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palisade: Biological Overview | References

The innermost layer of the Drosophila eggshell, the vitelline membrane, provides structural support and positional information to the embryo. It is assembled in an incompletely understood manner from four major proteins to form a homogeneous, transparent extracellular matrix. This study shows that RNAi knockdown or genetic deletion of a minor constituent of this matrix, Palisade, results in structural disruptions during the initial synthesis of the vitelline membrane by somatic follicle cells surrounding the oocyte, including wide size variation among the precursor vitelline bodies and disorganization of follicle cell microvilli. Loss of Palisade or the microvillar protein Cad99C results in abnormal uptake into the oocyte of sV17, a major vitelline membrane protein, and defects in non-disulfide cross-linking of sV17 and sV23, while loss of Palisade has additional effects on processing and disulfide cross-linking of these proteins. Embryos surrounded by the abnormal vitelline membranes synthesized when Palisade is reduced are fertilized but undergo developmental arrest, usually during the first 13 nuclear divisions, with a nuclear phenotype of chromatin margination similar to that described for wild-type embryos subjected to anoxia. These results demonstrate that Palisade is involved in coordinating assembly of the vitelline membrane and is required for functional properties of the eggshell (Elalayli, 2008).

Assembly of extracellular matrices as diverse as tendons, basement membranes, and the pearl oyster shell requires specific cell-matrix interactions, self-assembly processes, accessory proteins modulating assembly and morphology of networks, and post-secretory proteolysis and cross-linking to generate structures with physical and functional properties appropriate to a given tissue. The Drosophila eggshell provides a valuable genetic model for studying regulated matrix assembly during development (Waring, 2000). Synthesized by somatic follicle cells surrounding the oocyte, the eggshell plays roles in sperm entry, gas exchange, physical protection of the egg, and embryonic patterning. It has a complex architecture, with a homogeneous vitelline membrane layer immediately adjacent to the oocyte, surrounded sequentially by a wax layer, inner chorionic layer, endochorion, and exochorion. The vitelline membrane is of particular interest as, in addition to its structural roles, it serves as a stable repository for positional information used in locally activating Torso-like, a receptor tyrosine kinase that patterns the embryo termini, and may perform a similar role in establishment of the embryonic dorsoventral axis. Such roles may be analogous to the storage of growth factors and localized glycoproteins within the extracellular matrix of vertebrate tissues, and argue for the importance of understanding the molecular interactions involved in the assembly and function of this eggshell layer (Elalayli, 2008).

The vitelline membrane is the first eggshell layer to be synthesized, in Stages 8-10 of oogenesis. It is first visible as a discontinuous layer of precursor vitelline bodies within the perivitelline space, comprising aggregates of secreted eggshell proteins. The vitelline bodies are physically separated from one another by microvilli that extend from the oocyte and follicle cells, spanning the perivitelline space. Disorganization of the follicle cell microvilli caused by loss of the fly PCDH15 protocadherin orthologue, Cad99C, results in disordered vitelline membrane formation (D'Alterio, 2005; Schlichting, 2006), suggesting that the microvilli may act as a scaffold on which the vitelline bodies are assembled. These microvilli shorten in Stage 10B as the vitelline bodies coalesce to form a continuous vitelline membrane, which then thins over the remainder of oogenesis (Elalayli, 2008).

At a molecular level, four major structural proteins of the vitelline membrane have been identified (sV17, sV23, VM32E, VM34C) (Waring, 2000). These proteins are small (116-168 amino acids), rich in proline and alanine residues, and share a highly conserved 38 amino acid VM domain. Their assembly into the vitelline membrane involves stage-specific proteolytic processing (Pascucci, 1996) and disulfide cross-linking (Andrenacci, 2001; LeMosy, 2000; Manogaran, 2004; Petri, 1979) during oogenesis, and non-disulfide cross-linking occurring rapidly following ovulation and likely involving cross-linking of tyrosines (Heifetz, 2001; Petri, 1976). The VM domain is essential for disulfide cross-linking, containing the 3 cysteines found in each of these proteins. Partial deletions of this domain in sV23 and in VM32E result in proteins that are poorly secreted and unstable (sV23 only) and that are unable to stably incorporate into the matrix (Andrenacci, 2001; Manogaran, 2004). The later tyrosine-based cross-linking depends upon the earlier alignment of cross-linking sites during assembly of the vitelline membrane proteins. In particular, the N-terminal domain of sV23, although later cleaved away from the mature protein, appears to be critical for the initial alignment of vitelline membrane proteins into an array or polymer (Elalayli, 2008).

