The Crumbs protein is exclusively expressed on the apical membrane of all ectodermally derived epithelia concentrated at the borders between cells. Later, the protein can be detected on the apical and basolateral membranes (Knust, 1993 and Tepass, 1990).

The Crumbs mRNA is likewise concentrated at the apical periplasm. The expression of crumbs is correlated with epithelium with the exception of the peripheral nervous system. It is detectable at the blastoderm stage, and found in the stomatogastric nervous system as long as it displays an epithelial organization. The only non-epithelial organs expressing crumbs are the external sensory organs and the chordotonal organs. CRB is detectable for a short time in cells of the anterior and posterior midgut primordia (Tepass, 1990).

A Delta-Notch signaling border regulated by Engrailed/Invected repression specifies boundary cells in the Drosophila hindgut

The Drosophila hindgut develops three morphologically distinct regions along its anteroposterior axis: small intestine, large intestine and rectum. Single-cell rings of 'boundary cells' delimit the large intestine from the small intestine at the anterior, and the rectum at the posterior. The large intestine also forms distinct dorsal and ventral regions; these are separated by two single-cell rows of boundary cells. Boundary cells are distinguished by their elongated morphology, high level of both apical and cytoplasmic Crb protein, and gene expression program. During embryogenesis, the boundary cell rows arise at the juxtaposition of a domain of Engrailed- plus Invected-expressing cells with a domain of Delta (Dl)-expressing cells. Analysis of loss-of-function and ectopic expression phenotypes shows that the domain of Dl-expressing cells is defined by En/Inv repression. Further, Notch pathway signaling, specifically the juxtaposition of Dl-expressing and Dl-non-expressing cells, is required to specify the rows of boundary cells. This Notch-induced cell specification is distinguished by the fact that it does not appear to utilize the ligand Serrate and the modulator Fringe (Iwaki, 2002).

At its anterior, the hindgut joins the posterior midgut; at its posterior, it forms the anus. Along this AP axis, the hindgut of the mature embryo consists of three morphologically distinct domains: the wide, looping small intestine, the long and narrow large intestine, and the tapered rectum. Beginning at stage 13, these domains are demarcated at their junctions by rings of unusually high accumulation of the apical surface protein Crumbs (Crb). The ring at the small intestine/large intestine junction is designated the anterior boundary cell ring, and the ring at the large intestine/rectum junction is designated the posterior boundary cell ring (Iwaki, 2002).

Patterning of the hindgut in the DV axis is detected at stage 10 (germ band extension) when the hindgut develops an interiorly directed (dorsal) convexity. The side of the hindgut closest to the interior of the embryo is dorsal and expresses both En and Inv; that closest to the exterior is ventral and expresses dpp. By the completion of germ band retraction, the convexity at the anterior of the hindgut has shifted toward the left side of the embryo. Thus at the anterior of the hindgut, the initially dorsal, En- and Inv-expressing side comes to lie on the outer (left-facing) curve, while the initially ventral, Dpp-expressing side of the hindgut comes to lie on the inner (right-facing) curve; the DV relationship is retained at the posterior connection to the rectum. These initially DV patterned domains of the large intestine persist to the end of embryogenesis and into the larval stages; they are referred to as large intestine dorsal (li-d) and large intestine ventral (li-v). At each of the two boundaries between li-d and li-v, there is a single row of cells with high levels of Crb expression running the length of the large intestine, from the anterior boundary cell ring to the posterior boundary cell ring. These are designated the 'boundary cell rows'. In addition to their high level of Crb expression, the boundary cell rows and rings express the nuclear protein Dead ringer (Dri). Double antibody staining reveals that boundary cell rows at the border of the En/Inv-expressing li-d domain and the Dpp-expressing li-v domain express Dri in their nuclei and have strong Crb expression at their apical surfaces (Iwaki, 2002).

Interestingly, the Dri- and Crb-expressing boundary cells delimit both AP and DV boundaries in the hindgut. The rings form borders at the anterior and posterior ends of the large intestine, while the rows form borders between the dorsal (li-d) and ventral (li-v) regions of the large intestine. This study focusses primarily on the establishment and characteristics of the boundary cell rows (Iwaki, 2002).

Staining with both anti-Crb and anti-ßHEAVY Spectrin shows that the boundary cell rows are significantly more elongated along the AP axis than other hindgut epithelial cells. Staining of bynapro/+ embryos (containing a P-element insert in byn) with anti-ß-Gal antibody reveals that the nuclei of the cells of the boundary rows (identified by strong staining with anti-Crb) are also elongated in the AP axis (Iwaki, 2002).

The dramatically higher level of Crb expression in the boundary cells (both rings and rows) suggests that their apical surface may differ from that of other hindgut epithelial cells, and/or that, in the boundary cells, Crb may be present in cellular compartments in addition to the apical surface. Both of these expectations are borne out by a higher magnification examination of the boundary cells. In cross-sections of the large intestine viewed by electron microscopy, short microvilli on the apical surfaces of two cells on opposite sides of the hindgut lumen were observed; these cells most likely correspond to the boundary cell rows. The microvilli of the presumed boundary cell rows appear more organized and parallel than the irregular protrusions on the surfaces of the other cells of the hindgut epithelium. Because of their apical microvilli, the presumed boundary cell rows have a larger apical membrane surface and are expected to be labeled more strongly with anti-Crb. Consistent with this, cross-sections of anti-Crb-stained embryos viewed by light microscopy reveal two cells on opposite sides of the large intestine lumen with a higher level of Crb on their apical surfaces. In addition to their stronger apical labeling with anti-Crb, these presumed boundary cell rows also display an accumulation of Crb in their cytoplasm; this is strongest apical to the nucleus. The cytoplasmic accumulation of Crb suggests that Crb is produced at a higher level, or is more stable, in the boundary cells (Iwaki, 2002).

In conclusion, differences in gene expression demonstrate that the boundary cells are a separately patterned (fated) group of cells in the large intestine. The unique fate of the boundary cells is manifested both molecularly, in their expression of Dri and high cytoplasmic accumulation of Crb, and morphologically, in their marked AP elongation and development of apical microvilli (Iwaki, 2002).

The boundary cell rows form at the junction of the li-d and li-v domains, which express different genes. To investigate whether the spatially restricted gene expression observed in these domains is essential for establishment of boundary cell rows, embryos homozygous for loss-of-function alleles of en, inv, dpp, dri, Dl, Ser, Notch, or fng were examined. The presence or absence of boundary cells was assessed by anti-Crb staining, since this delineates their characteristic morphology, and also detects one of their unique differentiated features (i.e. the cytoplasmic accumulation of Crb) (Iwaki, 2002).

The data presented here support the following model. En/Inv is expressed in li-d and represses Dl in that domain; Dl expression is thereby restricted to the li-v domain. At the li-v/li-d transition, the Dl-expressing cells induce, by Notch signaling, a row of Dl-non-expressing cells to become a boundary cell row. Since En/Inv is not detected in differentiated boundary cells, Notch activation likely represses En/Inv expression. Notch activation also leads to Dri expression and an upregulation of Crb expression. While all of these transcriptional changes could be mediated by Su(H), they could also be further downstream (Iwaki, 2002).

Role of spastin in apical crumbs domain control along the rhabdomere elongation in Drosophila photoreceptor

Mutations in spastin are the most common cause of hereditary spastin paraplegia, a neurodegenerative disease. In this study, the role of spastin was examined in Drosophila photoreceptor development. The spastin mutation in developing pupal eyes causes a mild mislocalization of the apical membrane domain at the distal section, but the apical domain was dramatically reduced at the proximal section of the developing pupal eye. Since the rhabdomeres in developing pupal eyes grow from distal to proximal, this phenotype strongly suggests that spastin is required for apical domain maintenance during rhabdomere elongation. This role of spastin in apical domain modulation was further supported by spastin's gain-of-function phenotype. Spastin overexpression in photoreceptors caused the expansion of the apical membrane domain from apical to basolateral in the developing photoreceptor. Although the localizations of the apical domain and adherens junctions (AJs) were severely expanded, there were no defects in cell polarity. These results strongly suggest that spastin is essential for apical domain biogenesis during rhabdomere elongation in Drosophila photoreceptor morphogenesis (Chen, G. 2010).

In animal photoreceptor cells, the surface membrane is enlarged for the storage of opsin photopigment. Insect eyes use an actin-based structure for surface membrane enlargement, but mammalian eyes use microtubule-based structure. This study examined whether the Drosophila photoreceptor cells may have any microtubule-based structures. There is a distinctive structure which is specifically labeled by anti-acetylated-tubulin antibody in the developing photoreceptors of Drosophila. Given the specific localization of stable microtubules in developing pupal Drosophila photoreceptors, these subcellular structures might provide a functional role for photoreceptor morphogenesis (Chen, G. 2010).

Spastin is an ATPase that binds microtubules and localizes to the spindle pole and distal axon in mammalian cell lines. Furthermore, its Drosophila homolog, Drosophila spastin, has been shown to regulate microtubule stability and synaptic function at the Drosophila larval neuromuscular junction. Genetic analysis of the spastin mutation strongly indicates that spastin not only modulates the microtubules, but also modulates the apical Crb membrane domain during rhabdomere elongation. The apical membrane modulation activity of spastin was further confirmed by spastin overexpression which caused a dramatic expansion of the apical membrane domain (Chen, G. 2010).

Based on the highly concentrated localization of Spastin in the apical domains of the photoreceptors, it is proposed that the apically localized Spastin might control the apical Crb domains. This apical domain-specific function of Spastin is based on the following results; (1) Apical domain localization of Spastin, (2) Loss of spastin causes apical domain defects, and (3) Overexpression of spastin causes apical domain expansions. But, another possibility of the direct modulation of stable microtubules by Spastin cannot be excluded. Any subtle changes in stable microtubules by spastin might affect potential trafficking machinery which is responsible for the apical Crb targeting. But, these two possibilities are not necessarily mutually exclusive (Chen, G. 2010).

Spastin has microtubule-severing activities in vitro. Therefore, microtubule-severing activity of Spastin may facilitate the apical Crb domain, since loss of spastin caused loss of Crb, and gain of spastin caused the expansion of the Crb domain. Furthermore, this facilitating activity of Spastin for the apical domain could be independent from the main stable microtubule structures which are located far beneath the apical domains. This possibility is supported by the more direct influence on apical Crb, as the stable microtubules were not dramatically changed, relatively speaking, by either spastin mutants or spastin overexpression (Chen, G. 2010).

During the massive growth of the rhabdomeres in the pupal retina, many membrane materials, including Crb, will be targeted into the growing apical membranes. Spastin may participate in this material transport to the apical membrane domain during rhabdomere growth, although the initial targeting is spastin-independent. The outcome of this study will provide useful information for understanding the molecular genetic mechanism of spastic paraplegia. Although the spastin mutation subtly affects the main microtubules, this genetic approach will provide more convincing clues concerning the microtubule-based processes in photoreceptor morphogenesis (Chen, G. 2010).

Thus, analysis of the microtubule-modulating Spastin in Drosophila photoreceptors may provide important insights into the understanding of the functional basis of the microtubule-based structure and the microtubule-related genes involved the formation and development of photoreceptors. Evolutionary conservation in the structure and function of eye morphogenesis genes makes the Drosophila eye an excellent model for studying the genetic and molecular basis of retinal cell organization (Chen, G. 2010).

Future work will help to uncover other genes that might affect the microtubule cytoskeleton and cell polarity targeting during the extensive membrane growth phase of the pupal eye. Determining the role of Spastin in photoreceptor development will help in understanding retinitis pigmentosa, spastic paraplegia and other retinal degenerative diseases that involve mutations in crb and spastin. The finding that human mutations in CRB1 lead to retinitis pigmentosa emphasizes the importance of deciphering the molecular networks associated with Crb in the apical membrane domain of the Drosophila photoreceptor (Chen, G. 2010).

In summary, this study has examined the role of Spastin in the regulation of the apical Crb domain in developing photoreceptors. The data strongly suggests that Spastin plays important functions in the modulation of cell membrane domains including the apical domains of photoreceptors during pupal eye development. Because proper maintenance of the apical Crb domain is important for the massive growth observed in rhabdomeres at the apical region of photoreceptor cells, malfunction of spastin results in severe defects in the formation of functional photoreceptors (Chen, G. 2010).

The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth

Disruption of epithelial polarity is a key event in the acquisition of neoplastic growth. JNK signalling is known to play an important part in driving the malignant progression of many epithelial tumours, although the link between loss of polarity and JNK signalling remains elusive. In a Drosophila genome-wide genetic screen designed to identify molecules implicated in neoplastic growth, this study identified grindelwald (grnd; CG10176), a gene encoding a transmembrane protein with homology to members of the tumour necrosis factor receptor (TNFR) superfamily. This study shows that Grnd mediates the pro-apoptotic functions of Eiger (Egr), the unique Drosophila TNF, and that overexpression of an active form of Grnd lacking the extracellular domain is sufficient to activate JNK signalling in vivo. Grnd also promotes the invasiveness of RasV12/scrib-/- tumours through Egr-dependent Matrix metalloprotease-1 (Mmp1) expression. Grnd localizes to the subapical membrane domain with the cell polarity determinant Crumbs (Crb) and couples Crb-induced loss of polarity with JNK activation and neoplastic growth through physical interaction with Veli (also known as Lin-7). Therefore, Grnd represents the first example of a TNFR that integrates signals from both Egr and apical polarity determinants to induce JNK-dependent cell death or tumour growth (Andersen, 2015).

A genome-wide screen was carried to identify molecules that are required for neoplastic growth. The condition used for this screen was the disc-specific knockdown of avalanche, also known as syntaxin 7), a gene encoding a syntaxin that functions in the early step of endocytosis2. avl-RNAi results in ectopic Wingless (Wg) expression, neoplastic disc overgrowth, and a 2-day delay in larva-to-pupa transition. A collection of 10,100 transgenic RNA interference (RNAi) lines were screened for their ability to rescue the pupariation delay, and 121 candidate genes were identified. Interestingly, only eight candidate genes also rescued ectopic Wg expression and neoplastic overgrowth. These included five lines targeting core components of the JNK pathway (Bendless, Tab2, Tak1, Hemipterous and Basket. Using a puckered enhancer trap (puc-lacZ) as a readout for JNK activity, it was confirmed that JNK signalling is highly upregulated in avl-RNAi discs. One of the remaining lines targets CG10176, a gene encoding a transmembrane protein. Reducing expression of CG10176 by using two different RNAi lines was as efficient as tak1 silencing to restore normal Wg pattern and suppresses JNK signalling and neoplastic growth in the avl-RNAi background. Sequence analysis of GC10176 identified a cysteine-rich domain (CRD) in the extracellular part with homology to vertebrate TNFRs harbouring a glycosphingolipid-binding motif (GBM) characteristic of many TNFRs including Fas. CG10176 was named grindelwald (grnd) , after a village at the foot of Eiger, a Swiss mountain that lent its name to the unique Drosophila TNF, Egr. Immunostaining and subcellular fractionation of disc extracts confirmed that Grnd localizes to the membrane. Moreover, co-immunoprecipitation experiments showed that both Grnd full-length and Grnd-intra, a form lacking its extracellular domain, directly associate with Traf2, the most upstream component of the JNK pathway. This interaction is disrupted by a single amino acid substitution within a conserved Traf6-binding motif (human TRAF6 is the closest homologue to Traf2. Overexpression of Grnd-intra, but not full-length Grnd, is sufficient to induce JNK signalling, ectopic Wg expression and apoptosis, and Grnd-intra-induced apoptosis is efficiently suppressed in a hep (JNKK) mutant background, confirming that Grnd acts upstream of the JNK signalling cascade (Andersen, 2015).

