View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector DAvID STEIN PATTERN FORMATION DAVID STEIN PATTERN FORMATION The link between ovary and embryo The Drosophila gene nudel may encode a spatially restricted serine protease involved in producing the ligand for the receptor Toll and linking dorsal-ventral polarity in the egg chamber to the developing embryonic axis.

One of the important insights to have emerged from the signs of dorsal-ventral polarity exhibited by the embryo study of early pattern formation in the Drosophila embryo that lies within the polarized shell occur during gastrula- is that the basic embryonic axes have their origins in the tion, when cells that will go on to become mesoderm polarity established in the egg chamber in which the invaginate into a furrow on the presumptive ventral side oocyte develops [1]. During oogenesis, the female uses a of the embryo. This ventral furrow always forms along signalling cascade mediated by a receptor tyrosine kinase the curved ventral surface of the egg shell - thus the to establish dorsal-ventral polarity in the egg chamber polarity of the embryo and the egg shell are coordinated. [2]. This polarity is subsequently communicated to the embryo via a signaling pathway similar to the inter- A group of twelve 'maternal-effect' genes (mutations of leukin-1/NF-KB pathway used by the mammalian which are manifest in eggs or embryos produced by immune system [3]. A gap in our understanding of homozygous female flies) termed the 'dorsal group' is embryonic dorsal-ventral pattern has been in the integra- required for the establishment of correct embryonic dor- tion of these two signalling pathways. How does the fol- sal-ventral polarity, but not for egg polarity [4]. The Toll licle cell layer impose its dorsal-ventral polarity on the gene encodes a transmembrane receptor that is uniformly embryo? The product of the recently cloned nudel gene distributed in the embryonic membrane. Toll is thought may play a direct role in integrating the two systems, and to be activated on the ventral side of the embryo by the may do so as a component of the Drosophilaegg shell. binding of a localized ligand present in the perivitelline space that lies between the embryonic membrane and the The Drosophila egg chamber comprises the developing inner layer of the egg shell, the vitelline membrane. Fol- oocyte, its fifteen associated nurse cells and a surrounding lowing Toll activation, the ventralizing signal is propa- epithelium of somatically derived follicle cells (Fig. la). gated into the embryo and mediates the graded nuclear The first morphological manifestations of dorsal-ventral uptake of the Dorsal protein, which is a transcription fac- pattern in the egg chamber can be seen during stages 8 to tor. Highest levels of Dorsal protein are found in the 10 of oogenesis, when the oocyte nucleus assumes a nuclei on the ventral side of the embryo. dorsal-anterior location within the oocyte. The outside of the egg is also visibly polarized dorsoventrally, with Formation of the ligand that binds to Toll appears to be curved ventral and flattened dorsal surfaces and two achieved through the spatially regulated proteolytic appendages located dorsally. The first morphological processing of an inactive ligand precursor [5]. Three of

Fig. 1. The establishment of dorsal-ventral polarity in the egg chamber and oocyte. See text for details.