Of the major structural proteins, only sV23 has been studied as a null mutant resulting in 100% collapsed eggs that remain unfertilized (Savant, 1989). Several other genes are required for vitelline membrane assembly, although their roles remain unclear. Eggshell collapse and defects in vitelline membrane cross-linking are seen in a subset of mutations in three oocyte surface proteoglycans, Nasrat, Polehole, and Nudel, which have additional roles in embryonic patterning (Cernilogar, 2001; Degelmann, 1990; Hong, 1996; LeMosy, 2000; Turcotte, 2002). Two additional genes, yellow-g and alpha-methyldopa hypersensitive, are required for vitelline membrane integrity and have been postulated to have roles in tyrosine-based cross-linking (Claycomb, 2004; Konrad, 1993). Isolated loss of Nudel's protease activity, also required for dorsoventral patterning, leads to a specific and complete defect in tyrosine cross-linking, and a milder phenotype of vitelline membrane permeability and fragility without spontaneous eggshell collapse (LeMosy, 2000). With the exception of Nudel protease, known to become active only at the time of tyrosine cross-linking, it is not clear what processes of eggshell assembly are first affected by mutation of these proteins (Elalayli, 2008).

As a complement to ongoing studies in other labs of the major structural components of the vitelline membrane, the LeMosy lab has been concerned with identifying minor proteins that may perform important structural or regulatory functions during eggshell formation (Fakhouri, 2006), e.g., in the alignment of vitelline membrane proteins into an organized array. This paper describes one such protein that has been named Palisade because its loss results in disruption of the palisade-like arrangement of vitelline bodies and follicle cell microvilli within the perivitelline space during vitelline membrane secretion. Palisade is shown to be an integral component of the vitelline membrane whose knockdown or deletion affects the cross-linking and localization of major vitelline membrane proteins, consistent with a role in coordinating their assembly. Additionally, knockdown of Palisade results in a majority of embryos that initiate development but arrest prior to cellularization, supporting a developmental role for the vitelline membrane, possibly in gas exchange, that could not be visualized in previous mutations that result in early collapse of all eggs (Elalayli, 2008).

The palisade lies at 26A on chromosome 2, within a gene cluster that also includes the genes for two well-characterized major vitelline membrane components, sV23 and sV17 (Popodi, 1988). Previous studies have shown that psd mRNA is co-expressed with sV23 and sV17, although at 40- to 80-fold lower levels, and that the Palisade protein is found in enriched eggshell preparations (Fakhouri, 2006; Popodi, 1988). These findings are all consistent with Palisade being a component of the vitelline membrane (Elalayli, 2008).

Comparison of primary sequence features illustrates that Palisade differs in several respects from the major vitelline membrane proteins (Waring, 2000, and references therein). It is more than twice as large as the largest of these proteins, sV23, and lacks homology to the 38 amino acid VM domain found in all 4 major VM proteins. It is an acidic protein (pI 4.42), similar to VM32E (pI 4.98) but distinct from sV17, sV23, and VM34C (pIs 7.75 to 8.34), suggesting differences in its general physical properties from these proteins. Apart from its signal sequence, Palisade can be divided into three domains. At the N-terminus is a 164 amino acid domain containing 10 clustered asparagines separated only by a pair of cysteines; its mixed alpha and beta structure, 6 cysteines, and relative hydrophilicity suggest this could be a globular domain. At the C-terminus is a 125 amino acid domain enriched in serines and threonines that may be O-glycosylated (supported by NetOGlyc prediction of 15 potential mucin-like O-glycosylation sites in Palisade, 11 of which are within this domain), and containing 2 cysteines. Between these domains is a 77 amino acid predicted alpha-helical domain containing 7 copies of an imperfect 10 amino acid repeat with the consensus sequence, PAAPAYEAPA. This domain provides the one significant similarity to VM proteins, with an organization similar to that found in a central domain of sV23 containing 7 copies of an imperfect 8 amino acid repeat with the consensus sequence, PAAPAYSA; small degenerate motifs enriched in PAYS are also seen immediately N-terminal to the VM domain in VM32E and VM34C but not sV17 (Elalayli, 2008).