The Drosophila TNF Egr activates JNK signalling and triggers cell death or proliferation, depending on the cellular context. Therefore tests were performed to see whether Grnd is required for the small-eye phenotype generated by Egr-induced apoptosis in the retinal epithelium (via Egr overexpression). Inhibition of JNK signalling by reducing tak1 or traf2 expression, or by overexpressing puckered, blocks Egr-induced apoptosis and rescues the small-eye phenotype. In contrast to a previous report, RNAi silencing of wengen (wgn) , a gene encoding a presumptive receptor for Egr, does not rescue the small-eye phenotype. Furthermore, the small-eye phenotype is not modified in a wgn-null mutant background, confirming that Wgn is not required for Egr-induced apoptosis in the eye. By contrast, reducing grnd levels partially rescues the Egr-induced small-eye phenotype, producing a 'hanging-eye' phenotype that is not further rescued in a wgn-knockout mutant background. A similar phenotype was previously reported as a result of non-autonomous cell death induced by a diffusible form of Egr. This suggests that Grnd prevents Egr from diffusing outside of its expression domain. Co-immunoprecipitation experiments show that both full-length Grnd and Grnd-extra, a truncated form of Grnd lacking the cytoplasmic domain, associate with Egr through its TNF-homology domain. Although Grnd-extra can bind Egr, it cannot activate JNK signalling. Therefore, it was reasoned that Grnd-extra expression might prevent both cell-autonomous and non-autonomous apoptosis by trapping Egr and preventing its diffusion and binding to endogenous Grnd. Indeed, GMR-Gal4-mediated expression of grnd-extra fully rescues the Egr small-eye phenotype. To confirm that the removal of Grnd induces Egr-mediated non-autonomous cell death, wing disc clones were generated expressing egr alone, egr + tak1 RNAi, or egr + grnd RNAi. As expected, reducing tak1 levels in egr-expressing clones prevents their elimination by apoptosis. Similarly, reducing grnd levels prevents autonomous cell death, but also induces non-autonomous apoptosis. This suggests that Egr, like its mammalian counterpart TNF-α, can be processed into a diffusible form in vivo whose interaction with Grnd limits the potential to act at a distance. Flies carrying homozygous (grndMinos/Minos) or transheterozygous (grndMinos/Df) combinations of a transposon inserted in the grnd locus express no detectable levels of Grnd protein and are equally resistant to Egr-induced cell death. In addition, grndMinos/Minos mutant flies are viable and display no obvious phenotype, suggesting that Grnd, like Egr, participates in a stress response to limit organismal damage. Collectively, these data demonstrate that Grnd is a new Drosophila TNF receptor that mediates most, if not all, Egr-induced apoptosis (Andersen, 2015).

TNFs probably represent a danger signal produced in response to tissue damage to rid the organism of premalignant tissue or to facilitate wound healing. Disc clones mutant for the polarity gene scribbled (scrib) induce an Egr-dependent response resulting in the elimination of scrib mutant cells by JNK-mediated apoptosis. To test the requirement for Grnd in this process, scrib-RNAi and scrib-RNAi + grnd-RNAi clones obtained 72 h after heat shock induction were compared. As expected, scrib-RNAi cells undergo apoptosis and detach from the epithelium. By contrast, scrib-RNAi clones with reduced grnd expression survive, indicating that Grnd is required for Egr-dependent elimination of scrib-RNAi cells. Similar results were obtained by generating scrib mutant clones in the eye disc (Andersen, 2015).

In both mammals and flies, TNFs are double-edged swords that also have the capacity to promote tumorigenesis in specific cellular contexts. Indeed, scrib minus eye disc cells expressing an activated form of Ras (RasV12) exhibit a dramatic tumour-like overgrowth and metastatic behaviour, a process that critically relies on Egr. RasV12/scrib-/- metastatic cells show a strong accumulation of Grnd and Mmp1, and invade the ventral nerve cord. Primary tumour cells reach peripheral tissues such as the fat body and the gut, where they form micro-metastases expressing high levels of Grnd. Reducing grnd levels in RasV12/scrib-/- clones is sufficient to restore normal levels of Mmp1 and abolish invasiveness in a way similar to that observed in an egr mutant background. Therefore, Grnd is required for the Egr-induced metastatic behaviour of RasV12/scrib-/- tumorous cells. Similarly, reducing grnd, but not wgn levels, strongly suppresses Mmp1 expression in RasV12/dlg-RNAi cells and limits tumour invasion, indicating that Wgn does not have a major role in the progression of these tumours (Andersen, 2015).

Perturbation of cell polarity is an early hallmark of tumour progression in epithelial cells. In contrast to small patches of polarity-deficient cells, for example, scrib mutant clones, organ compartments or animals fully composed of polarity-deficient cells become refractory to Egr-induced cell death and develop epithelial tumours. The formation of these tumours requires JNK/MAPK signalling, but not Egr, suggesting Egr-independent coupling between loss of polarity and JNK/MAPK-dependent tumour growth. In line with these observations, it was noticed that, in contrast to Grnd, Egr is not required to drive neoplastic growth in avl-RNAi conditions. This suggests that, in addition to its role in promoting Egr-dependent functions, Grnd couples loss of polarity with JNK-dependent growth independently of Egr. Disc immunostainings revealed that Grnd co-localizes with the apical determinant Crb in the marginal zone, apical to the adherens junction protein E-cadherin (E-cad) and the atypical protein kinase C (aPKC). In avl-RNAi discs, Grnd and Crb accumulate in a wider apical domain. Apical accumulation of Crb is proposed to be partly responsible for the neoplastic growth induced by avl knockdown, since overexpression of Crb or a membrane-bound cytoplasmic tail of Crb (Crb-intra) mimics the avl-RNAi phenotype. Therefore whether Grnd might couple the activity of the Crb complex with JNK-mediated neoplastic growth was examined. Indeed, reducing grnd levels, but not wgn, in ectopic crb-intra discs suppresses neoplastic growth as efficiently as inhibiting the activity of the JNK pathway. Notably, Yki activation is not rescued in these conditions, illustrating the ability of Crb-intra to promote growth independently of Grnd by inhibiting Hippo signalling through its FERM-binding motif (FBM). Indeed, neoplastic growth and polarity defects induced by a form of Crb-intra lacking its FBM (CrbΔFBM-intra) are both rescued by Grnd silencing. As expected, the size of ectopic crbΔFBM-intra;grnd-RNAi discs is reduced compared to the size of ectopic crb-intra; grnd-RNAi discs (Andersen, 2015).

Crb, Stardust (Sdt; PALS1 in humans), and Pals1-associated tight junction protein (Patj) make up the core Crb complex, which recruits the adaptor protein Veli (MALS1-3 in humans). In agreement with previous yeast two-hybrid data, this study found that Grnd binds directly and specifically to the PDZ domain of Veli through a membrane-proximal stretch of 28 amino acids in its intracellular domain. Grnd localization is unaffected in crb and veli RNAi mutant clones. However, reducing veli expression rescues the patterning defects and disc morphology of ectopic crb-intra mutant cells, suggesting that Grnd couples Crb activity with JNK signalling through its interaction with Veli. Interestingly, aPKC-dependent activation of JNK signalling also depends on Grnd. aPKC is capable of directly binding and phosphorylating Crb, which is important for Crb function. This suggests that aPKC, either directly or through Crb phosphorylation, activates Grnd-dependent JNK signalling in response to perturbation of apico-basal polarity (Andersen, 2015).

These data are consistent with a model whereby Grnd integrates signals from Egr, the unique fly TNF, and apical polarity determinants to induce JNK-dependent neoplastic growth or apoptosis in a context-dependent manner. Recent work reveals a correlation between mammalian Crb3 expression and tumorigenic potential in mouse kidney epithelial cells. The conserved nature of the Grnd receptor suggests that specific TNFRs might carry out similar functions in vertebrates, in which the link between apical cell polarity and tumour progression remains elusive (Andersen, 2015).

The transmembrane protein Crumbs displays complex dynamics during follicular morphogenesis and is regulated competitively by Moesin and aPKC

The transmembrane protein Crumbs (Crb) functions in apical polarity and epithelial integrity. To better understand its role in epithelial morphogenesis, this study examined Crb localization and dynamics in the late follicular epithelium of Drosophila. Crb was unexpectedly dynamic during middle-to-late stages of egg chamber development, being lost from the marginal zone (MZ) in stage 9 before abruptly returning at the end of stage 10b, then undergoing a pulse of endocytosis in stage 12. The reappearance of MZ Crb was necessary to maintain an intact adherens junction and MZ. Although Crb has been proposed to interact through its juxtamembrane domain with Moesin (Moe), a FERM domain protein that regulates the cortical actin cytoskeleton, the functional significance of this interaction is poorly understood. This study found that whereas the Crb juxtamembrane domain was not required for adherens junction integrity, it was necessary for MZ localization of Moe, aPKC and F-actin. Furthermore, Moe and aPKC functioned antagonistically, suggesting that Moe limits Crb levels by reducing its interactions with the apical Par network. Additionally, Moe mutant cells lost Crb from the apical membrane and accumulated excess Crb at the MZ, suggesting that Moe regulates Crb distribution at the membrane. Together, these studies reveal reciprocal interactions between Crb, Moe and aPKC during cellular morphogenesis (Sherrard, 2015).

Crumbs is an essential regulator of cytoskeletal dynamics and cell-cell adhesion during dorsal closure in Drosophila

The evolutionarily conserved Crumbs protein is required for epithelial polarity and morphogenesis. This study identified a novel role of Crumbs as a negative regulator of actomyosin dynamics during dorsal closure in the Drosophila embryo. Embryos carrying a mutation in the FERM (protein 4.1/ezrin/radixin/moesin) domain-binding motif of Crumbs die due to an overactive actomyosin network associated with disrupted adherens junctions. This phenotype is restricted to the amnioserosa and does not affect other embryonic epithelia. This function of Crumbs requires DMoesin, the Rho1-GTPase, class-I p21-activated kinases and the Arp2/3 complex. Data presented here point to a critical role of Crumbs in regulating actomyosin dynamics, cell junctions and morphogenesis (Flores-Benitez, 2015).

Effects of Mutation or Deletion

The insertion of Crumbs into the plasma membrane is both necessary and sufficient to confer apical character on a membrane domain. Overexpression of crumbs results in an enormous expansion of the apical plasma membrane and the concomitant reduction of the basolateral domain. This is followed by the redistribution of beta Heavy-spectrin, a component of the membrane cytoskeleton, and by the ectopic deposition of cuticle and other apical components into these areas. Strikingly, overexpression of the membrane-bound cytoplasmic portion of crumbs alone is sufficient to produce this dominant phenotype (Wodarz, 1995).

A mutation in crb deleting a small cytoplasmic region, behaves as a null allele. In embryos homozygous for crb8F105, the Crumbs protein is diffusely distributed in the cytoplasm instead of being apically localized as in wild-type; this mislocation occurs before any morphologically detectable cellular phenotype becomes manifest, suggesting that apical targeting of proteins is affected in crb mutant embryos. This resulet suggests that the polarizing ativity of Crumbs resides in its cytoplasmic domain (Wodarz, 1993).

Mutations in crumbs lead to severe disruptions in the organization of ectodermally derived epithelia and in some cases to cell death (see Reaper) in these tissues (Tepass, 1990).

Loss-of-function mutations in the Drosophila genes crumbs and stardust are embryonic lethal and cause a breakdown of ectodermally derived epithelia during organogenesis, leading to formation of irregular cell clusters and extensive cell death in some epithelia. The mutant phenotype develops gradually and affects, to different extents, the various epithelia. Mutations in stardust produce a phenotype nearly identical to that associated with crumbs mutations, suggesting that both genes are functionally related. Double mutant combinations and gene dosage studies suggest that both genes are part of a common genetic pathway, in which stardust acts downstream of crumbs. The gene function is completely abolished by a crumbs mutation that causes production of a protein with a truncated cytoplasmic domain. Instead of being apically localized as in wild-type, the mutant Crumbs protein is diffusely distributed in the cytoplasm. This occurs before any morphologically detectable cellular phenotype is visible, suggesting that targeting of proteins is affected in crumbs mutant embryos. (Knust, 1993).

In sdt mutant embryos CRB is present only during gastrulation, but becomes undetectable during germ band extension [Image]; the protein is again visible during early organogenesis, at the time when the sdt mutant phenotype becomes apparent. In sdt mutant embryos, CRB is associated with the apical membrane only in well-differentiated epithelial cells, but it is expressed diffusely in the cytoplasm of cells which have lost epithelial morphology. Mosaic experiments suggest that sdt is required cell autonomously, in contrast to the crb requirement, which appears to be non-cell-autonomous. It seems that sdt acts downstream of crb and is activated by the latter (Tepass, 1993).

Deletion of reaper protects embryos from apoptosis caused by x-irradiation and developmental defects. Mutation of the gene crumbs leads to widespread defects in the development of the epithelial tissues, followed by massive cell death during embryogenesis. reaper deletion blocks the massive ectopic death seen in crumbs mutant embryos (White, 1994).

The mutations bazooka and sdt belong to a group in which mutant embryos show severe abnormalities in the differentiation of the larval cuticles, including the genes crumbs (crb) and shotgun (shg). Although the similarity in the late phenotypes of these mutants shows that the respective genes are all required for the same process, i.e., epithelial differentiation, it is difficult to determine whether all these genes act in a common pathway. Nevertheless, the genes crb and sdt show an interesting genetic interaction. Using chromosomal duplications, it has been shown that the phenotype of crb (null) embryos can be rescued by an additional copy of sdt but not vice versa (Tepass, 1993). Based on these findings, a model has been proposed that positions sdt downstream of crb in a regulatory hierarchy (Tepass, 1993). This model is complicated by the fact that sdt regulates Crb protein distribution (Tepass, 1993). A more attractive model might be that sdt functions in a parallel pathway, and, in sufficient dosage, bypasses the requirement for crb (Muller, 1996).

Localization of apical epithelial determinants by the basolateral PDZ protein Scribble

Loss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. Cells in epithelial sheets are characterized by columnar or cuboidal shape, strong cell-cell adhesion, and pronounced apicobasal polarity. However, tumors of epithelial origin lose these characteristics as they progress from benign growth to malignant carcinoma, and this loss is associated with poor clinical prognosis. Evidence is provided that a group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation. Scribbled (Scrib) is a cell junction-localized protein required for polarization of embryonic and imaginal disc and follicular epithelia. The tumor suppressor scrib was isolated in a screen for maternal effect mutations that disrupt aspects of epithelial morphogenesis such as cell adhesion, shape and polarity. scrib encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein. The structure of the embryonic cuticle was used to reflect the organization of the underlying epithelial epidermis that secretes it. The wild-type cuticle forms a smooth, continuous sheet, but embryos that are maternally and zygotically mutant for scrib produce a corrugated cuticular surface that is riddled with holes, hence the name scribbled (Bilder, 2000).

