemble those caused by βPS integrin depletion, only defects in the attachment of the AS with the underlying yolk cells are found in lanA mutant embryos. Mutations in wing blister (wb), the only other fly α-laminin, cause defects in GBR and in the attachment between AS cells and the posterior germ band. These data suggest that LanA and Wb have a redundant function and that Scarf most probably promotes BM localization of not only LanA but also Wb. Consistent with the proposed redundancy, halving the dose of lanA or wb increased the frequency of the βPS integrin loss-of-function cuticle phenotype. The expression of Wb protein in AS cells using the available antibodies and the role of Scarf in the polarized distribution of other BM proteins, such as Perlecan or Collagen IV, were not addressed as these two proteins were not expressed in AS cells (Sorrosal, 2010).

    These results indicate that Scarf depletion causes defects in the BM localization of LanA and in epithelial apico-basal polarity. The defects resemble those observed in follicle cells (that is, epithelial cells covering the fly oocyte) mutant for crag, a gene encoding for a protein localized in early and recycling endosomes and proposed to regulate protein transport and membrane deposition of BM proteins. Zygotic removal of crag induced anomalous localization of LanA on the apical side of AS cells. Zygotic or both maternal and zygotic removal of crag produced cuticles with a weak dorsally wrinkled phenotype, suggesting that Crag has a redundant function with another protein in the fly embryo and that the low BM levels of LanA observed in these embryos are sufficient to exert their function. Interestingly, halving the dose of scarf expression gave rise to a large proportion of crag cuticles with phenotypes similar to those caused by scarf depletion (Sorrosal, 2010).

    Antibodies against Scarf were raised to analyse its subcellular localization. As the antibodies did not work properly at embryonic stages and did not detect endogenous levels of Scarf protein, the wing imaginal disc, a monolayered epithelium, was used to analyse its subcellular localization. Scarf was not detected in wild-type wing cells. When scarf and green fluorescent protein (GFP) were expressed in a restricted domain in the wing disc, Scarf was detected not only in the GFP-labelled scarf-producing cells, but also on the apical side of the epithelium at long distances from the source. When atrial natriuretic factor-GFP (a fusion between the secreted rat atrial natriuretic peptide and GFP) was expressed, the protein was also observed at long distances from the source; however, it did not accumulate on the apical side of the epithelium. Scarf was found to localize in small punctate structures throughout the cytoplasm of scarf non-producing cells. These structures corresponded either to Rab5-positive early endosomes, to Rab7-positive late endosomes, or to Rab11-positive recycling endosomes. Together, these results indicate that Scarf is secreted apically and is internalized through endocytosis by non-producing cells. To confirm that Scarf is a secreted protein, Drosophila S2 cells were transfected with either a scarf-myc-tagged transgene or a transgene driving the expression of a myc-tagged membrane-tethered form of Scarf (Scarf-CD2). Scarf was isolated not only from the protein extract of the scarf-myc-expressing cells but also from the supernatant. Transfected Scarf-CD2 and endogenous actin were not observed in the supernatant (Sorrosal, 2010).

    This study has characterized the role of scarf, a JNK-regulated gene, in GBR and DC, two morphogenetic processes that rely on cell-matrix interactions between the AS epithelium and neighbouring cell populations. Evidence that Scarf is a secreted protein expressed in LE cells and involved in promoting the localized accumulation of LanA in the BM of AS cells. Although low levels of Scarf were detected in AS cells, the cuticle phenotype of scarf mutant embryos and the defects observed in BM localization of LanA and epithelial integrity of AS cells were rescued largely by driving expression of a scarf transgene only in ectodermal cells, indicating that Scarf is acting as a secreted protein. Three alternative hypotheses might explain the role of Scarf and Crag in the polarized localization of LanA to the BM. As Crag and Scarf are localized specifically on the apical side of the epithelium, they would have a repulsive role, inhibiting the targeting of LanA-containing vesicles with apical membranes. Alternatively, LanA might be secreted apically and basally and be stabilized preferentially on the basal side or degraded on the apical side. As Scarf encodes for a putative serine protease-like protein without catalytic activity, Scarf would function, in this scenario, to facilitate the effect of a cascade of serine proteases involved in the degradation of LanA in the apical domain of epithelial cells. Finally, in scarf mutant cells, LanA might be transported from the basal to the apical side through transcytosis, a mechanism responsible for the formation of a small apical BM cap over pre-invasive epithelial cells during Drosophila oogenesis (Sorrosal, 2010).

    Another secreted serine protease-like protein without catalytic activity, Masquerade, regulates cell-matrix interactions at the somatic-muscle attachment in the fly embryo, a process that also depends on integrin and laminin activity. It is speculated that a number of non-functional serine protease-like proteins, such as Masquerade and Scarf, are regulated temporally and spatially to promote the appropriate localization of BM proteins in a context-dependent manner to facilitate cell-matrix interactions and to ensure the maintenance of epithelial integrity. Similarly, among the large class of vertebrate functional and non-functional serine protease-like proteins involved in remodelling the extracellular matrix, some of them might exert a similar action (Sorrosal, 2010).