Using a combination of RNAi knockdown and homologous recombination knockout approaches, Palisade was shown to be an integral component of the vitelline membrane that is essential for multiple aspects of vitelline membrane biogenesis and function. The psd knockdown and psd null mutants give very similar results in most assays, but each also provides information that could not be obtained by using only the other approach. The psd null exhibits normal chorion structure, indicating that psd is not essential for chorion formation even if some Palisade is normally deposited in this eggshell layer. Comparison of the knockdown and null suggests that non-specific RNAi effects may contribute to the appearance of oocyte F-actin cytoskeletal defects and possibly to a greater defect in sV23 disulfide cross-linking than seen in the psd null. The psd knockdown demonstrates a hypomorphic phenotype affecting embryonic nuclei that was confirmed to be specific by careful examination of the psd null, and also provides confirmation of unpublished work from the Stein lab that the PG45 Gal4 driver gives the best available knockdown in follicle cells (Elalayli, 2008).

Palisade is required early in the assembly of the vitelline membrane, as judged by three criteria: the morphologic abnormalities in the vitelline bodies and follicle cell microvilli in Stage 10 of oogenesis; the early and continuing uptake of sV17-related epitopes into the oocyte beginning in Stage 10; and the partial defect in proteolytic processing of sV17 and sV23. These abnormalities could result from a primary defect either in interactions among the vitelline membrane proteins or in matrix assembly on a potential microvillar scaffold, either of which in turn could cause the later-observed defects in disulfide- and non-disulfide cross-linking of sV17 and sV23 (as described for the N-terminal domain of sV23 by Manogaran (2004). It is not yet understood how the major VM proteins are assembled into the homogeneous vitelline membrane, or how the microvilli on follicle cells and the oocyte are involved in this assembly, so it is difficult at this time to propose a model or straightforward experiments to determine the mechanism by which Palisade functions. Two main questions relevant to mechanism are raised by the novel observations that have been made, however, which may influence future work. Why is sV17 taken up into the oocyte, while sV23 appears not to be? What do the similarities and differences between the psd null and Cad99C null effects on vitelline membrane assembly suggest about a relationship between Palisade and Cad99C (Elalayli, 2008)?

Immunostaining experiments suggest that there is a striking uptake of sV17 into the oocyte in psd null and Cad99C null egg chambers, but no uptake of sV23. Less interesting possibilities to explain this difference between these proteins are that sV17 is more sensitive than sV23 to any perturbations in vitelline membrane assembly, or that the polyclonal sV17 antibody is particularly reactive with a fragment or conformation of sV17 that is highly represented in the endocytosed fraction of this protein. These possibilities are diminished by, in the first case, the finding that sV23 null egg chambers do not show this characteristic uptake of sV17, and, in the second case, the observation that, at least in the Cad99C null egg chambers, the overall complement of sV17 processed forms appears identical to that of wild-type egg chambers. Alternatively, if taken at face value, the data suggest that sV17 requires Palisade and Cad99C, but not sV23, for stable incorporation into the vitelline membrane, while sV23 does not require Palisade or Cad99C for its stable incorporation into this matrix. In the case of the Cad99C null, sV17 has undergone apparently complete disulfide cross-linking, suggesting that the sV17 represented within oocyte vesicles has formed a large disulfide-bonded network independently of sV23. Consistent with a model in which sV17 and sV23 initially undergo separate assembly processes, Gail Waring's lab has found that in wild-type Stage 10 egg chambers sV17 and sV23 are found in distinct small disulfide-bonded complexes that could represent homo-oligomers or complexes with other matrix components (G. Waring, personal communication to Elalayli, 2008). In the absence of Palisade or Cad99C, the sV17 network may never form proper linkages with other vitelline membrane proteins to stabilize it in the matrix (Elalayli, 2008).