To place Scrib within the known pathway for Drosophila epithelial polarity determination, the effect of scrib mutations on Crumbs (Crb) was examined. Crb is an apically localized transmembrane protein that is necessary and sufficient to confer apical character on plasma membrane. In scrib embryos, Crb shows unrestricted localization in both apical and basolateral regions. Whether scrib mutants are identical to a gain-of-function crb phenotype was examined by comparing them with embryos in which GAL4-driven Crb is present throughout the cell membrane. Ectopic Crb that is produced in this manner is sufficient to phenocopy several aspects of scrib embryos, including mislocalization of apical proteins and the cuticle pattern. These data indicate that a major function of Scrib in epithelial polarity is to exclude Crb from the basolateral domain. Since ectopic Crb does not cause the epithelial morphology and multilayering defects seen in scrib embryos, Scrib may be required for the localization of additional epithelial determinants as well (Bilder, 2000).

Analysis of the morphological and polarization phenotypes exhibited by scrib embryos shows that Scrib is a critical component of epithelial architecture in the Drosophila ectoderm, and suggests that its function is closely linked to that of Crb. Scrib is not required for the early localization of basal Discs lost (now redefined as Drosophila Patj) or apical Armadillo during blastoderm formation, and scrib mutants do not exhibit the defective cellularization or precipitous loss of cell adhesion seen when Discs lost or Armadillo, respectively, are depleted in the embryo. The increasingly severe cell shape, polarity and epithelial organization defects of scrib embryos are first manifested after gastrulation, coincident with the onset of defects in crb and stardust embryos. Loss of Crb results in loss of apical proteins from the plasma membrane and a failure to consolidate early adherens junction material into an apical band of zonula adherins (ZAs), while in scrib embryos early adherens junctions become misdistributed basolaterally. Together with the similarities between scrib loss-of-function and crb gain-of-function phenotypes, these data place Scrib and Crb in a pathway required for the progression from the initially differentiated blastoderm membrane domains into a fully polarized epithelium with mature junctions (Bilder, 2000).

These results show that the junctional protein Scrib specifically restricts apical membrane determinants to the apical cell surface. This restriction allows the proper segregation of apical and basolateral domain components, and the appropriate placement of the adherens junction, resulting in full epithelial polarization. How does Scrib, a putative scaffolding protein whose localizaton bounds the apical domain, dictate the proper confinement of apical proteins? Two models suggest themselves. Scrib could assemble a diffusion barrier that physically separates apical and basolateral compartments, similar to the 'fence' function proposed for the vertebrate tight junction. To date, such a barrier has been shown to exist only for lipid diffusion in the outer leaflet of the plasma membrane. If scrib mutations disrupt such a mechanical barrier, then secondary retention systems must serve to maintain basolateral protein restriction from the apical cell surface. An alternative is that Scrib has a role in the polarized targeting of transport vesicles carrying apical proteins. The junctional complex, and in particular the tight junction, has been proposed to be a key sorting site for a subset of Golgi-derived vesicles. In this model, Scrib might interact with the 'exocyst', a secretory targeting apparatus localized to the tight junction and involved in polarized segregation of transmembrane proteins. PDZ domain proteins have been implicated at several different sites of the protein trafficking pathway, and occasional punctate intracellular staining of Scrib is reminiscent of vesicles. Distinction between these models will rely on the identification of binding partners for Scrib (Bilder, 2000).

Adherens junctions inhibit asymmetric division in the Drosophila epithelium

The asymmetry of neuroblast cell divisions might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. By disrupting adherens junctions the symmetric epithelial division of epidermal cells can be changed into asymmetric division. The adenomatous polyposis coli (APC) tumor suppressor protein is recruited to adherens junctions, and both APC and microtubule-associated EB1 homologs are required for the symmetric epithelial division along the planar axis. These results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions (Lu, 2001).

Drosophila neuroblasts delaminate from a polarized epithelial layer in the ventral neuroectoderm and divide asymmetrically along the apical-basal axis to produce larger apical neuroblasts and smaller basal ganglion mother cells. Inscuteable (Insc) as a central protein in organizing neuroblast division. Insc provides positional information that couples mitotic spindle orientation with the basal localization of cell-fate determinants such as Numb and Prospero together with their respective adaptor proteins Partner of Numb (Pon) and Miranda (Lu, 2001 and references therein).

The apical localization of Insc involves both a Baz-dependent initiation step and a maintenance step that requires Baz and Partner of Inscuteable (Pins). The expression of Baz and Pins in both neuroblasts and neuroepithelial cells suggests that these cells share certain apical-basal polarity information. Consistent with this notion is the observation that, when Pon is expressed ectopically in epithelial cells it is localized to the basal cortex, as in neuroblasts. Unlike neuroblasts, however, epithelial cells divide symmetrically along the planar axis and segregate ectopic Pon equally between the two daughter cells. These observations raise further questions: do epithelial cells have the ability to couple spindle orientation with protein localization, and segregate proteins asymmetrically between two unequally sized daughter cells? If so, what prevents them from executing this asymmetric division (Lu, 2001)?

To characterize epithelial division by monitoring it in live embryos, transgenic embryos expressing Pon and tau proteins fused with green fluorescent protein (GFP) were used. During epithelial cell cycle, tau-GFP-labelled mitotic spindle is formed along the planar axis of the embryo, and Pon-GFP is initially uniformly associated with the cortex and then localized to a basal crescent. The mitotic spindle remains oriented along the planar axis throughout mitosis. After cytokinesis, the Pon-GFP crescent is bisected by the cleavage furrow and is equally distributed between two equally sized daughter cells. This in vivo analysis shows that the machinery for basal protein localization is intact in epithelial cells, but it is uncoupled from spindle orientation (Lu, 2001).

Double-stranded (ds) CRB RNA was injected into transgenic embryos expressing Pon-GFP and tau-GFP. In about 70% (n = 200) of crb(RNAi) embryos, the organization of the ectodermal epithelium is disrupted, with epithelial cells losing their columnar shape, adopting rounded morphology, and becoming separated from each other. Live imaging of epithelial divisions in these embryos reveals that nearly all the epithelial cells show a tight coupling between the positioning of Pon-GFP crescents and the orientation of the mitotic spindle. Pon-GFP crescents were found at basal and lateral positions and less frequently at apical positions on the cell cortex, and one of the spindle poles was positioned underneath the Pon-GFP crescent (Lu, 2001).

After cytokinesis, Pon-GFP was segregated to one of the two similarly sized daughter cells. Asymmetric segregation of Pon-GFP to one of two similarly sized daughter cells was also observed in crb zygotic mutant embryos. Immunostaining of crb(RNAi) embryos with antibodies against Asense, Prospero and Insc indicates that epithelial cells do not express these neuronal markers, suggesting that the ability of these cells to undergo asymmetric division is not a result of cell-fate change (Lu, 2001).

Overexpression of the membrane-bound cytoplasmic tail of Crb (Crb-intra) causes similar disorganization of the epithelium as seen in crb mutants. The effect of overexpressing Crb-intra on epithelial division was examined. As observed in crb(RNAi) embryos, epithelial cells overexpressing Crb-intra show coupling of the mitotic spindle with the Pon-GFP crescent and asymmetric segregation of Pon-GFP to one of the daughter cells. Thus, when the formation of the adherens junction is disrupted, epithelial cells switch from a symmetric to an asymmetric division pattern (Lu, 2001).

In addition to its function in localizing Insc and regulating division axis in the neuroblasts, Baz is also required for the formation of adherens junction and the maintenance of epithelial polarity. The function of Baz in epithelial division was examined. The baz(RNAi) embryos showed overall disruption of epithelium organization similar to that observed in crb(RNAi) embryos. Unlike in crb(RNAi) embryos, however, epithelial cells in baz(RNAi) embryos divide in a symmetric fashion, with Pon-GFP distributed uniformly around the cell cortex throughout mitosis and the mitotic spindle orients in random directions. After cytokinesis, two equally sized daughter cells are produced and Pon-GFP is equally distributed between them (Lu, 2001).

Daughter cell size asymmetry in neuroblast division is largely unaffected in baz(RNAi) embryos. In crb(RNAi) epithelial cells Baz can still be localized into a crescent but the crescent is mispositioned and Pon-GFP is always localized to the opposite side of the Baz crescent. This suggests that, although mispositioned, Baz is still functional in directing Pon-GFP localization in crb(RNAi) embryos. To test whether the coupling of Pon-GFP localization with spindle orientation observed in crb(RNAi) embryos is Baz dependent, double RNAi was performed by co-injecting a mixture of baz and crb dsRNAs. Epithelial divisions in the co-injected embryos appeared similar to baz single-injected embryos, with Pon-GFP segregated equally between two equally sized daughter cells. It is therefore concluded that epithelial cells depend on Baz to couple spindle orientation with protein localization when the adherens junction is disrupted (Lu, 2001).

Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis

The apical transmembrane protein Crumbs is a central regulator of epithelial apical-basal polarity in Drosophila. Loss-of-function mutations in the human homolog of Crumbs, CRB1 (RP12), cause recessive retinal dystrophies, including retinitis pigmentosa. Crumbs and CRB1 localize to corresponding subdomains of the photoreceptor apical plasma membrane: the stalk of the Drosophila photoreceptor and the inner segment of mammalian photoreceptors. These subdomains support the morphogenesis and orientation of the photosensitive membrane organelles -- rhabdomeres and outer segments, respectively. Drosophila Crumbs is required to maintain zonula adherens integrity during the rapid apical membrane expansion that builds the rhabdomere. Crumbs also regulates stalk development by stabilizing the membrane-associated spectrin cytoskeleton, a function mechanistically distinct from its role in epithelial apical-basal polarity. It is proposed that Crumbs is a central component of a molecular scaffold that controls zonula adherens assembly and defines the stalk as an apical membrane subdomain. Defects in such scaffolds may contribute to human CRB1-related retinal dystrophies (Pellikka, 2002a).

The rhabdomere of photo-receptor cells (PRCs) of Drosophila is a rod-shaped array of microvilli that contains the photopigment. The rhabdomere is surrounded by the stalk membrane that connects it to the zonula adherens (ZA), which separates apical and basolateral membranes. The architecture of PRCs is established during metamorphosis when the apical membranes of PRCs turn by 90o and undergo a dramatic distal-to-proximal expansion as the rhabdomere and stalk are formed. The apical membranes of mammalian PRCs show a similar subdivision into the outer segment, which contains the photopigment, and the surrounding inner segment, which connects to the ZA and the basolateral membrane (Pellikka, 2002a).

Human CRB1 and Crb exhibit a conserved domain architecture and their cytoplasmic domains show 54% identity. Alignment of the cytoplasmic domains of Crb, CRB1, a Caenorhabditis elegans Crb homolog, and two additional putative human Crb-like proteins (CRB2 and CRB3) reveals two conserved regions, including a carboxy-terminal PDZ-domain binding motif (ERLI), through which Crb interacts with the PDZ domain proteins Discs lost (Dlt) and Stardust (Sdt). This study investigated the distribution of Crb and CRB1 in PRCs and the function of Crb in PRC morphogenesis (Pellikka, 2002a).

This analysis identified two roles of Crb in PRC morphogenesis. (1) Crb is required to maintain the integrity of the ZA during its rapid 100-fold expansion in PRCs. In early embryos, Crb is needed for the final step of ZA formation when patches of adherens junction material fuse into a continuous band. Crb probably functions similarly in embryonic ZA formation and during the expansion of the ZA in PRCs. It is speculated that Crb establishes a mechanism at the transition zone of apical and lateral membranes that anchors adherens junction material and thereby supports ZA formation. ZA integrity in crb mutant embryos and PRCs shows recovery at later stages, and crb mutant cells of the imaginal disc display a normal ZA, suggesting that other mechanisms, which functionally overlap with Crb, contribute to ZA formation and maintenance. The Bazooka complex, composed of Bazooka (the Drosophila homolog of C. elegans Par-3 and mammalian ASIP), Drosophila Par-6 and atypical protein kinase 3, is a candidate for such a role because it co-localizes with Crb at the apical membrane and is required for ZA formation in early embryos. Defects in ZA integrity could contribute to retinal dystrophy in patients carrying CRB1 mutations. Functional redundancies among the mechanisms that stabilize cellular architecture may explain the slow progression of human CRB1-associated retinal degeneration (Pellikka, 2002a).

(2) Crb is a central regulator of stalk membrane biogenesis. Several arguments suggest that this role of Crb is independent from its role in ZA formation. (1) Within the stalk membrane, Crb does not localize immediately adjacent to the ZA as in undifferentiated epithelial cells. (2) Overexpression of Crb enlarges the stalk membrane of PRCs but does not transform basolateral membrane to apical membrane or disrupt the ZA as seen with overexpression of Crb in undifferentiated epithelia. (3) The gain-of-function effect that results from overexpression of Crb can be reproduced by expression of CrbextraTM-GFP but not by expression of Crbintra. In contrast, overexpression of Crb and Crbintra but not of CrbextraTM-GFP in undifferentiated epithelia such as the larval eye imaginal disc compromises apical-basal polarity and ZA integrity. (4) karst;crb (karst codes for ßH-Spectrin) double mutants have dramatically reduced stalk membranes but normal ZAs. These findings suggest that in supporting stalk membrane formation, Crb has adopted a role in PRC morphogenesis that is mechanistically distinct from its role in epithelial polarity and ZA formation. These results also reveal for the first time a mechanism that subdivides the apical membrane of epithelial cells into two different domains that support distinct structures and physiologies (Pellikka, 2002a).

The membrane-associated spectrin cytoskeleton can determine membrane topology and stability. These findings demonstrate such a role for the spectrin cytoskeleton of PRCs, because the lack of ßH-Spectrin causes shortening and reduced folding of the stalk membrane. Loss of ßH-Spectrin may destabilize the stalk membrane causing rapid endovesiculation, as is seen when the spectrin cytoskeleton of red blood cells is compromised. Recent work has shown that coated pit budding is accompanied by a local loss of spectrin, and blocking spectrin remodelling results in the retention of coated pits at the cell surface. Coated pits often associate with the folds within the stalk membrane of PRCs, indicating that these folds are the site of frequent endocytosis. It is hypothesized that a rate increase of endocytosis in response to lack of ßH-Spectrin could remove the membrane folds and thus straighten and shorten the stalk membrane. Conversely, stabilization of the apical spectrin cytoskeleton by Crb overexpression may prevent budding of coated pits and thus enlarge the stalk membrane. Crb must also interact with factors other than ßH-Spectrin to support stalk formation, because the loss of Crb leads to a much greater reduction in stalk size than the loss of ßH-Spectrin, and because the kst null mutant phenotype is dominantly enhanced by a crb mutation. Moreover, because the extracellular domain of Crb promotes stalk formation independently of the cytoplasmic domain, Crb is likely to interact with other integral membrane or extracellular factors that in turn can stabilize the stalk membrane. It is proposed therefore that Crb and ßH-Spectrin are components of an extensive molecular scaffold that integrates extracellular, integral membrane and cytoplasmic factors, and specifies topology and size of the stalk membrane (Pellikka, 2002a).