    Cellular mechanics of germ band retraction in Drosophila

    Germ band retraction involves a dramatic rearrangement of the tissues on the surface of the Drosophila embryo. As germ band retraction commences, one tissue, the germ band, wraps around another, the amnioserosa. Through retraction the two tissues move cohesively as the highly elongated cells of the amnioserosa contract and the germ band moves so it is only on one side of the embryo. To understand the mechanical drivers of this process, a series of laser ablations was designed that suggest a mechanical role for the amnioserosa. first, it was found that during mid retraction, segments in the curve of the germ band are under anisotropic tension. The largest tensions are in the direction in which the amnioserosa contracts. Second, ablating one lateral flank of the amnioserosa reduces the observed force anisotropy and leads to retraction failures. The other intact flank of amnioserosa is insufficient to drive retraction, but can support some germ band cell elongation and is thus not a full phenocopy of ush mutants. Another ablation-induced failure in retraction can phenocopy mys mutants, and does so by targeting amnioserosa cells in the same region where the mutant fails to adhere to the germ band. It is concluded that the amnioserosa must play a key, but assistive, mechanical role that aids uncurling of the germ band (Lynch, 2013).

    Elongated cells drive morphogenesis in a surface-wrapped finite-element model of germband retraction

    During Drosophila embryogenesis, the germband first extends to curl around the posterior end of the embryo and then retracts back; however, retraction is not simply the reversal of extension. At a tissue level, extension is coincident with ventral furrow formation, and at a cellular level, extension occurs via convergent cell neighbor exchanges in the germband, whereas retraction involves only changes in cell shape. This study investigated this process using a whole-embryo, surface-wrapped cellular finite-element model. This model represents two key epithelial tissues-amnioserosa and germband-as adjacent sheets of two-dimensional cellular finite elements that are wrapped around an ellipsoidal three-dimensional approximation of an embryo. The model reproduces the detailed kinematics of in vivo retraction by fitting just one free model parameter, the tension along germband cell interfaces; all other cellular forces are constrained to follow ratios inferred from experimental observations. With no additional parameter adjustments, the model also reproduces quantitative assessments of mechanical stress using laser dissection and failures of retraction when amnioserosa cells are removed via mutations or microsurgery. Surprisingly, retraction in the model is robust to changes in cellular force values but is critically dependent on starting from a configuration with highly elongated amnioserosa cells. Their extreme cellular elongation is established during the prior process of germband extension and is then used to drive retraction. The amnioserosa is the one tissue whose cellular morphogenesis is reversed from germband extension to retraction, and this reversal coordinates the forces needed to retract the germband back to its pre-extension position and shape. In this case, cellular force strengths are less important than the carefully established cell shapes that direct them (McCleery, 2019).

    Cytocortex-dependent dynamics of Drosophila Crumbs controls junctional stability and tension during germ band retraction

    During morphogenesis epithelia undergo dynamic rearrangements, which requires continuous remodelling of junctions and cell shape, but at the same time mechanisms preserving cell polarity and tissue integrity. Apico-basal polarity is key to localise the machinery that enables cell shape changes. The evolutionarily conserved Drosophila Crumbs protein is critical for maintaining apico-basal polarity and epithelial integrity. How Crumbs is maintained in a dynamically developing embryo remains largely unknown. In this study quantitative fluorescence techniques were applied to show that during germ band retraction, Crumbs dynamics correlates with the morphogenetic activity of the epithelium. Genetic and pharmacological perturbations revealed that the mobile pool of Crumbs is fine-tuned by the actomyosin cortex in a stage dependent manner. Stabilisation of Crumbs at the plasma membrane depends on a proper link to the actomyosin cortex via an intact FERM-domain binding site in its intracellular domain, loss of which leads to increased junctional tension and higher DE-cadherin turnover, resulting in impaired junctional rearrangements. These data define Crumbs as a mediator between polarity and junctional regulation to orchestrate epithelial remodelling in response to changes in actomyosin activity (Bajur, 2019).


    Bajur, A. T., Iyer, K. V. and Knust, E. (2019). Cytocortex-dependent dynamics of Drosophila Crumbs controls junctional stability and tension during germ band retraction. J Cell Sci. PubMed ID: 31300472

    Lynch, H. E., Crews, S. M., Rosenthal, B., Kim, E., Gish, R., Echiverri, K. and Hutson, M. S. (2013). Cellular mechanics of germ band retraction in Drosophila. Dev Biol. 384(2): 205-13. PubMed ID: 24135149

    McCleery, W. T., Veldhuis, J., Bennett, M. E., Lynch, H. E., Ma, X., Brodland, G. W. and Hutson, M. S. (2019). Elongated cells drive morphogenesis in a surface-wrapped finite-element model of germband retraction. Biophys J. PubMed ID: 31229244

    Schock, F. and Perrimon, N. (2002). Cellular processes associated with germ band retraction in Drosophila. Dev. Bio. 248: 29-39. PubMed ID: 12142018

    Sorrosal, G., Pérez, L., Herranz, H., and Milán, M. (2010). Scarface, a secreted serine protease-like protein, regulates polarized localization of laminin A at the basement membrane of the Drosophila embryo. EMBO Rep. 11(5): 373-9. PubMed ID: 20379222

    date revised: 15 September 2019

    Genes involved in organ development

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