1360 © Current Biology 1995, Vol 5 No 12 DISPATCH 1361 the genes acting upstream of Toll, namely easter, snake and egg chambers, the nudel transcript was observed to be gastrulation defective ([4] and Rob DeLotto, personal asymmetrically distributed, with a higher level on the communication), have been found to encode putative ventral side of the follicle. The demonstration that the secreted serine proteases; Easter and Snake can be detec- putative nudel transcript was altered in nudel mutant ted as inactive precursors in the perivitelline fluid [6,7]. females confirmed the transcript's identity. By constructing gain-of-function mutant forms of Easter and Snake that do not require proteolytic cleavage for The nudel gene encodes an extremely large protein of activation, and injecting these into embryos produced by 2 616 amino acids, with a predicted molecular weight of females carrying mutations in one of the dorsal group of 292 kDa. It carries a potential secretion signal at the genes, it has been possible to order the three serine pro- amino terminus as well as a large number of possible teases in a pathway: Gastrulation defective-> Snake-> target sites for glycosylation. The predicted Nudel Easter [7,8]. protein contains 11 copies of an approximately 40- residue cysteine-rich sequence that was first found in the The spiitzle gene is the best candidate for encoding the ligand-binding domain of the low density lipoprotein ligand for Toll. Spaitzle is a secreted protein that may (LDL) receptor, termed the A repeat. Nudel also contains require processing by the Easter protease for its activity six copies of a novel nine-residue motif of the sequence [5]. How is spatially restricted ligand formation WI(I/L)D (using the single-letter amino-acid code), accomplished? One clue is the finding that three of the which the authors call the WIID repeat. Perhaps most dorsal group of genes - nudel, pipe and windbeutel - informative is the presence of two separate regions that must be expressed in the somatic cells of the fly (probably have homology to the catalytic domains of serine in the follicular epithelium) to function normally. [9,10]. proteases. One of these protease domains is incomplete This suggests that the three genes might provide the link and lacks the catalytic triad of amino acids required for between the follicle cell layer and the embryo. enzymatic activity in other proteases. The other, located in the middle of the nudel open reading frame, is more In a recent publication that provides the first molecular similar to serine proteases and does contain the catalytic information about the somatically required dorsal group triad. This second protease-like domain shows most of genes, Hong and Hashimoto [11] describe the cloning similarity to plasma kallikreins (34 % identity). of the nudel gene. Conventional methods were used to isolate the genomic region containing nudel and to Hong and Hashimoto [11] propose that the Nudel identify a follicle-cell-specific transcript as the nudel gene protein acts as the spatial organizer of the protease product. In a large proportion (71 %) of stage 9 and 10 cascade that leads to the localized formation of the ligand

Fig. 2. The establishment of dorsal-ventral polarity in the embryo. See text for details. 1362 Current Biology 1995, Vol 5 No 12