This analysis reveals a function for the extracellular part of the Crb protein. The dominant negative effect of Crbextra expression on stalk formation suggests that the inter-rhabdomeral space contains an important ligand that interacts with the extracellular part of Crb and is titrated out by Crb extra. Although Crb needs to be anchored in the stalk membrane to support stalk formation, cytoplasmic interactions are apparently not essential for this activity, as demonstrated by the effect of CrbextraTM-GFP expression. The critical activity that supports stalk formation resides in the extracellular part of Crb that includes thirty EGF and four LG domains, which represent protein and carbohydrate interaction domains. Also the extracellular domain of human CRB1 may mediate critical interactions, because many mutations that give rise to retinal dystrophies are missense mutations that affect different EGF or LG domains of CRB1. The localization of CRB1 in the inner segment supports the notion that the Drosophila stalk and the inner segment of vertebrate PRCs are homologous plasma-membrane domains that rely on similar mechanisms to support their structural integrity. The localization of CRB1 in the periphery of the cone outer segments is reminiscent of the localization of the tetraspanins peripherin/RDS and ROM1 in rod and cone PRCs. Like these tetraspanins, CRB1 may contribute to the maintenance of cone outer segments (Pellikka, 2002a).

A role for CRB1 in colour vision may have been masked by its function in general retinal maintenance, which is apparent in CRB1-related pathologies. Further analysis of Crb and CRB1 and their interacting factors will shed light on the mechanisms that organize epithelial surface domains, and should help to understand the mechanisms of retinal differentiation and maintenance in humans (Pellikka, 2002a).

Drac1 and Crumbs participate in amnioserosa morphogenesis during dorsal closure in Drosophila

Dorsal closure of the Drosophila embryo involves morphological changes in two epithelia, the epidermis and the amnioserosa, and is a popular system for studying the regulation of epithelial morphogenesis. The small GTPase Rac1 has been implicated in the assembly of an actomyosin contractile apparatus, contributing to cell shape change in the epidermis during dorsal closure. Evidence is presented that Rac1 and Crumbs, a determinant of epithelial polarity, are involved in setting up an actomyosin contractile apparatus that drives amnioserosa morphogenesis by inducing apical cell constriction. Expression of constitutively active Rac1 causes excessive constriction of amnioserosa cells and contraction of the tissue, whereas expression of dominant-negative Rac1 impairs amnioserosa morphogenesis. These Rac1 transgenes may be acting through their effects on the amnioserosa cytoskeleton, since constitutively active Rac1 causes increased staining for F-actin and myosin, whereas dominant-negative Rac1 reduces F-actin levels. Overexpression of Crumbs causes premature cell constriction in the amnioserosa, and dorsal closure defects are seen in embryos homozygous for hypomorphic crumbs alleles. The ability of constitutively active Rac1 to cause contraction of the amnioserosa is impaired in a crumbs mutant background. It is proposed that amnioserosa morphogenesis is a useful system for studying the regulation of epithelial morphogenesis by Rac1 (Hardin, 2002).

Expression of dominant negative Drac1N17 in the amnioserosa slows morphogenesis of this tissue which remains as a squamous epithelium for a longer period than in wild-type embryos. In Drac1N17-expressing embryos, where amnioserosa morphogenesis is lagging, the movement of the epidermis is also slowed, and the embryos have a larger dorsal hole than wild-type embryos of similar age. It is thought that the impaired movement of the epidermis in such embryos is caused by lack of morphogenesis in the amnioserosa. These results are strong evidence that active cell shape changes in the amnioserosa are required for normal dorsal closure. Examination of wild-type embryos has shown that this cell shape change in the amnioserosa begins with apical constriction of cells at the anterior and posterior ends of the amnioserosa. These cells have elevated levels of myosin, F-actin and phosphotyrosine, suggesting that an apically localized actomyosin contractile apparatus is driving their constriction. Early in dorsal closure, the middle cells in between the two clusters of apically constricted cells do not show elevated levels of F-actin or myosin but do change shape, losing their original elongation perpendicular to the A-P axis of the embryo. The middle cells may be stretching passively, in response to tension from the cell constrictions occurring at both ends of the amnioserosa. By the end of dorsal closure, the middle cells are both elongated along the A-P axis and apically constricted, and it is conceivable that late in dorsal closure they undergo an active cell shape change as their neighbors did earlier (Hardin, 2002).

Excessive Drac1 activity induces a dramatic contraction of the amnioserosa such that it shrinks to occupy less than half the dorsal hole, and this is accompanied by elevated levels of myosin, F-actin, and phosphotyrosine in this tissue. It is thought that Drac1V12 is driving premature and excessive amnioserosa cell constriction through its effects on the cytoskeleton. It is proposed that Drac1 participates in amnioserosa morphogenesis by driving the assembly of an apical actomyosin contractile apparatus that constricts the amnioserosa cells, first at the ends of the tissue and possibly later in the middle. Contraction of an apical actomyosin belt has been implicated in diverse types of epithelial morphogenesis including Drosophila gastrulation, which shows some similarity to amnioserosa morphogenesis in that both processes involve apical construction of a monolayer of cells that then invaginates (Hardin, 2002).

Cell ablation has been used to address the contributions of the epidermis and amnioserosa to dorsal closure. This work has demonstrated that the amnioserosa is under tension, since ablation of cells in the amnioserosa causes the tissue to recoil away from the wound site, and the leading edge is pushed back away from the dorsal midline. It is concluded that there is active cell shape change in the amnioserosa that contributes to dorsal closure, rather than the tissue being simply compressed by the movement of the leading edge. The finding that the recoiling of the amnioserosa after wounding pushes back the leading edge is consistent with the result that impairing amnioserosa morphogenesis through Drac1N17 expression hinders leading edge migration (Hardin, 2002).

Overexpression of Crb in the amnioserosa leads to contraction of the tissue and failure of dorsal closure. This phenotype was examined in more detail; excessive Crb activity induces a premature constriction of cells at the ends of the amnioserosa. Five P-element-induced crb alleles were identified that are hypomorphic mutations, causing defects in dorsal closure and germband retraction. One of these crb mutations, crbS010409, was characterized in detail. Embryos homozygous for crbS010409 show a dorsal closure defect similar to that seen with expression of Drac1N17 in the amnioserosa: amnioserosa morphogenesis is impaired, but the leading edge cytoskeleton is intact. In contrast to amorphic crb alleles, the epidermis is not disorganized in crbS010409 mutants and it secretes cuticle. Amnioserosa morphogenesis and germband retraction may be particularly sensitive to the level of Crb activity. It is thought that Crb activity in the amnioserosa is required for amnioserosa morphogenesis, although the possibility cannot be excluded that loss of Crb activity elsewhere in the embryo is affecting this process (Hardin, 2002).

Drac1 may act through Crb in regulating the cytoskeleton, since the constitutively active Drac1V12-induced phenotype of excessive contraction of the amnioserosa is weakened in a crbS010409 mutant background. This weaker Drac1V12 phenotype of premature constriction of the end cells of the amnioserosa is very similar to that caused by Crb overexpression. There may be sufficient Crb in the crbS010409 mutant embryos for Drac1V12 to be able to prematurely constrict cells at the ends of the amnioserosa but not to excessively contract the tissue. Crb overexpression does not appear to require Drac1 to cause premature constriction of amnioserosa cells, since it can achieve this in the presence of Drac1N17. The excessive contraction of the amnioserosa caused by Drac1V12 expression in embryos with wild-type Crb activity, and the dumbbell-shaped amnioserosa induced by Crb overexpression, could both result from excessive constriction of amnioserosa cells to produce a tissue that only occupies a fraction of the dorsal hole. Such excessive constriction may be driven by ectopic accumulation of a normally apically localized actomyosin contractile apparatus. A role for Crb in defining the location of the actomyosin contractile apparatus is consistent with the idea that Crb defines the range of the apical membrane cytoskeleton. The actin-crosslinking protein ßHeavyH)-spectrin normally has an apicolateral distribution, but upon overexpression of Crb is also found at the basolateral membrane, indicating a redistribution of the membrane cytoskeleton. ßH-spectrin is required for apical constriction of follicle cells during Drosophila oogenesis and may participate in organization of an actomyosin contractile apparatus. It is conceivable that the ectopic localization of (ßH)-spectrin domain following Crb overexpression could be accompanied by an ectopic accumulation of F-actin and myosin. Future goals in studying Drac1-Crb function in amnioserosa morphogenesis will include addressing the nature of the interaction between the two proteins and defining which portion(s) of the Crb protein are required. The short cytoplasmic domain of Crb appears sufficient to execute all Crb functions studied to date. No definitive role has been found for the large extracellular domain, although there is evidence that the Drosophila and human Crb proteins have non-cell-autonomous functions (Hardin, 2002).

Although Drac1 and Crb both generate premature contraction of the amnioserosa when their activity is experimentally upregulated in this tissue, their phenotypic effects are not identical. Drac1V12 expression drives constriction of all amnioserosa cells early in closure, whereas, at the same stage, Crb overexpression only promotes constriction of the end cells. A plausible explanation for this is that constriction of the middle cells requires Drac1 to activate Crb-independent processes and that Crb function is necessary but not sufficient for middle cell constriction. Crb overexpression in the amnioserosa causes disruption of the leading edge cytoskeleton and a failure of cell shape change in the epidermis, suggesting that a signal from the amnioserosa required for dorsal closure is disrupted. That communication between the amnioserosa and the epidermis is a component of dorsal closure is demonstrated by the observation that JNK signaling in the amnioserosa is required for phosphotyrosine accumulation at the leading edge and dorsalward migration of the epidermis and by the observation that leading edge cells are specified wherever an interface of amnioserosa and dorsal epidermis occurs. Drac1V12 expression in the amnioserosa does not disrupt the leading edge cytoskeleton or prevent closure of the epidermis, and this result suggests that Drac1V12 cannot activate a function of Crb that influences communication between the amnioserosa and the epidermis (Hardin, 2002).

Drosophila Crumbs is required to inhibit light-induced photoreceptor degeneration

Mutations in the human transmembrane protein CRB1 are associated with severe forms of retinal dystrophy, retinitis pigmentosa 12 (RP12), and Leber's congenital amaurosis (LCA). The Drosophila homolog, crumbs, is required for polarity and adhesion in embryonic epithelia and for correct formation of adherens junctions and proper morphogenesis of photoreceptor cells. Mutations in Drosophila crumbs have been shown to result in progressive, light-induced retinal degeneration. Degeneration is prevented by expression of p35, an inhibitor of apoptosis, or by reduction of rhodopsin levels through a vitamin A-deficient diet. In the dark, rhabdomeres survive but exhibit morphogenetic defects. It is the extracellular portion of the Crumbs protein that is essential to suppress light-induced programmed cell death, while proper morphogenesis depends on the intracellular part. It is concluded that human and Drosophila Crumbs proteins are functionally conserved to prevent light-dependent photoreceptor degeneration. This experimental system is now ideally suited to study the genetic and molecular basis of RP12- and LCA-related retinal degeneration (Johnson, 2002).

The pattern of Crumbs expression in the adult eye was analyzed. Immunohistochemistry reveals that Crumbs protein is found in a circumferential stripe at the apical sides of the photoreceptor cells, bordering the ZA, which is visualized as a circumferential belt by anti-Armadillo staining. Within the apical domain, Crumbs is localized to the apical stalk membrane, which connects the ZA with the rhabdomeres. The rhabdomeres are also apical derivatives containing highly pleated stacks of microvilli, rich in F-actin, that carry the photosensitive pigment rhodopsin and the phototransduction complexes (Johnson, 2002).

Since, in Drosophila, hereditary retinal degenerations are light dependent in several cases, mosaic eyes containing large crb mutant clones of flies kept under constant illumination were examined. The Drosophila eye is composed of about 800 ommatidia, cylindrical, barrel-like structures, containing eight photoreceptor cells in their center that are arranged in a stereotypic manner. When flies carrying crb11A22 mosaic eyes are kept in constant light for 7 days, the retina shows massive degeneration. This phenotype strictly depends on continous exposure to light, since, in flies kept under standard laboratory conditions, i.e., in artificial low light, no degeneration of photoreceptors occurs. crb mosaic flies raised under these conditions show a slightly variable, mutant phenotype. Their rhabdomeres are thicker and shorter compared to wild-type and are often found in close contact with other rhabdomeres of the same ommatidium. Serial cross-sections and horizontal sections stained with FITC-phalloidin, which highlights the F-actin bundles of the rhabdomeres, reveal that the rhabdomeres fail to reach the basal lamina and extend from the distal pole near the lens to only about one third of the normal length. In addition, the stalk membrane is reduced in length. However, the tightly stacked 'semicristalline' internal structure of the rhabdomere is unaffected. The catacomb-like rhabdomere base is also unaffected in mutant photoreceptor cells. In many ommatidia, ZAs are visible in the distal regions of the cells. Control of morphogenesis seems to be cell autonomous, since ommatidia composed of wild-type and mutant cells only exhibit defects in the mutant cells (Johnson, 2002).

To analyze the temporal course of degeneration, eyes carrying mutant clones were sectioned at different time points after light exposure. Eyes kept for only 1 day in constant illumination do not show any sign of photoreceptor degeneration but exhibit only the morphogenetical defects described above. After 5 days in constant light, each ommatidium contains one or two photoreceptor cells with signs of degeneration. In ommatidia exposed to constant light for 7 days, most of the photoreceptor cells are degenerating. Signs of degeneration include the devolution of the highly pleated microvillar rhabdomere structure, concomitant with the loss of the catacomb-like rhabdomere base. Affected nuclei round up, and the nucleolus, nucleoplasm, and cytoplasm appear condensed. These features are indicative of PCD. Assays were conducted to determine whether PCD was the mechanism involved: would overexpression of the baculovirus-derived p35 survival protein inhibit light-induced photoreceptor cell death in crb11A22 mutant ommatidia? It is well established that PCD involves a conserved cascade of cysteine proteases, the caspases, many of which can be inhibited by the ectopic expression of p35 protein. During Drosophila eye development, all stages of endogenous, pattern-forming PCD can be inhibited by using a transgene expressing p35. Similarly, age-related and light-induced retinal degeneration in flies mutant for ninaERH27 (rhodopsin1), rdgC306 (rhodopsin phosphatase), norpAEE5 (phospholipase C), or arr2(P261S) (arrestin) can be prevented by overexpressing p35 survival protein. Overexpression of p35 protein rescues light-induced degeneration of crb11A22 photoreceptor cells, prevents devolution of the rhabdomeres, and preserves the rhabdomere bases. Even when exposed to constant illumination for 14 days, the rescue by p35 expression is similar, implicating PCD as the downstream consequence of crb-induced retinal degeneration. Together these results suggest that crb mediates two functions in photoreceptor cells. One function controls the proper morphogenesis of the rhabdomeres and allows the formation of highly elongated (>100 μm) cells. The other function is required after eclosion for survival of photoreceptor cells when exposed to light (Johnson, 2002).