The psd and Cad99C nulls demonstrate an overlapping spectrum of phenotypes. The similarity of disorganization of the vitelline bodies and follicle cell microvilli in Stage 10 of oogenesis in these mutants was initially striking, although they later diverge in that only the Cad99C null vitelline membranes retain obvious full-thickness breaks that are not repaired later in oogenesis (D'Alterio, 2005; Schlichting, 2006). Both mutants show greater effects on sV17 than on sV23, including the similar movement into the oocyte and a greater defect in non-disulfide cross-linking of sV17. The psd null has additional effects on the major vitelline membrane proteins, including a modest defect in proteolytic processing of both sV17 and sV23 and failure of these incompletely processed forms to be incorporated into a disulfide-bonded network, and a greater defect than Cad99C in the non-disulfide cross-linking of sV23. These results suggest that Palisade and Cad99C could both act in a similar process, such as coordinating the assembly of the vitelline bodies on a microvillar scaffold, with the structural abnormalities of that scaffold being more profound in the absence of Cad99C and with Palisade being additionally required for interactions among the major vitelline membrane proteins. At this time, there is no clear evidence that Palisade and Cad99C directly interact or function in a common process. Flies hemizygous for both psd and Cad99C null alleles do not exhibit any decrease in hatch rate, suggesting that there is no significant defect in the vitelline membrane when both Palisade and Cad99C are present at half their normal levels. While there is no difference in the amount of Cad99C in psd null Stage 10 egg chambers compared to wild-type, a modest but variable decreases is seen in the amount of Palisade in Cad99C null Stage 10 or 14 egg chambers compared to wild-type. This result suggests that Palisade may be less stable in the absence of Cad99C but does not demonstrate a direct interaction. Nonetheless, the results suggest that Palisade may be an important molecule to examine in tests of models proposed by researchers of Cad99C that the vitelline bodies and follicle cell microvilli make important functional contacts within the perivitelline space that stabilize the microvillar structure and/or modulate the biogenesis of the vitelline membrane (Elalayli, 2008).

Embryos surrounded by a psd knockdown vitelline membrane generally arrest during the first 13 nuclear divisions prior to cellularization and show an unusual nuclear morphology of chromatin margination that somewhat resembles the phenotype of anoxic arrest. Although detailed molecular analyses would be required to compare the physiology and cell biology of psd knockdown and anoxic embryos, one possibility is that the abnormally assembled vitelline membrane surrounding these embryos blocks the normal passage of oxygen into the perivitelline space. Studies in Manduca sexta have shown that subchorion layers of the insect eggshell, including the inner chorionic layer, wax layer and vitelline membrane, provide significant resistance to the free delivery of oxygen to the embryo (Woods, 2005), so alterations in these layers might increase, as well as decrease, this resistance. Either insufficient oxygen delivery or excessive water loss across the psd knockdown vitelline membrane would be incompatible with the metabolic needs of the embryo, and eventually could result in developmental arrest and death via non-apoptotic mechanisms. Such an embryonic role for the vitelline membrane has not been explored previously because most described mutants affecting this structure, including the psd null, result in absent or very limited embryonic development (Elalayli, 2008).

Search PubMed for articles about Drosophila Palisade

Andrenacci, D., et al. (2001). Specific domains drive VM32E protein distribution and integration in Drosophila eggshell layers. J. Cell Sci. 114: 2819-2829. PubMed ID: 11683415

Cernilogar, F. M., et al. (2001). Drosophila vitelline membrane cross-linking requires the fs(1)Nasrat, fs(1)polehole and chorion genes activities. Dev. Genes Evol. 211: 573-580. PubMed ID: 11819114

Claycomb, J. M., et al. (2004). Gene amplification as a developmental strategy: isolation of two developmental amplicons in Drosophila. Dev. Cell 6: 145-155. PubMed ID: 14723854