for Toll (Fig. 2). Ventrally enriched Nudel protein may oocyte. Presumably, localized Gurken protein activates locally activate one or more of the other dorsal group the receptor, Torpedo, in the follicle cells of the dorsal proteases. Alternatively, Nudel could provide the scaffold side of the egg chamber, transmitting a signal that for anchoring a stable complex of the proteases. The induces them to assume a dorsal fate. Follicle cells that Gastrulation defective protein is the most likely prote- fail to receive the signal will develop a ventral fate. olytic target of Nudel, as genetic epistasis tests have placed gastrulation defective upstream of easter and snake [8]. If nudel's asymmetric expression is crucial in defining Consistent with this idea, the key residues in the active embryonic polarity, its pattern of expression would be site of the conserved Nudel protease domain suggest a expected to change in the follicles of females carrying trypsin-like cleavage specificity, which corresponds to mutations that alter egg chamber polarity. Unfortunately, that predicted to be required for the activation of the strong gurken alleles affect the anterior-posterior polarity Gastrulation defective protein (Rob Delotto personal as well as the dorsal-ventral polarity of the egg chamber communication). [13,15], making it difficult to interpret the pattern of expression of nudel [11]. And although no change in Both of the models suggested above require that Nudel nudel expression pattern was detected in torpedo mutant protein is present in the egg and has access to the peri- follicles, this might be due to the weak ventralizing vitelline fluid, where ligand formation occurs. One way phenotype of torpedo mutations. However, nudel expres- this could be achieved is if Nudel is a component of the sion was reported to be uniform, failing to exhibit vitelline membrane. The peak expression of nudel RNA ventral enrichment, in the egg chambers produced by occurs at the same time as all previously studied vitelline females carryingfs(1)K10, a mutation that dorsalizes egg membrane constituents are secreted. The temperature- chambers. Infs(1)K10 oocytes, gurken RNA is not con- sensitive period of the mutant ndl9/nd146 allelic combina- fined to the dorsal side [12] but instead is present in a tion extends from a point during oogenesis just prior to ring around the anterior cortex. As a result, the Gurken the expression of vitelline membrane proteins until signal is received by follicle cells all around the circumfer- roughly two hours after egg deposition [11]. Thus, ence. This results in the dorsalization of the follicular although the nudel gene is expressed by the follicle cells, epithelium, and indirectly leads to uniform expression of its activity continues to be required after the egg has been nudel; nudel expression was also apolar in pipe mutant laid, consistent with its postulated presence in the egg. follicles, suggesting that the wild-type function of pipe Eggs produced by nudel mutant females from which the might be to enhance nudel expression ventrally. chorion has been removed are extremely flaccid, suggest- ing that the vitelline membrane is defective. Thus, if One intriguing aspect of the effect of gurken-torpedo Nudel is indeed a component of the vitelline membrane, signalling on follicle cell fate is the recent demonstration it may play a role in structural integrity of the mem- that mutations in gurken do not lead to a simple expansion brane, as well as in embryonic patterning. Interestingly, of the ventral region in affected embryos [16]. Rather, one mutant allele of nudel that dorsalizes the embryo but the embryos produced by gurken mutant females exhibit does not cause a fragile egg phenotype has a mutation two high points of nuclear Dorsal protein, positioned predicted to affect a glycine residue near the active site ventrolaterally and separated by a region of decreased the protease domain [11]. This change would be ex- nuclear Dorsal protein. Using a variety of molecular pected to impair protease activity but apparently does not markers, Roth and Schiipbach [16] demonstrated that affect vitelline membrane structure. these embryos develop a pattern duplication with two regions of mesoderm separated by a region of neuro- How is the spatial regulation of nudel expression in the ectoderm. It is not clear whether this pattern duplication follicular epithelium achieved? A number of maternal occurs in the follicle cell layer, in the perivitelline space, genes are required for the development of polarity of the or in the cytoplasm of the embryo. In this regard it would egg chamber [2]. Among these, mutants in the genes be interesting to learn whether gurken mutant follicles torpedo and gurken produce egg chambers that are contain two distinct regions of high nudel expression. ventralized. Torpedo is the Drosophila homologue of the vertebrate epidermal growth factor (EGF) receptor, and The elucidation of Nudel's action should help us to Gurken resembles transforming growth factor oc(TGF-ox) understand the mechanism that orients the serine protease and is likely to be the ligand for Torpedo (Fig. lb) [12]. cascade that produces the ligand for Toll. A necessary step Analysis of genetic mosaics has demonstrated that torpedo in that task will be determination of the distribution of function is required in the somatic follicle cells, whereas Nudel protein. Determining whether Nudel has serine gurken is required in the germ-line cells. During proteolytic activity and, if so, which protein acts as its oogenesis, the gurken transcript becomes localized in a substrate is also an immediate concern. Two other dorsal perinuclear position at the dorsal-anterior corner of the group genes, pipe and windbeutel, remain to be cloned, and oocyte [12], intimately associated with the oocyte their analysis should provide important additional infor- nucleus. Immunostaining indicates that the Gurken pro- mation about the role of nudel as well as clues to the tein becomes enriched along the dorsal surface of the mechanism by which the torpedo-gurken signaling cascade oocyte [13,14], accumulating in the cytoplasm and integrates patterning along the dorsal-ventral axes of the plasma membrane at the dorsal-anterior corner of the follicle and the embryo. Given the sophisticated tool box DISPATCH 1363 of techniques available to the Drosophila researcher, the 9. Stein D, Roth S, Vogelsang E, Nusslein-Volhard C: The polarity of the dorsoventral axis in the Drosophila embryo is defined by an answers to these questions should not be long in coming. extracellular signal. Cell 1991, 65:725-735. 10. Schupbach T, Clifford RJ, Manseau LJ, Price JV: Dorsoventral Acknowledgements: I thank Leslie Stevens for her valuable com- signaling processes in Drosophila oogenesis. In Cell-Cell Interac- ments on the manuscript and Rob DeLotto for communicating tions in Early Development. Edited by Gerhart J. New York: Wiley- unpublished results. Liss; 1991:163-174. 11. Hong CC, Hashimoto C: An unusual mosaic protein with a protease domain encoded by the nudel gene is involved in defining References embryonic polarity in Drosophila. Cell 1995, 82:785-794. 1. St Johnston D, Nusslein-Volhard C: The origin of pattern and 12. Neumann-Silberberg FS,Schupbach T: The Drosophila dorsoventral polarity in the Drosophila embryo. Cell 1992, 68:201-219. patterning gene gurken produces a dorsally localized RNA and 2. SchOpbach T, Roth S: Dorsoventral patterning in Drosophila encodes a TGF-like protein. Cell 1993, 75:165-174. oogenesis. Curr Opin Genet Dev 1994, 4:502-507. 13. Roth S, Neuman-Silberberg S, Barcelo G, Schdpbach T: cornichon 3. Wasserman, S: A signal transduction pathway regulating the and the EGF receptor signaling process are necessary for both ante- activity of the Rel-like proteins dorsal and NF-KB. Mol Biol Cell rior-posterior and dorsal-ventral pattern formation in Drosophila. 1993,4:767-771. Cell 1995, 81:967-978. 4. Chasan R, Anderson KV: Maternal control of dorsal-ventral polarity 14. Serano TL, Karlin-McGinness M, Cohen RS: The role of fs(l)K10 in and pattern in the embryo. In The Development of Drosophila the localization of the mRNA of the TGFs homolog gurken within Melanogaster, Volume 1. Edited by Bate M, Martinez Arias A. New the Drosophila oocyte. Mech Dev 1995, 51:183-192. York: Cold Spring Harbor Press; 1993:387-424. 15. Gonzalez-Reyes A, Elliot E, St Johnston D: Polarization of both 5. Roth S: Proteolytic generation of a morphogen. Curr Biol 1994, major body axes in Drosophila by gurken-torpedo signalling. 4:755-757. Nature 1995, 375:654-658. 6. Stein DS, Nsslein-Volhard C: Multiple extracellular activities in 16. Roth S, Schopbach T: The relationship between ovarian and embry- Drosophila egg perivitelline fluid are required for establishment of onic dorsoventral patterning in Drosophila. Development 1994, embryonic dorsal-ventral pattern. Cell 1992, 68:429-440. 120:2245-2257. 7. Chasan R, in Y, Anderson KV: Activation of the easter zymogen is regulated by five other genes to define dorsal-ventral polarity in David Stein, Department of Molecular Genetics, Albert the Drosophila embryo. Development 1992, 115:607-616. 8. Smith CL, DeLotto R: Ventralizing signal determined by protease Einstein College of Medicine, Bronx, New York 10461, activation in Drosophila embryogenesis. Nature 1994, 368:548-551. USA.