Since Crumbs is not expressed in the rhabdomere, it cannot directly participate in the signal transduction process, which raises the question of how Crumbs controls cell survival. Recently, a mechanism has been put forward that explains retinal degeneration in a subset of retinal degeneration mutations (arr2, norpA, rdgB, and rdgC) as a result of abnormally stable, light-induced meta-rhodopsin/arrestin complexes. In mutant eyes, these stable complexes are internalized by clathrin-mediated endocytosis. The accumulation of internal meta-rhodopsin/arrestin complexes triggers PCD through an unknown mechanism. In these mutants, PCD is prevented if internalization is inhibited in a shibire (D-dynamin) mutant background or when larvae are raised on a vitamin A-deficient medium. The depletion of vitamin A reduces the amount of rhodopsin to about 3% of its normal amount, which leads to the development of normal, though smaller, rhabdomeres in wild-type flies (Johnson, 2002).

To test whether light-induced degeneration in crb mutant eyes is based on a similar mechanism, crb11A22 mosaic flies were raised on this vitamin A-deficient medium and exposed to continuous room light. Mutant photoreceptor cells show smaller, thinner rhabdomeres due to the reduced amount of rhodopsin. They also show morphogenetic defects similar to those of crb11A22 ommatidia raised on standard medium and kept in the dark. However, only minor signs of photoreceptor degeneration were found. This finding is consistent with the possibility that PCD in illuminated crb11A22 photoreceptor cells is due to the increased internalization of meta-rhodopsin/arrestin complexes. Proof of this will require a direct determination of the number of complexes formed, their localization, and the possible involvement of endocytosis in crb-mediated degeneration (Johnson, 2002).

For the embryo, it is well documented that the short intracellular domain of Crumbs is crucial for proper function since its truncation leads to a complete loss of function phenotype. The cytoplasmic domain recruits the MAGUK protein Stardust and the four PDZ-domain protein Discs Lost into a subapical protein scaffold (SAC). Overexpression of the membrane-bound cytoplasmic domain (Crumbsintra) is sufficient to achieve a partial rescue of crb embryos, and the degree of rescue is comparable to that obtained by overexpression of the full-length Crumbs protein. In order to determine which part of the Crumbs protein is necessary to prevent retinal degeneration, clones were induced that were homozygous mutant for the crb8F105 allele. The crb8F105 allele encodes a protein that lacks the C-terminal 23 amino acids of the cytoplasmic domain and is completely nonfunctional in the embryo. The morphogenetic phenotype observed in mosaic eyes of crb8F105 is comparable to that of the crb11A22 null allele kept in the dark. In contrast to crb11A22, however, crb8F105 mutant ommatidia do not show major signs of degeneration, even after 14 days of exposure to light. This indicates that either the extracellular domain or the N-terminal portion of the cytoplasmic tail of Crumbs prevents light-induced degeneration. To discriminate between these two possibilities, crb8F105 and crb11A22 mutant ommatidia were analyzed in the presence of a transgene expressing the membrane-bound cytoplasmic domain of Crumbs. This transgene largely rescues the morphogenetic defects in mosaic eyes of both alleles under low-light conditions. This is manifested by the fact that many rhabdomeres are elongated to reach the basal lamina. In contrast, crb11A22 photoreceptor cells exposed to light still undergo retinal degeneration despite the expression of the cytoplasmic domain. It is concluded that the intracellular domain of Crumbs is not sufficient to prevent light-induced photoreceptor degeneration and suggest a function for the extracellular domain in the prevention of PCD. Interestingly, all mutations mapped to the CRB1 gene in RP12 or LCA patients have been localized to the extracellular portion of the protein and lead either to amino acid exchanges, frame shifts, or stop codons. While this may simply reflect the fact that the intracellular domain is a small target for mutagenesis, the large number of RP12 cases showing exclusive amino acid exchanges extracellularly indicate a requirement for an intact extracellular CRB1 domain (Johnson, 2002).

The results in Drosophila indicate that the depletion of rhodopsin through a vitamin A-deficient diet prevents the light-induced crb11A22 degeneration of the retina. A randomized study of ungenotyped RP patients, given high-dose oral vitamin A supplementation, suggested a modest slowing of the disease progression. This finding may indicate that, at least for CRB1-induced RP12 and LCA cases, a high-dose vitamin A supplementation might be counterproductive rather than beneficial in slowing disease progression. Both assumptions on the progression of the disease should be tested in a vertebrate animal model, i.e., a knockout mouse. The Drosophila system, however, can now be exploited to unravel the underlying mechanism of crb-dependent retinal degeneration (Johnson, 2002).

Crumbs stabilises epithelial polarity during tissue remodelling

The apicobasal polarity of epithelia depends on the integrated activity of apical and basolateral proteins, and is essential for tissue integrity and body homeostasis. Yet these tissues are frequently on the move as they are sculpted by active morphogenetic cell rearrangements. How does cell polarity survive these stresses? This question was analyzed in the renal tubules of Drosophila, a tissue that undergoes dramatic morphogenetic change as it develops. This study shows that whereas the Bazooka and Scribble protein groups are required for the establishment of tubule cell polarity, the key apical determinant, Crumbs, is required for cell polarity in the tubules only from the time when morphogenetic movements start. Strikingly, if these movements are stalled, polarity persists in the absence of Crumbs. Similar rescue of the ectodermal phenotype of the crumbs mutant when germ-band extension is reduced suggests that Crumbs has a specific, conserved function in stabilising cell polarity during tissue remodelling rather than in its initial stabilisation. A requirement was also identified for the exocyst component Exo84 during tissue morphogenesis, which suggests that Crumbs-dependent stability of epithelial polarity is correlated with a requirement for membrane recycling and targeted vesicle delivery (Campbell, 2009).

Epithelial cell rearrangements are accompanied, and may be driven by, changes in the spatial arrangement of their intercellular contacts. Contacts with new neighbours are engineered by the reorganisation of intercellular junctions through vesicle recycling; zonula adherens (ZAs) shrink as the interface disappears between cells that lose contact and expand as borders develop between new neighbours. These changes involve dynamin-dependent endocytosis of ZA components, rab-mediated vesicle trafficking and the targeted recycling of vesicles to specific membrane domains (Classen, 2005; Langevin, 2005). This study shows that Crb is specifically required to maintain apicobasal polarity and the integrity of ZAs as these cell activities occur. If cell rearrangements are halted or reduced in crb mutants, there is extensive rescue of epithelial integrity. This leads to proposal of a model in which the Crb complex is dispensable for the establishment of cell polarity in embryonic epithelia but that as soon as morphogenetic cell rearrangements start, the Crb complex acts both to stabilise apical proteins and to restrict the spread of basolateral proteins (Campbell, 2009).

The requirement for E-cadherin in different tissues shows a similar dependence on the degree of morphogenetic activity. It has been shown that the zygotic Drosophila E-cadherin mutant phenotype can be rescued in dynamic tissues, for example in the neurectoderm and Malpighian tubules, by suppressing morphogenetic cell movements (Campbell, 2009).

It is tempting to speculate that Crb acts by targeting recycling vesicles of ZA components in order to maintain junctional integrity in the elongating renal tubules. Without Crb ZAs are lost and membrane domains no longer remain distinct, leading to the collapse of cell polarity. Alternatively, lack of Crb could result in loss of cell polarity in morphogenetically active tissues and, as a consequence, ZAs cannot be maintained. In this case the primary requirement for Crb during cell movement would be to maintain the apical localisation of Baz/Par-6/aPKC, thereby also ensuring the normal distribution of basolateral proteins (Campbell, 2009).

The exo84 mutant phenotype in remodelling tissues could be explained by a depletion of apical Crb caused by reduced exocyst-mediated delivery, resulting in a phenotype reminiscent of crb mutant embryos. This view is supported by recent findings showing that expression of dominant-negative Cdc42 in stage 9-11 embryos results in loss of Crb and other proteins from the apical membrane and disruption of cadherin-based adhesion in the morphogenetically active ventral neuroectoderm. Furthermore, mutations in crb enhance the phenotype induced by dominant-negative Cdc42 expression in this tissue (Harris, 2008). That study suggests that Cdc42 normally acts to repress endocytosis of apical proteins including Crb, so that inactivation of Cdc42 results in defects in cell polarity, leading to the loss of ZAs and tissue disruption. Whether Crb ensures ZA plasticity during cell rearrangements by restricting excessive endocytosis of apical proteins indirectly via stabilisation of Baz and/or Par-6 and Cdc42, or in a more direct way by regulating the recycling of junctional proteins remains to be determined. Similar defects in ZA integrity were also observed in the epithelium of the developing adult dorsal thorax upon loss of Cdc42 function. This study has shown that Cdc42-Par-6-aPKC control endocytosis by the cytoskeletal regulators Arp2/3 and Cip4 and WASP. These results complement and support recent data showing that Cdc42, together with PAR-3, PAR-6 and PKC, are required for membrane trafficking in C. elegans coelomocytes and human HeLa cells (Campbell, 2009).

Members of the Crb complex also play a critical role in ZA stability and apical membrane delivery or stabilisation during photoreceptor development, when the ZAs enlarge and the apical domain selectively expands as the rhabdomere forms. These morphogenetic events require the targeted delivery and retention of large amounts of membrane. Here too, it is not yet clear whether Crb acts directly on the stability of ZA components or indirectly, by controlling other polarity proteins. Although Drosophila Par-6 is delocalised in crb mutant photoreceptor cells, other data suggest that the Crb complex regulates ZA integrity and trafficking of apical membrane via stabilisation of the membrane-associated cytoskeleton, including βH-spectrin (Campbell, 2009).

Vertebrate homologues of members of the fly Crb complex appear to have conserved roles in the control of epithelial integrity. Loss of oko meduzy, one of the five zebrafish crb orthologues, of the sdt orthologue nagie oko or of mosaic eyes, a regulator of Crb, perturbs polarity and morphogenesis of the retinal neuroepithelium and the heart. In addition, RNAi-mediated knock down of the mammalian Sdt orthologue, Pals1, in Madin Darby canine kidney (MDCK) cells in culture prevents proper delivery of E-cadherin to the cell surface, a phenotype strikingly similar to that of crb or sdt mutant epithelia in Drosophila (Campbell, 2009).

These observations suggest that, as in the fly, vertebrate epithelia affected by the loss of Crb are those that undergo morphogenetic reorganisation, including cell shape change. Loss of human CRB1 is associated with retinitis pigmentosa and Leber congenital amaurosis, resulting in retinal degeneration and blindness, a phenotype with striking similarity to flies with crb mutant photoreceptor cells, which exhibit light-dependent retinal degeneration. In mice mutant for Crb1, photoreceptor cells develop normally but later their ZAs degenerate so that photoreceptors in certain areas of the retina are displaced, followed by cell death. Further experiments will elucidate whether the defects observed in morphogenetically active epithelia and photoreceptor cells are based on a common cell biological function of the Crb complex in these two cell types (Campbell, 2009).

Crumbs is required to achieve proper organ size control during Drosophila head development

Crumbs (Crb) is a conserved apical polarity determinant required for zonula adherens specification and remodelling during Drosophila development. Interestingly, crb function in maintaining apicobasal polarity appears largely dispensable in primary epithelia such as the imaginal discs. This study shows that crb function is not required for maintaining epithelial integrity during the morphogenesis of the Drosophila head and eye. However, although crb mutant heads are properly developed, they are also significantly larger than their wild-type counterparts. In the eye, this is caused by an increase in cell proliferation that can be attributed to an increase in ligand-dependent Notch (N) signalling. Moreover, in crb mutant cells, ectopic N activity correlates with an increase in N and Delta endocytosis. These data indicate a role for Crb in modulating endocytosis at the apical epithelial plasma membrane, which is shown to be independent of Crb function in apicobasal polarity. Overall, this work reveals a novel function for Crb in limiting ligand-dependent transactivation of the N receptor at the epithelial cell membrane (Richardson, 2010).

This demonstrates a novel function for crb in the proper control of head and eye size during Drosophila development. This function is not restricted to the fly head and eye, but also extends to other tissues such as the wing. In the case of the eye, the data indicate that this is linked to a function for Crb in limiting ligand-dependent transactivation of N. In support of this model, a significant increase in endosomes positive for NICD, NECD and Dl was observed in crb mutant eye discs, that correlates with excessive cell proliferation. The eye overgrowth phenotype associated with the loss of crb function is correlated with an increase in NECD/Dl co-endocytosis. The data further indicate that this increase is dependent on the S2 cleavage of N. There is currently no evidence for the requirement of the S2 cleavage to promote endocytosis of N with Dl in cis. Moreover, Dl in cis is thought to inhibit N activation. It is therefore concluded that during eye development, Crb limits ligand-dependent transactivation of the N receptor (Richardson, 2010).

Mutations in the crb gene are associated with a failure to properly polarise the ectoderm along the apicobasal axis in the gastrulating embryo. In human, three Crb orthologues, CRB1, 2 and 3, have been described to date. Interestingly, CRB1 can be differentially spliced to produce the CRB1b isoform, which contains only the extracellular domain, whereas CRB3 lacks the conserved extracellular domain and comprises just the TM and intracellular domains. This suggests independent function for the extracellular and intracellular domains. The function of crb in establishing apicobasal polarity can be rescued in the Drosophila embryo using its intracellular domain anchored to the plasma membrane via the TM domain. Interestingly, crb function in the developing pupal photoreceptor has been linked to stalk membrane endocytosis through the connection of Crb to βHeavy-spectrin. This idea is supported by the finding that overexpression of Crb in either the gastrulating fly ectoderm or fly pupal photoreceptor leads to an increase the length of the apical and stalk membranes, respectively. Interestingly, when examining crb mutant adult photoreceptors, clathrin-coated-like pits were detected in the region of the stalk membrane, which are not normally readily detectable in the WT. Importantly, overexpression of the extracellular domain of Crb linked only to its TM domain is sufficient to cause a striking increase in the length of the stalk membrane in the developing fly photoreceptor. Consistent with this finding is the observation that this transgene can rescue the overgrowth phenotype in crb mutant heads, a phenotype that is correlated with an increased in N/Dl endocytosis. Moreover, the data indicate that Crb function in regulating N activity does not depend on the ability for Crb to interact with βHeavy-spectrin, Moesin, Yurt, Sdt, Patj, or Lin7. This suggests that the extracellular domain of Crb might bind to a component of the extracellular matrix or with itself. It is therefore probable that in the WT, both the extracellular and intracellular domain could synergise to limit endocytosis at the apical membrane (Richardson, 2010).

During Drosophila eye development, N activation at the D/V boundary is thought to promote cell proliferation within the eye primordium via activation of the JAK-STAT pathway. Consistent with this model, overexpression of the NICD in the developing eye discs leads to overgrowth of the eyes. Indeed, flies heterozygous for N, Dl and Ser have smaller eyes than WT flies. However, the width of the corresponding head capsules remains unchanged, arguing that N activity is not required during the head capsule growth. These data suggest that crb function in this epithelium might not be related to N activity, but is instead linked to that of another growth signalling pathway. The data in the eye strongly argue that loss of crb function causes ectopic activation of the N signalling pathway. Moreover, the overgrowth of crb mutant eye tissue can be suppressed by inhibiting the N pathway. Finally, this analysis of crb mutant clones during oogenesis, together with the inhibition of the S2 cleavage, indicates that crb limits ligand-dependent transactivation of N (Richardson, 2010).