D'Alterio, C., et al. (2005). Drosophila melanogaster Cad99C, the orthologue of human Usher cadherin PCDH15, regulates the length of microvilli. J. Cell Biol. 171: 549-558. PubMed ID: 16260500

Degelmann, A., Hardy, P. A. and Mahowald, A. P. (1990). Genetic analysis of two female-sterile loci affecting eggshell integrity and embryonic pattern formation in Drosophila melanogaster. Genetics 126: 427-434. PubMed ID: 2123163

Elalayli, M., et al. (2008). Palisade is required in the Drosophila ovary for assembly and function of the protective vitelline membrane. Dev. Biol. 319(2): 359-69. PubMed ID: 18514182

Fakhouri, M. et al. (2006). Minor proteins and enzymes of the Drosophila eggshell matrix. Dev. Biol. 293: 127-141. PubMed ID: 16515779

Heifetz, Y., Yu, J. and Wolfner, M. F. (2001). Ovulation triggers activation of Drosophila oocytes. Dev. Biol. 234: 416-424. PubMed ID: 11397010

Hong, C. C. and Hashimoto, C. (1996). The maternal nudel protein of Drosophila has two distinct roles important for embryogenesis. Genetics 143: 1653-1661. PubMed ID: 8844153

Konrad, K. D., Wang, D. and Marsh, J. L. (1993). Vitelline membrane biogenesis in Drosophila requires the activity of the a-methyl dopa hypersensitive gene (l(2)amd) in both the germline and follicle cells. Insect Mol. Biol. 1: 179-187

LeMosy, E. K. and Hashimoto, C. (2000). The Nudel protease of Drosophila is required for eggshell biogenesis in addition to embryonic patterning. Dev. Biol. 217: 352-361. PubMed ID: 10625559

Manogaran, A. and Waring, G. L. (2004). The N-terminal prodomain of sV23 is essential for the assembly of a functional vitelline membrane network in Drosophila. Dev. Biol. 270: 261-271. PubMed ID: 15136154

Pascucci, T., Perrino, J., Mahowald. A. P. and Waring, G. L. (1996). Eggshell assembly in Drosophila: processing and localization of vitelline membrane and chorion proteins. Dev. Biol. 177: 590-598. PubMed ID: 8806834

Petri, W. H., Mindrinos, M. N. and Lombard, M. F. (1979). Independence of vitelline membrane and chorion cross-linking in the Drosophila melanogaster eggshell. Dev. Biol. 83: 23a

Petri, W. H., Wyman, A. R. and Kafatos, F. C. (1976). Specific protein synthesis in cellular differentiation. III. The eggshell proteins of Drosophila melanogaster and their program of synthesis. Dev. Biol. 49: 185-199. PubMed ID: 815116

Popodi, E., Minoo, P., Burke, T. and Waring, G. L. (1988). Organization and expression of a second chromosome follicle cell gene cluster in Drosophila. Dev. Biol. 127: 248-256. PubMed ID: 3132408

Savant, S. S. and Waring, G. L. (1989). Molecular analysis and rescue of a vitelline membrane mutant in Drosophila. Dev. Biol. 135: 43-52. PubMed ID: 2504634

Schlichting, K., Wilsch-Brauninger, M., Demontis, F. and Dahmann, C. (2006). Cadherin Cad99C is required for normal microvilli morphology in Drosophila follicle cells. J. Cell Sci. 119: 1184-1195. PubMed ID: 16507588

Turcotte, C. L. and Hashimoto, C. (2002). Evidence for a glycosaminoglycan on the Nudel protein important for dorsoventral patterning of the Drosophila embryo. Dev. Dyn. 224: 51-57. PubMed ID: 11984873

Waring, G. L. (2000). Morphogenesis of the eggshell in Drosophila. Intnl. Rev. Cytol. 198: 67-108. PubMed ID: 10804461

Woods, H. A., Bonnecaze, R. T. and Zrubek, B. (2005). Oxygen and water flux across eggshells of Manduca sexta. J. Exp. Biol. 208: 1297-1308. PubMed ID: 15781890

date revised: 4 February 2009

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