THE OCTOBER 1995 ISSUE (VOL. 5, NO. 5) OF CURRENT OPINION IN GENETICS AND DEVELOPMENT

includes the following reviews, edited by Steven McKnight and Ueli Schibler, on Differentiation and gene regulation:

Molecular mechanisms of cell-type determination in budding yeast by Alexander D. Johnson Control of mating and development in Ustilago maydis by Regine Kahmann, Tina Romeis, Michael Balker and J6rg Kdmper C/EBPa: a critical regulator of genes governing integrative metabolic processes by Gretchen J. Darlington, Naidy Wang and Richard W. Hanson Regulation of adipocyte gene expression and differentiation by peroxisome proliferator activated receptor y by Peter Tontonoz, Erding Hu and Bruce M. Spiegelman Intestinal epithelial cell differentiation: new insights from mice, flies and nematodes by Theodore C. Simon and Jeffrey I. Gordon Prolactin-mediated gene activation in mammary epithelial cells by Bernd Groner and Fabrice Gouilleux The role of BSAP (Pax-5) in B-cell development by Meinrad Busslinger and Pavel Urbinek New perspectives on eye evolution by Georg Halder, Patrick Callaerts and Walter J. Gehring Axis formation in zebrafish by Wolfgang Driever Homeobox genes in vertebrate gastrulation by Edoardo Boncinelli and Antonello Mallamaci MADS-box genes in plant ontogeny and phylogeny: Haeckel's 'biogenetic law' revisited by Giinter Thei3en and Heinz Saedler Self-incompatibility in flowering plants by John E Golz, Adrienne E. Clarke and Ed Newbigin Complementation cloning of mammalian transcriptional regulators: the example of MHC class II gene regulators by Viktor Steimle and Bernard Mach Species specificity of transcription by RNA polymerase I by Jutta Heix and Ingrid Grummt The genetics of olfaction by Irene C. Griff and Randall R. Reed Molecular control of circadian rhythms by Michael Rosbash