Trafficking, and in particular endocytosis, plays a major role in modulating the N signalling pathway. A steady level of N at the cell surface is achieved by a balance between its activation on the way to the plasma membrane and its endocytosis and degradation. Ligand activation also requires the endocytosis and recycling of the ligand back to the cell surface in the signal-sending cell. Upon ligand binding, the transendocytosis of NECD bound to its ligand into the signal-sending cell allows the S2 cleavage of N, and recent work indicates that proteolytic cleavage of N by the γ-secretase complex occurs in the endocytic compartment. In normal conditions, the S2 cleavage of N is required for its subsequent S3/S4 cleavage by the γ-secretase complex. The increase in the number of NECD/Dl endosomes observed in the absence of crb function cannot be explained by Crb's link to the γ-secretase complex and indicates that in the developing eye disc, crb is also required to limit ligand-dependent transactivation of N (Richardson, 2010).

How does Crb act to regulate ligand-dependent N signalling activity? Structural analysis of Crb, N and Dl shows that these proteins all contain extracellular domains that contain multiple EGF-like repeats. This raises the possibility that the extracellular domain of Crb could interact with N and/or Dl via their EGF-like repeats, thus preventing N-Dl binding. Such specificity towards the N pathway is supported by the observation that there is no significant increase in Hrs-positive endosomes in the absence of crb function. Interestingly, other EGF repeat-containing proteins have been shown to inhibit N signalling; Dlk, for example, interacts and inhibits Notch1 in mammalian cells (Baladron, 2005). Alternatively, Crb might limit the rate of endocytosis of N or Dl. One outcome of this could be that Crb limits Dl activation by regulating its endocytosis and subsequent recycling back to the plasma membrane in order for it to become competent for signalling. Another possibility is that Crb might limit the endocytosis of NECD and Dl into the signal-sending cell, which is proposed to be required for the S2 cleavage of N. An increase in either of these endocytic events due to Crb loss-of-function could result in ectopic N activity. It is difficult to differentiate signal-sending versus signal-receiving cells in the context of the early developing eye epithelium, which prevents determination of whether crb function is required in one of these cell types or in both. In addition, it will be interesting to determine how exactly the extracellular domain of Crb modulates endocytosis at the apical membrane, and whether, as suggested by the present study, this might target specific signalling pathways such as the N pathway. Finally, given that crb itself is a transcriptional target of the N pathway, its ability to limit the activity of the γ-secretase complex, together with its function in limiting ligand-dependent transactivation of N, is likely to provide a very robust negative-feedback loop mechanism to regulate N activity during organogenesis (Richardson, 2010).

Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors

Photoreceptor morphogenesis in Drosophila requires remodelling of apico-basal polarity and adherens junctions (AJs), and includes cell shape changes, as well as differentiation and expansion of the apical membrane. The evolutionarily conserved transmembrane protein Crumbs (Crb) organises an apical membrane-associated protein complex that controls photoreceptor morphogenesis. Expression of the small cytoplasmic domain of Crb in crb mutant photoreceptor cells (PRCs) rescues the crb mutant phenotype to the same extent as the full-length protein. This study shows that overexpression of the membrane-tethered cytoplasmic domain of Crb in otherwise wild-type photoreceptor cells has major effects on polarity and morphogenesis. Whereas early expression causes severe abnormalities in apico-basal polarity and ommatidial integrity, expression at later stages affects the shape and positioning of AJs. This result supports the importance of Crb for junctional remodelling during morphogenetic changes. The most pronounced phenotype observed upon early expression is the formation of ectopic apical membrane domains, which often develop into a complete second apical pole, including ectopic AJs. Induction of this phenotype requires members of the Par protein network. These data point to a close integration of the Crb complex and Par proteins during photoreceptor morphogenesis and underscore the role of Crb as an apical determinant (Muschalik, 2011).

Strikingly, CrbFLAGintra can only affect photoreceptor cell (PRC) shape and adhesion when expressed during late larval and early pupal development. During this period, PRCs undergo substantial morphogenetic changes to adopt their final shape. It is noteworthy that the epithelial cells of the imaginal disc are already well polarised, with an elaborated ZA encircling the apices of the cells. Therefore, the transition from a larval epithelial cell into the highly modified PRC does not require establishment of polarity, but rather mechanisms that control remodelling of polarity and AJs. This study shows that early expression of CrbFLAGintra interferes with this process. Similar conclusions were drawn from studies in the Malpighian tubules, where proper Crb levels are essential for maintenance of polarity and epithelial integrity only during the process of tube elongation, which depends on major cell rearrangements. Once most of the morphogenetic changes and remodelling of the ZA have been completed, PRCs are less susceptible to elevated CrbFLAGintra levels. This is reflected by the observation that cells in which the intracellular domain of Crb is expressed during late pupal development and in the adult, exhibit a normal polarised shape, although junctional and polarity proteins are severely mislocalised in these cells. Two explanations might account for this difference. First, the apical and basolateral membrane domain, as well as the ZA, might be more stable at later stages, so that ectopic apical and junctional components recruited by CrbFLAGintra are unable to affect apico-basal polarity and AJs. Second, some of the downstream factors required for ectopic apical pole formation might no longer be available at later stages. In fact, Baz is removed from the ZA at ~60% of pupal development and becomes enriched in the rhabdomere, similar to aPKC. Furthermore, Par-6 can be found at the basolateral membrane in adult PRCs. Although the polarised shape is unaffected, PRCs overexpressing CrbFLAGintra during later stages display defects in ZA positioning and show an increase in stalk membrane length, the development of which is regulated by crb (Muschalik, 2011).

Loss-of-function studies show that crb is not required for the development of an apical pole, yet, as shown in this study, overexpression of its cytoplasmic tail is sufficient to induce formation of ectopic apical membranes. This raises the question of how ectopic apical poles develop under these conditions. The results, from localisation studies and genetic interactions, indicate that, once initiated, development of an ectopic apical membrane domain relies on the same events and requires identical components to those required for formation of the original apical domain. It is suggested that CrbFLAGintra assembles a new Crb-dependent membrane-associated protein platform at the basolateral membrane domain, enabling the recruitment of effector proteins essential to develop apical features. One of these is βH-spec, which might stabilise the CrbFLAGintra complex by linking it to the underlying spectrin-based membrane skeleton. In fact, removal of one copy of kst strongly suppresses the overexpression phenotype and F-actin accumulates at CrbFLAGintra-positive membranes. In addition, the actin-based cytoskeleton is likely to be directly involved in the formation of ectopic rhabdomeres, as rhabdomeres are composed of microvilli and the terminal web, both of which are actin-rich structures (Muschalik, 2011).

In addition to βH-spec, Par-6 and aPKC are also recruited into the CrbFLAGintra complex and both are required to mediate the CrbFLAGintra-induced overexpression phenotype, as demonstrated by genetic interactions. Furthermore, by using different hypomorphic alleles of aPKC, the function of aPKC in this process could be shown to depend on its ability to bind Par-6 and the presence of an intact kinase domain. In the embryonic epidermis, aPKC ensures apical identity by phosphorylation of the tumour suppressor Lgl, thereby excluding it from the apical domain and restricting its activity to the basolateral side of the cells. Lgl, in contrast, prevents Baz from promoting apical membrane characteristics basolaterally. It is proposed that, upon overexpression of CrbFLAGintra, Lgl is removed from CrbFLAGintra-positive sites through phosphorylation by aPKC, which weakens basolateral membrane identity. The observation that other basolateral markers are absent from ectopic rhabdomeres and diminished at membranes surrounding ectopic rhabdomeres supports this assumption. Furthermore, removal of Lgl from the basolateral membrane upon overexpression of CrbFLAGintra would be consistent with the finding that the lgl loss-of-function phenotype of PRCs mimics the CrbFLAGintra overexpression phenotype. This is similar to the situation in Drosophila embryonic epithelia, and suggests that there is a conserved mechanism for both cell types. Moreover, it might explain why lowering the dose of lgl does not cause an enhancement of the overexpression phenotype. By contrast, an enhancement was found with yrt, which negatively regulates Crb activity, demonstrating that the experimental approach is suitable for the identification of enhancers. Besides Lgl, aPKC also phosphorylates Baz, as shown in the Drosophila follicle epithelium, the embryonic epidermis and PRCs. Phosphorylation of Baz is required to exclude it from the apical membrane, thereby restricting AJs to more basal positions. Apical exclusion of Baz also requires Crb, which prevents binding of Baz to Par-6. It is suggested that the following scenario occurs upon CrbFLAGintra overexpression. First, removal of Lgl from the basolateral membrane enables Baz to spread basolaterally. However, under these conditions, Baz becomes immediately excluded from CrbFLAGintra-positives sites by the same mechanisms occurring at the original apical domain. Delocalisation of Baz, in turn, affects AJs and alters the adhesive properties of the cells, as Baz localisation defines the position of the ZA. The model is consistent with observations from genetic interactions, which have shown that simultaneous expression of CrbFLAGintra and a non-phosphorylatable version of Baz (GFP-Baz-S980A) strongly suppressed the CrbFLAGintra overexpression phenotype. This suppression could be the result of Baz S980A either binding to aPKC-Par-6, or to Sdt, therefore preventing aPKC-Par-6 or Sdt from binding to CrbFLAGintra. Alterations in PRC adhesion might also explain the disruption of the basal lamina and the elimination of PRCs. As no obvious decrease in cell number was noticed at 45-55% of pupal development, elimination is likely to occur during late pupal development (Muschalik, 2011).

Formation of distinct membrane domains also requires polarised protein trafficking. The ectopic localisation of Rh1 and Spam (Eyes shut) upon overexpression of CrbFLAGintra during late larval and pupal development suggests that the apical secretory machinery becomes reorganised under these conditions. In Drosophila PRCs, delivery of various apical proteins, including Rh1, depends on the small GTPase Rab11 and the exocyst component Sec6. A redistribution of these proteins upon overexpression of CrbFLAGintra in developing PRCs might account for the delivery of apical transport vesicles to CrbFLAGintra-positive membranes, which facilitates the formation of a second apical pole. In case of cells with reversed apico-basal polarity the majority of apical vesicles might be targeted to the ectopic apical pole so that the original apical membrane domain receives only minor amounts of apical proteins, with it eventually adopting basolateral membrane identity (Muschalik, 2011).

Another crucial component in polarised vesicle delivery and targeting are phosphoinositides. In developing Drosophila PRCs, PtdIns(3,4,5)P3 is enriched at the apical membrane, whereas PtdIns(4,5)P2 predominantly localises at the ZA. Studies in MDCK (Madin-Darby canine kidney) cells have shown that ectopic localisation of either of the above two phosphoinositides is sufficient to cause a switch from one membrane identity to the other. Strikingly, Baz recruits the lipid phosphatase PTEN (phosphatase and tensin homolog) to the AJs of PRCs and embryonic epidermal cells, and Baz is delocalised upon CrbFLAGintra expression in pupal PRCs. Mutations in, or overexpression of, PTEN cause severe morphogenetic defects, including loss of PRCs and absence or splitting of rhabdomeres, phenotypes that are also observed upon overexpression of CrbFLAGintra. Given these data, it is tempting to speculate that ectopic CrbFLAGintra and its associated proteins cause a modification in the lipid composition of the basolateral membrane domain, thereby remodelling the polarity of PRCs (Muschalik, 2011).

Retromer controls epithelial cell polarity by trafficking the apical determinant Crumbs

The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described in this study, Crb was determined to be an interaction partner and cargo of the retromer complex (See Retromer-mediated sorting). Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals. Loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. These data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. A novel function is also shown for retromer in maintaining epithelial cell polarity (Pocha, 2011a).

This study aimed to identify factors that interact with the cytoplasmic domain of the type I transmembrane protein Crumbs (Crb) and are involved in its trafficking. A strategy was devised to present the Crb cytoplasmic tail on liposomes, a method uniquely suited to recruit and identify coats, because it mimics the native configuration of a receptor tail at the membrane/cytosol interface (Pocha, 2011a).

Proteoliposomes have been used successfully to identify coat complexes and their accessory proteins; however, these studies were restricted to short, chemically synthesized peptides, which severely limited the length of the cytoplasmic tail. To overcome this, this study redesigned the recruitment assay enabling the use of tails expressed and purified from E. coli. A bacterial expression plasmid was designed containing an N-terminal tandem affinity tag followed by a tobacco etch virus (TEV) protease cleavage site and a single cysteine for the chemical coupling to liposomes, to which the cytoplasmic tail of mouse Crb2 (amino acids R1246 to I1282) was fused (Pocha, 2011a).

Because the levels of many transmembrane proteins are regulated by sorting decisions in the early (sorting) endosome, phosphatidylinositol 3-phosphate, the predominant inositol phospholipid of early endosomes, was incorporated into proteoliposomes to selectively enrich endosomal trafficking proteins. These proteoliposomes were used for recruiting cytosolic coat components and other interactors from brain extract], followed by protein identification by tandem mass spectrometry (MS/MS). Crb2 was chosen, because it is the predominant Crb gene expressed in the vertebrate brain. Importantly, the tails of all Crb proteins are highly conserved, suggesting that their trafficking mechanisms may also be conserved. Mass spectroscopic analysis confirmed that large amounts of Crb2 (∼600 MS2 spectra) were coupled onto the liposomes. The most abundant protein isolated (as determined by MS2 spectra) with an established role in the recognition and trafficking of transmembrane cargoes was the retromer subunit Vps35. In addition, Vps26B was identified. Western blotting confirmed the presence of Vps35 in our Crb2 recruitment reactions and showed it to be highly enriched relative to two independent controls (Pocha, 2011a).

The mammalian retromer is composed of a cargo recognition subcomplex containing Vps35, Vps26, and Vps29 and a membrane interacting subcomplex consisting of SNX1/SNX2 and SNX5/SNX6 heterodimers. Because both Vps35 and Vps26 are crucial for cargo recognition and binding, the recruitment data suggest that Crb2 is a retromer cargo (Pocha, 2011a).

To probe the hypothesis that Crb is a retromer cargo, internalization assays were performed by overexpressing Flag-hCrb2 in HeLa cells and analyzing the uptake of anti-Flag antibodies, visualizing compartments through which Crb2 traffics. Previous studies using the classical retromer cargo, the cation-independent mannose-6-phosphate receptor (ciMPR), have shown that retromer subunits and cargo decorate tubules that emanate from endosomes and travel toward the trans-Golgi network (TGN). This study observed colocalization of Crb2 with Vps35 on intracellular vesicles and tubules as well as an overlap with ciMPR- and galactosyltransferase (GalT) label. These data suggest that in HeLa cells, Crb2 travels in retromer-decorated tubules and can traffic via the TGN. However, it should be noted that it does not accumulate there like other retromer cargoes (e.g., ciMPR). Instead, Crb2 appears to undergo rapid transport back to the plasma membrane. RNA interference (RNAi) suppression of Vps35 in HeLa cells displays enhanced localization of Crb2 in lysosomal structures positive for Lamp-I, a phenotype described previously for other retromer cargoes. These data are all in line with Crb being a potential retromer cargo (Pocha, 2011a).

To study the functional interaction between Crb and retromer in Drosophila, a previously generated null allele of Vps35, Vps35MH20 was used. As a result of strong maternal contribution, animals homozygous for this allele reach the third larval instar, allowing analysis of Crb in homozygous mutants. Because retromer is required for the retrieval of receptors from endosomes and thus the prevention of their lysosomal degradation, total Crb levels were analyzed and found to be reduced in Vps35MH20 heterozygote third-instar larvae compared to stage-matched wild-type (WT) larvae and dramatically reduced in Vps35MH20 homozygotes. Analysis of the mRNA levels of Crb showed that loss of Vps35 has very little effect on crb transcripts, suggesting that the dramatic reduction in Crb protein that was seem is due to posttranscriptional regulation of Crb by Vps35 (Pocha, 2011a).

This led to an investigation of Crb at a cellular level. For this, two different epithelia, wing discs of third-instar larvae and the follicle epithelium, were chosen. Clones of Vps35MH20 mutant cells in wing disc epithelia, labeled with GFP using the mosaic analysis with a repressible cell marker (MARCM) system, were induced by heat shock-Flp at early larval stages. Crb localizes to the subapical region of wing disc epithelial cells. In agreement with results from western blot analysis, Crb staining is decreased in Vps35MH20 clones. Quantification of the fluorescence intensity in the clone and in surrounding tissues revealed that there is an ∼50% reduction in Crb signal within Vps35MH20 clones. The wing discs of Vps35MH20 homozygous animals are small and show variable morphological defects, presumably as a result of defective Wingless secretion. Analysis of Crb localization (by immunofluorescence) and protein levels (by western blotting) in Vps35MH20 hetero- and homozygous wing discs corroborated the data that were obtained using Vps35MH20 clones and larval lysate, respectively (Pocha, 2011a).

The stability of the cargo-selective retromer subcomplex is dependent on the presence of all of its components [8 and 16]. To show that the loss of Crb is due to loss of retromer function rather than just the loss of Vps35, the effect was compared of Vps26 and Vps35 knockdown in the posterior compartment of the wing disc using engrailed-Gal4 to drive UAS-Vps26RNAi and UAS-Vps35RNAi. Hedgehog expression, which is unperturbed by loss of retromer, served to label the posterior compartment. Expression of either RNAi construct resulted in a clear reduction of Crb staining in the posterior compartment (∼50% reduction in fluorescence). Expression of engrailed-Gal4 alone had no effect on Crb. From these data, it is concluded that the retromer cargo recognition subcomplex is required for the maintenance of Crb levels (Pocha, 2011a).

To further analyze the relation between Crb and retromer, the follicular epithelium, which surrounds the germline cysts of the Drosophila ovary, was examined. Previous work has identified key roles for Crb in polarization of the follicular epithelium. Crb localizes to the entire apical membrane of the follicle epithelial cells, with very little detectable in the cytoplasm. Vps35MH20 clones show strong reduction in Crb staining and protein loss from the apical membrane. Interestingly, although Crb staining at the apical membrane is strongly reduced, it is not detected at increased levels within the cytoplasm, suggesting that Crb is not merely mislocalized but reduced at the protein level, as shown in larvae. The cytoplasmic domain of Crb organizes an apical, membrane-associated protein complex by recruiting the scaffolding proteins Stardust (Sdt), DPATJ, and DLin-7. Therefore, the apical localization of Sdt in the follicular epithelium was assessed, and at was found to be heavily reduced in Vps35MH20 clones. Probing whole larval lysates from third-instar WT and Vps35MH20 hetero- and homozygotes for Sdt confirmed that at the protein level, like Crb, Sdt shows a dose dependence on Vps35. Thus, retromer function in maintaining Crb levels and function is conserved between wing and follicle epithelia (Pocha, 2011a).

Interestingly, in some Vps35MH20 clones, the strict monolayer structure of the epithelium is disrupted and the tissue appears multilayered, an indication of polarity defects and characteristic of loss of Crb at early stages of follicle development, whereas loss at later stages results only in the mislocalization of other polarity proteins, without affecting tissue integrity. Multilayering was observed in 19% of Vps35MH20 clones in follicles between stages 7 and 10 and did not appear to be dependent on clone size or position. Given that various links between Crb and Notch have been reported, tests were performed to see whether the multilayering phenotype observed in the follicle epithelium upon loss of Vps35MH20 could be the result of defective Notch signaling. The expression of Notch and Hindsight, a transcription factor downstream of Notch signaling that represses proliferation in the follicle epithelium, were examined in Vps35MH20 mutant clones. Both showed wild-type expression, suggesting that Notch signaling is not affected by loss of retromer, similar to previous findings in the wing disc (Pocha, 2011a).

To test whether the loss of Crb in retromer mutants is due to missorting of Crb to the lysosome, follicles harboring Vps35MH20 clones were incubated in leupeptin, a potent inhibitor of lysosomal proteases. After a 3 hr incubation, a dramatic accumulation of Crb was observed in punctae within the cytoplasm of Vps35MH20 cells, a phenomenon that was not seen in WT tissue or in follicles containing Vps35MH20 clones that were incubated in control medium lacking leupeptin. Additionally, colocalization of these intracellular Crb punctae with LysoTracker was observed. Together with the reduction of Crb protein levels and constant crb mRNA levels in Vps35MH20 larvae and tissue, these data strongly suggest that retromer ablation leads to lysosomal degradation of Crb, as observed for other retromer cargoes (Pocha, 2011a).

To test whether retromer functions after endocytosis of Crb, internalization of Crb from the plasma membrane was blocked by expression of a dominant-negative construct of shibire (dynamin) or by incubating follicles in dynasore, a dynamin inhibitor. In Vps35MH20 clones, this resulted in the accumulation of Crb at the plasma membrane, confirming that retromer is indeed transporting Crb after internalization from the plasma membrane (Pocha, 2011a).

Crb is required, together with atypical protein kinase C (aPKC), to restrict Bazooka/Par3 to the zonula adherens, an adhesion belt at the apex of epithelial cells, in the follicle epithelium, and in photoreceptor cells, thus excluding it from the apical membrane and specifying the border between apical and lateral domains. In previous studies, it was shown that the localization of aPKC and Par6 was dependent on Crb. To test whether loss of retromer phenocopies the loss of Crb, aPKC and Par6 localization were examined in follicles containing Vps35MH20 clones. Indeed, the level of both proteins is reduced at the apical surface in Vps35MH20 clones. Interestingly, unlike Crb and the Crb complex member Sdt, Par6 and aPKC protein levels are not reduced in Vps35MH20 mutant larvae. Therefore, it is likely that the loss of Par6 and aPKC from the apical membrane of Vps35MH20 clones in the follicle epithelium is due to loss of cell polarity in the absence of Crb rather than loss of the proteins themselves (Pocha, 2011a).

To test this, Crb was overexpressed in Vps35MH20 clones. Because overexpression of Crb causes defects in epithelial cell polarity, Crb overexpression was induced using GABFc204 Gal4, a follicle epithelium-specific driver that starts expression late in follicle development (stage 8). Thereby, it was possible to rescue the apical localization of Par6 and Sdt. This rescue did not appear to be dependent on clone size or location. From these data, it is concluded that the loss of polarity observed in retromer mutant clones is the direct result of loss of Crb (Pocha, 2011a).

The identification of Crb as a retromer cargo confirms the hypothesis that one crucial step in the regulation of Crb occurs at the early (sorting) endosome and, importantly, fills a gap in the current understanding of Crb trafficking. Previous reports showed that transport of Crb to the plasma membrane is reliant on Rab11, the exocyst and Cdc42 in Drosophila embryonic epithelia. Internalization of Crb from the plasma membrane into endosomes is mediated by the syntaxin Avalanche and Rab5. This study has shown that retromer is responsible for sorting Crb away from the degradative pathway and into a recycling one, thus allowing a high level of control over the amount of cellular Crb, previously shown to be vital for maintaining epithelial polarity and integrity, as demonstrated by numerous loss- and gain-of-function studies. Interestingly, retromer was previously shown to play a role in the apical delivery of the polymeric immunoglobulin receptor (pIgR) in Madin-Darby canine kidney cells. However, as for Crb, it remains unclear whether this transport occurs via the TGN, via recycling endosomes, or through alternative pathways. The exact trafficking itinerary of Crb following recycling by retromer remains unclear and may depend upon the purpose of Crb recycling (Pocha, 2011a).

Which function of Crb is the prime target of retromer-driven retrieval? Is this a Crb level-sensing mechanism, in which retromer regulates the amount of protein at the plasma membrane, which is crucial for cell homeostasis? To date, all known functions of Crb require an intact Crb complex. By controlling the recycling of Crb and thereby its level at the plasma membrane, retromer could define the amount of Crb available for complex formation. Alternatively, it is tempting to speculate that Crb, much like Wntless (Wls), acts as a transport receptor and that apical delivery of its (yet to be identified) ligand or many ligands is the main purpose of its recycling to the TGN. These are fascinating hypotheses that will be the focus of future research (Pocha, 2011a).

Retromer regulates apical-basal polarity through recycling Crumbs

Epithelial cells are characterized by an 'apical–basal' polarization. The transmembrane protein Crumbs (Crb) is an essential apical determinant which confers apical membrane identity. Previous studies indicated that Crb did not constantly reside on the apical membrane, but was actively recycled. However, the cellular mechanism(s) underlying this process was unclear. This study shows that in Drosophila, retromer, which was a retrograde complex recycling certain transmembrane proteins from endosomes to trans-Golgi network (TGN), regulates Crb in epithelial cells. In the absence of retromer, Crb was mis-targeted into lysosomes and degraded, causing a disruption of the apical–basal polarity. It was further shown that Crb co-localizes and interacts with retromer, suggesting that retromer regulated the retrograde recycling of Crb. These data uncover the role of retromer in regulating apical–basal polarity in epithelial cells and identify retromer as a novel regulator of Crb recycling (Zhou, 2011).

It is important to understand how apical–basal polarity is regulated during development. This study has uncovered a role of retromer in regulating apical–basal polarity in epithelial cells. The analysis showed that this regulation was achieved through stabilizing the apical determinant Crb, which acted as a novel transmembrane cargo of retromer. It is proposed that retromer regulates apical–basal polarity by mediating the retrograde transportation of Crb from endosomes to TGN. This study analysed the regulation of Crb in Drosophila embryos. Taken together with the studies of Pocha (2011), it is proposed that the recycling of Crb by retromer is a general regulation mechanism in all major epithelial cell types in Drosophila (Zhou, 2011).

A previous study suggested a role of recycling endosomes in regulating Crb recycling (Roeth, 2009). A question of interest lies in the relationship between recycling endosomes- and retromer-mediated Crb recycling. Retromer mediates the transportation from early/late endosomes to TGN. It is as yet unknown whether recycling endosomes- and retromer-mediated recycling are two routes in parallel or whether recycling endosomes serve as a stop during retromer-mediated early/late endosomes to TGN transportation. One intriguing observation is that in Rab11 defective embryos (expressing dominant negative protein), the Crb defect mainly occurred in the ventral ectoderm but much less in the dorsal ectoderm (Roeth, 2009). However, at the same stage, this study found that the retromer mutant embryos had a wider range of Crb defect, which occurred not only in the ventral ectoderm but also largely in the dorsal ectoderm and head epithelium. The weaker Crb defect in Rab11 defective embryos might result from the usage of dominant negative instead of null mutants. However, another interesting possibility is that the retromer-mediated recycling is a general mechanism of Crb recycling in all epithelial cells and the recycling endosome-mediated recycling contributes additionally to Crb recycling in the ventral ectoderm, where a high rate of Crb recycling may occur. In other words, in the ventral ectoderm, the recycling endosome route and the retromer route might work in parallel to recycle Crb. Further work is needed to explore the relationship of recycling endosome- and retromer-mediated recycling (Zhou, 2011).

Apical deficiency triggers JNK-dependent apoptosis in the embryonic epidermis of Drosophila

Epithelial homeostasis and the avoidance of diseases such as cancer require the elimination of defective cells by apoptosis. This study investigated how loss of apical determinants triggers apoptosis in the embryonic epidermis of Drosophila. Transcriptional profiling and in situ hybridisation show that JNK signalling is upregulated in mutants lacking Crumbs or other apical determinants. This leads to transcriptional activation of the pro-apoptotic gene reaper and to apoptosis. Suppression of JNK signalling by overexpression of Puckered, a feedback inhibitor of the pathway, prevents reaper upregulation and apoptosis. Moreover, removal of endogenous Puckered leads to ectopic reaper expression. Importantly, disruption of the basolateral domain in the embryonic epidermis does not trigger JNK signalling or apoptosis. It is suggested that apical, not basolateral, integrity could be intrinsically required for the survival of epithelial cells. In apically deficient embryos, JNK signalling is activated throughout the epidermis. Yet, in the dorsal region, reaper expression is not activated and cells survive. One characteristic of these surviving cells is that they retain discernible adherens junctions despite the apical deficit. It is suggested that junctional integrity could restrain the pro-apoptotic influence of JNK signalling (Kolahgar, 2011).

This study has shown that apical, but not basolateral, disruption leads to apoptosis in a developing epithelium. It was also showed that JNK signalling is a key intermediate in the signal transduction mechanism that triggers apoptosis in response to the loss of apical determinants. Apical disruption leads to activation of JNK signalling, which in turn activates transcription of the pro-apoptotic gene rpr. Moreover, rpr expression is not activated in apically disrupted embryos that are prevented from activating JNK signalling. Interestingly, in bicoid-deficient embryos and other segmentation mutants, cell fate misspecification requires activation of a different pro-apoptotic gene, hid. Therefore, distinct quality control pathways might exist to ensure that different forms of defective cells are removed from developing epithelia. JNK signalling has been shown to mediate apoptosis in a variety of other situations, including after DNA damage. However, JNK signalling does not necessarily cause apoptosis. Indeed, this pathway modulates many other cell activities, such as proliferation, differentiation and morphogenesis. What conditions determine whether JNK triggers apoptosis or not is an important issue. Another obvious question raised by the current findings concerns the nature of the mechanism that triggers JNK signalling following the loss of apical determinants (Kolahgar, 2011).

This study has shown that, in the embryonic epidermis, JNK signalling is activated by the loss of apical, not basolateral, determinants. In fact, reduction of lgl activity prevents JNK activation in crb mutant embryos. Similarly, Scrib knockdown prevents JNK activity in the mouse mammary epithelium, suggesting that the loss of the basolateral domain could have a general anti-JNK (and perhaps anti-apoptotic) activity. Although JNK activation has been documented in tissues that lack a basolateral determinant, it is suggested that this might be an indirect consequence of cell competition, which triggers JNK signalling, or of the specific experimental conditions (partial reduction of Puc activity). Overall, the results suggest the existence of an apical domain-dependent activity that modulates JNK signalling in the embryonic epidermis of Drosophila. This activity could be similar to that postulated to be at work in cultured MDCK cells, but is likely to be distinct from the process that leads to apoptosis in response to mosaic disruption of the basolateral domain in imaginal discs (Kolahgar, 2011).

The mechanism underlying the activation of JNK signalling by loss of apical determinants remains unknown. For example, it is uncertain at this point whether there is an apically localised activity that directly modulates JNK signalling or whether a more indirect route is at work (paths 1 and 2 respectively; see Signalling upstream and downstream of JNK). Since apical organisation is required for the establishment of adherens junctions, it is conceivable that the effect of apical disruption on JNK signalling is mediated by junctional disruption. This possibility is compatible with the absence of ectopic JNK activation in basolateral mutants, in which E-cadherin remains localised to patches at the cell surfac. However, one would have to invoke that slight junctional disruption is sufficient to trigger JNK signalling, as this pathway is upregulated in the dorsal region of crb mutants, where the extent of junctional disruption is relatively mild. It has not been possible to discriminate between paths 1 and 2, partly because of the current difficulty in eliminating adherens junctions from early Drosophila embryos. Future work will require novel means of interfering specifically with adherens junctions. Considering the lack of involvement of Egr, it will also be necessary to identify the upstream components of JNK signalling that respond to epithelial disruption (Kolahgar, 2011).

Overexpression of Puc, a feedback inhibitor of JNK signalling, prevents apoptosis in the ventral epidermis of crb embryos. This is clear evidence that JNK signalling is required for apical deficit to trigger apoptosis. However, it is well established that JNK signalling does not necessarily lead to apoptosis. This is particularly well illustrated by the situation at the dorsal edge of wild-type embryos, where JNK is highly active without triggering apoptosis. Moreover, in crb mutants, a 6- to 10-cell-wide band of dorsal tissue is refractory to the pro-apoptotic influence of JNK signalling. Therefore, additional conditions must be met for JNK signalling to activate rpr expression and trigger apoptosis. This study has identified two situations when refractory cells succumb to the pressure of JNK signalling (see Signalling upstream and downstream of JNK). One involves the reduction of Puc and the other the removal of zygotic E-cadherin activity. The first situation suggests that endogenous Puc can limit the ability of JNK to activate rpr expression and trigger apoptosis. The important role of Puc in preventing cell death is also highlighted by the extensive apoptosis seen in embryos lacking both maternal and zygotic Puc activity. Puc could act solely by limiting the extent of JNK signalling, thus preventing the very high level of signalling required for rpr expression. Alternatively, or in addition, Puc could have an activity that specifically prevents certain genes, such as rpr, from being spuriously activated. In any case, it is likely that the regulatory relationships between JNK, Puc and apoptosis are influenced by the cellular context (e.g., the state of adherens junctions (Kolahgar, 2011).

JNK signalling triggers rpr expression (and apoptosis) more readily if adherens junctions are weakened or disrupted. Therefore, junctional integrity could also protect epithelial cells from the pro-apoptotic effects of JNK signalling. Dorsoventral differences in junctional integrity and remodelling have been noted in the embryonic epidermis of Drosophila and these might explain why these two regions respond differently to the loss of crb. The results suggest that residual junctional integrity in the dorsal epidermis prevents JNK signalling from activating rpr expression. It is conceivable that a protective signal emanates from adherens junctions. Alternatively, junctional disruption could interfere with the ability of Puc to rein in the effect of JNK signalling on rpr expression. Although differential junctional dynamics between the dorsal and ventral epidermis could determine the propensity to undergo apoptosis, the possibility cannot be excluded that other dorsoventral determinants are at work too (Kolahgar, 2011).

This study has shown that loss of apical polarity leads to apoptosis by activating JNK signalling and causing junctional disruption. It is expected that this response, which is readily detectable in the crb mutant condition, might reflect a process that ensures the removal of abnormal and damaged cells during epithelial homeostasis. It is hoped that understanding the machinery that links epithelial disruption to JNK signalling and the transcription of pro-apoptotic genes will suggest means of reactivating this pathway in pathological situations (Kolahgar, 2011).

Crumbs limits oxidase-dependent signaling to maintain epithelial integrity and prevent photoreceptor cell death

Drosophila melanogaster Crumbs (Crb) and its mammalian orthologues (CRB1-3) share evolutionarily conserved but poorly defined roles in regulating epithelial polarity and, in photoreceptor cells, morphogenesis and stability. Elucidating the molecular mechanisms of Crb function is vital, as mutations in the human CRB1 gene cause retinal dystrophies. This study reports that Crb restricts Rac1-NADPH oxidase-dependent superoxide production in epithelia and photoreceptor cells. Reduction of superoxide levels rescued epithelial defects in crb mutant embryos, demonstrating that limitation of superoxide production is a crucial function of Crb and that NADPH oxidase and superoxide contribute to the molecular network regulating epithelial tissue organization. It was further shown that reduction of Rac1 or NADPH oxidase activity or quenching of reactive oxygen species prevents degeneration of Crb-deficient retinas. Thus, Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1-NADPH oxidase complex activity. Collectively, these results elucidate an important mechanism by which Crb functions in epithelial organization and the prevention of retinal degeneration (Chartier, 2012).

Crumbs regulates polarity and prevents light-induced degeneration of the simple eyes of Drosophila, the ocelli

The evolutionary conserved transmembrane protein Crumbs (Crb) regulates morphogenesis of photoreceptor cells in the compound eye of Drosophila and prevents light-dependent retinal degeneration. This study examine the role of Crb in the ocelli, the simple eyes of Drosophila. Crb is expressed in ocellar photoreceptor cells, where it defines a stalk membrane apical to the adherens junctions, similar as in photoreceptor cells of the compound eyes. Loss of function of crb disrupts polarity of ocellar photoreceptor cells, and results in mislocalisation of adherens junction proteins. This phenotype is more severe than that observed in mutant photoreceptor cells of the compound eye, and resembles more that of embryonic epithelia lacking crb. Similar as in compound eyes, crb protects ocellar photoreceptors from light induced degeneration, a function that depends on the extracellular portion of the Crb protein. These data demonstrate that the function of crb in photoreceptor development and homeostasis is conserved in compound eyes and ocelli and underscores the evolutionarily relationship between these visual sense organs of Drosophila (Mishra, 2012).

Novel neuroprotective function of apical-basal polarity gene crumbs in amyloid β 42 (aβ42) mediated neurodegeneration

Alzheimer's disease (AD), a progressive neurodegenerative disorder with no cure to date, is caused by the generation of amyloid-β-42 (Aβ42) aggregates that trigger neuronal cell death by unknown mechanism(s). This study has developed a transgenic Drosophila eye model where misexpression of human Aβ42 results in AD-like neuropathology in the neural retina. An apical-basal polarity gene crumbs (crb) was identified as a genetic modifier of Aβ42-mediated-neuropathology. Misexpression of Aβ42 caused upregulation of Crb expression, whereas downregulation of Crb either by RNAi or null allele approach rescued the Aβ42-mediated-neurodegeneration. Co-expression of full length Crb with Aβ42 increased severity of Aβ42-mediated-neurodegeneration, due to three fold induction of cell death in comparison to the wild type. Higher Crb levels affect axonal targeting from the retina to the brain. The structure function analysis identified intracellular domain of Crb to be required for Aβ42-mediated-neurodegeneration. This study has demonstrated a novel neuroprotective role of Crb in Aβ42-mediated-neurodegeneration (Steffensmeier, 2013).

Seamless tube shape is constrained by endocytosis-dependent regulation of active moesin
Most tubes have seams (intercellular or autocellular junctions that seal membranes together into a tube), but 'seamless' tubes also exist. In Drosophila, stellate-shaped tracheal terminal cells make seamless tubes, with single branches running through each of dozens of cellular extensions. Mutations in braided impair terminal cell branching and cause formation of seamless tube cysts. This study shows that braided encodes Syntaxin7 and that cysts also form in cells deficient for other genes required either for membrane scission (shibire) or for early endosome formation (Rab5, Vps45, and Rabenosyn-5). These data define a requirement for early endocytosis in shaping seamless tube lumens. Importantly, apical proteins Crumbs and phospho-Moesin accumulate to aberrantly high levels in braided terminal cells. Overexpression of either Crumbs or phosphomimetic Moesin induced lumenal cysts and decreased terminal branching. Conversely, the braided seamless tube cyst phenotype was suppressed by mutations in crumbs or Moesin. Indeed, mutations in Moesin dominantly suppressed seamless tube cyst formation and restored terminal branching. It is proposed that early endocytosis maintains normal steady-state levels of Crumbs, which recruits apical phosphorylated (active) Moe, which in turn regulates seamless tube shape through modulation of cortical actin filaments (Schottenfeld-Roames, 2014).

Hydroxylated sphingolipid biosynthesis regulates photoreceptor apical domain morphogenesis

Apical domains of epithelial cells often undergo dramatic changes during morphogenesis to form specialized structures, such as microvilli. This study addressed the role of lipids during morphogenesis of the rhabdomere, the microvilli-based photosensitive organelle of Drosophila photoreceptor cells. Shotgun lipidomics analysis performed on mutant alleles of the polarity regulator crumbs, exhibiting varying rhabdomeric growth defects, revealed a correlation between increased abundance of hydroxylated sphingolipids and abnormal rhabdomeric growth. This could be attributed to an up-regulation of fatty acid hydroxylase transcription. Indeed, direct genetic perturbation of the hydroxylated sphingolipid metabolism modulated rhabdomere growth in a crumbs mutant background. One of the pathways targeted by sphingolipid metabolism turned out to be the secretory route of newly synthesized Rhodopsin, a major rhabdomeric protein. In particular, altered biosynthesis of hydroxylated sphingolipids impaired apical trafficking via Rab11, and thus apical membrane growth. The intersection of lipid metabolic pathways with apical domain growth provides a new facet to understanding of apical growth during morphogenesis (Hebbar, 2020).

Despite the importance of intact microvilli for the homeostasis of many epithelia, regulators controlling microvilli formation are only partially understood. This study used the rhabdomere of Drosophila PRCs, a highly expanded and elaborated apical membrane, as a model to dissect mechanisms controlling the formation of microvilli. It was shown that increased transcription of fatty acid 2-hydroylase (fa2h), followed by increased levels of hydroxylated sphingolipids, is associated with defective rhabdomere morphogenesis. This is, at least partially, due to impaired apical trafficking of Rh1, an important structural component of the rhabdomeral membrane. These results also contribute to understanding of the role of crb in rhabdomere morphogenesis. They imply that increased levels of hydroxylated sphingolipids particularly impact one aspect of rhabdomeral growth, namely the addition of new microvilli, resulting in the extension of the rhabdomere along the proximodistal axis. Rhabdomeral thickness, determined by the length of the microvilli, is not obviously affected by changes in these lipids (Hebbar, 2020).

The connection between hydroxylated sphingolipid metabolism and apical domain morphogenesis is not unprecedented. Increased hydroxylation of sphingolipids was observed to be one of the major lipidomic changes during the polarization of MDCK cells, and inhibition of sphingolipid biosynthesis resulted in a reduced number of apical microvilli on their apical surface. Likewise, the Caenorhabditis elegans orthologue of fa2h, fath-1, was identified in a screen for genes affecting intestinal polarity. Until now, however, the link between hydroxylated sphingolipids and their metabolism and polarization of epithelial cells was limited to circumstantial evidence. This study now clearly demonstrate that manipulating hydroxylated sphingolipid metabolism, at least in a sensitized genetic background, is sufficient to modulate rhabdomere/apical domain extension (Hebbar, 2020).

How do hydroxylated sphingolipids and their metabolism affect rhabdomeric/apical domain growth? Given the low abundance of hydroxylated sphingolipids in the overall fly eye lipidome, it is considered unlikely that they play any major structural role in rhabdomere morphogenesis, e.g., by promoting the stability of Rhodopsin in the rhabdomeric membrane. The conclusion is favored that afa2h and thereby hydroxylated sphingolipids play a role as regulators of apical trafficking. This conclusion is based on two findings: (1) less newly synthesized Rh1 is delivered to the rhabdomeres upon overexpression of afa2h, as revealed by blue light-induced chromophore supply (BLICS) assays, which mimic Rh1 pulse-chase experiments; and (2) a similar defect in Rh1 trafficking has been observed in crb mutant PRCs , which have increased levels of afa2h expression. In particular, an effect on post-Golgi trafficking of Rh1 via the Rab11-mediated pathway was identified. Accumulation of cytoplasmic vesicles is observed upon genetic perturbation of Rab11 and/or genes that encode for Rab11 interacting proteins. However, no obvious accumulation of similar vesicles was observed in crb mutant PRCs. Thus, the trafficking defect observed upon afa2h overexpression cannot be attributed to a general increase/decrease in Rab11 compartments, but rather alludes to a defect in sorting apical cargo. These results are consistent with studies in C. elegans, where apical compartments labeled with Rab11 and Rab7 are affected upon loss of fath-1 (Li, 2018; Hebbar, 2020 and references therein).

Hydroxylated sphingolipids, as glycosphingolipids, are proposed to regulate the sorting of apically directed vesicles by combining different polarity cues (Zhang, 2011). In line with this, it is proposed that increased afa2h transcription (and hence an increase in hydroxylated sphingolipids) prevents apical membrane (rhabdomere) growth by inhibiting trafficking of Rh1 via the apical Rab11 compartments. By regulating the amount of Rh1 delivered to the rhabdomere, afa2h modulates the amount of rhabdomeric membrane in Drosophila PRCs, and hence has an impact on rhabdomere growth. Whether afa2h additionally promotes stability of Rhodopsin in the rhabdomeric membrane via the presence of more hydroxylated sphingolipids could not be addressed here. Interestingly, exogenously labeled hydroxylated fatty acids (the precursors of hydroxylated sphingolipids) preferentially distributed to apically localized membrane-bound compartments in the epithelial cells of the C. elegans gut (Li et al, 2018) (Hebbar, 2020).

Unexpectedly, afa2h overexpression or knockdown in an otherwise wild-type background does not result in an obvious rhabdomeric extension phenotypes. This may be because Rh1 trafficking depends on the concerted action of Rab1, Rab6, Rab11, Rip11, and MyoV as well as the Rab-effector proteins such as Parcas (Rab11GEF) and Rab11 interacting proteins such as dRip11, and to the amount of F-actin. Although sphingolipids can induce changes in the cytoskeleton, no overt changes in the staining of F-actin were observed in the rhabdomere. Therefore, it is speculated that increased afa2h acts to fine-tune growth by slowing down delivery of Rh1 and hence addition of new rhabdomeric membrane (Hebbar, 2020).

Yet, in a sensitized background, i.e., in cells homozygous mutant for crb, which have impaired Rh1 trafficking or upon carotenoid depletion, which also reduces Rh1 levels, afa2h overexpression now decreases Rh1 delivery via Rab11. Crb normally limits oxidative stress, and the resulting oxidative status (low oxidative stress) normally limits afa2h expression. In crb mutants, however, an altered oxidative status (increased oxidative stress) causes an up-regulation of afa2h transcription, which, in turn, results in severely reduced Rh1 delivery and improperly extended rhabdomeres. The afa2h dependence of rhabdomeric growth described in this study is only one aspect of the pleiotropic cellular response to an altered redox status of increased oxidative stress signaling due to loss of crb. Interestingly, loss of crb in epithelial cells of larval salivary glands impairs Rab6-, Rab11-, and Rab30-dependent apical trafficking and hence apical membrane homeostasis. Whether this is also caused by increased oxidative stress and/or defects in sphingolipid metabolism remains to be analyzed (Hebbar, 2020).

In conclusion, this work elucidates an interplay between oxidative stress, lipid metabolism, and apical domain growth during PRC morphogenesis. It will be interesting to investigate whether this link between cell metabolism and apical membrane growth and morphogenesis is a more widespread phenomenon in epithelial biology (Hebbar, 2020).


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crumbs: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 5 August 2023

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