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PubMed1996

A sperm-supplied factor required for embryogenesis in C. elegans.

The paternal-effect embryonic-lethal gene, spe-11, is required for normal development of early C. elegans embryos. Spe-11 embryos fail to complete meiosis, form a weak eggshell, fail to orient properly the first mitotic spindle, and fail to undergo cytokinesis. Here we report cloning and sequencing of the spe-11 gene, which encodes a novel protein. As predicted by the paternal-effect mutant phenotype, the gene is expressed during spermatogenesis but is not detectable in females undergoing oogenesis, and the protein is present in mature sperm. To investigate whether SPE-11's essential function is during spermatogenesis or whether sperm-delivered SPE-11 functions in the newly fertilized embryo, we engineered animals to supply SPE-11 to the embryo through the oocyte rather than through the sperm. We found that maternal expression is sufficient for embryonic viability. This result demonstrates that SPE-11 is not required during spermatogenesis, and suggests that SPE-11 is a sperm-supplied factor that participates directly in development of the early embryo. In contrast to the many known maternal factors required for embryogenesis, SPE-11 is the first paternally contributed factor to be genetically identified and molecularly characterized.

Amino Acid Sequence↗
PubMed1996

The TGF-beta signaling pathway is essential for Drosophila oogenesis.

We examine roles of signaling by secreted ligands of the TGF-beta family during Drosophila oogenesis. One family member, the DPP ligand encoded by the decapentaplegic (dpp) gene, is required for patterning of anterior eggshell structures. This requirement presumably reflects the expression pattern of dpp in an anterior subset of somatic follicle cells: the centripetally migrating and the nurse cell-associated follicle cells. Similar requirements are also revealed by mutations in the saxophone (sax)-encoded receptor, consistent with the idea that DPP signaling is, at least in part, mediated by the SAX receptor. A loss of germline sax function results in a block in oogenesis associated with egg chamber degeneration and a failure of the transfer of nurse cell contents to the oocyte, indicating that TGF-beta signaling is required for these events. Some phenotypes of sax mutations during oogenesis suggest that SAX responds to at least one other TGF-beta ligand as well in the posterior follicle cells.

Actins↗
PubMed1996

The Drosophila pipsqueak gene encodes a nuclear BTB-domain-containing protein required early in oogenesis.

Mutations at the pipsqueak locus affect early patterning in the Drosophila egg and embryo. We have cloned pipsqueak and found that it is a large and complex gene, encoding multiple transcripts and protein isoforms. One protein, PsqA, is absent in all of the mutants that we have examined. We show that PsqA is a nuclear protein present in the germ cells and somatically derived follicle cells throughout oogenesis and that it is required prior to stage one of oogenesis. PsqA contains a BTB (POZ) domain at its amino terminus; additionally, we have identified an evolutionarily conserved motif of unknown function present four times in tandem at the C terminus of the protein. PZ pipsqueak mutants produce a putative fusion protein containing the pipsqueak BTB domain fused to sequences resident on the PZ element (H. Horowitz and C. Berg, 1995 Genetics 139, 327-335). We demonstrate here that expression of this fusion protein in wild-type flies has a dominant effect, resulting in infertility and eggshell defects. These dominant phenotypes are discussed in light of current theories on the role of the BTB domain in protein-protein interactions.

Alternative Splicing↗
PubMed1997

The Drosophila G-protein-coupled receptor kinase homologue Gprk2 is required for egg morphogenesis.

G protein signaling is a widely utilized form of extracellular communication that is mediated by a family of serpentine receptors containing seven transmembrane domains. In sensory neurons, cardiac muscle and other tissues, G protein-coupled receptors are desensitized through phosphorylation by a family of kinases, the G protein-coupled receptor kinases (GRKs). Desensitization allows a cell to decrease its response to a given signal, in the continued presence of that signal. We have identified a Drosophila mutant, gprk2(6936) that disrupts expression of a putative member of the GRK family, the G protein-coupled receptor kinase 2 gene (Gprk2). This mutation affects Gprk2 gene expression in the ovaries and renders mutant females sterile. The mutant eggs contain defects in several anterior eggshell structures that are produced by specific subsets of migratory follicle cells. In addition, rare eggs that become fertilized display gross defects in embryogenesis. These observations suggest that developmental signals transduced by G protein-coupled receptors are regulated by receptor phosphorylation. Based on the known functions of G protein-coupled receptor kinases, we speculate that receptor desensitization assists cells that are migrating or undergoing shape changes to respond rapidly to changing external signals.

Amino Acid Sequence↗
PubMed1997

maelstrom is required for an early step in the establishment of Drosophila oocyte polarity: posterior localization of grk mRNA.

We describe a mutant, maelstrom, that disrupts a previously unobserved step in mRNA localization within the early oocyte, distinct from nurse-cell-to-oocyte RNA transport. Mutations in maelstrom disturb the localization of mRNAs for Gurken (a ligand for the Drosophila Egf receptor), Oskar and Bicoid at the posterior of the developing (stage 3-6) oocyte. maelstrom mutants display phenotypes detected in gurken loss-of-function mutants: posterior follicle cells with anterior cell fates, bicoid mRNA localization at both poles of the stage 8 oocyte and ventralization of the eggshell. These data are consistent with the suggestion that early posterior localization of gurken mRNA is essential for activation of the Egf receptor pathway in posterior follicle cells. Posterior localization of mRNA in stage 3-6 oocytes could therefore be one of the earliest known steps in the establishment of oocyte polarity. The maelstrom gene encodes a novel protein that has a punctate distribution in the cytoplasm of the nurse cells and the oocyte until the protein disappears in stage 7 of oogenesis.

Amino Acid Sequence↗
PubMed1997

The Drosophila toucan (toc) gene is required in germline cells for the somatic cell patterning during oogenesis.

We have characterized a new gene, called toucan, that is expressed and required in germline cells to promote proper differentiation of the somatic follicle cells. toucan mutant ovaries are defective in (i) the enclosure of newly formed germline cysts by the follicle cells, (ii) the formation of interfollicular stalks, (iii) the migration of the follicle cells over the oocyte and (iv) the formation of the eggshell. Overexpression of a toucan cDNA in the germline leads to the production of longer interfollicular stalks than wild-type ovaries, a phenotype that is the exact opposite of the toucan mutant phenotype. This observation shows that the formation of the interfollicular stalks depends not only on interactions among the somatic cells but also requires a germline signal. Moreover, dominant interactions have been observed between toucan and certain alleles of the daughterless, Notch and Delta genes, each of which is required in the somatic cells for the formation of egg chambers. toucan encodes for a large protein with a coiled-coil domain but has no other homology with known proteins. We propose that toucan participates in the production or localization of a germline-specific signal(s) that is required for the patterning of the follicular epithelium.

Animals↗
PubMed1999

The Drosophila chiffon gene is required for chorion gene amplification, and is related to the yeast Dbf4 regulator of DNA replication and cell cycle.

The Drosophila chorion genes encode the major protein components of the chorion (eggshell) and are arranged in two clusters in the genome. To meet the demand for rapid chorion synthesis, Drosophila ovary follicle cells amplify the chorion gene clusters approximately 80-fold. Amplification proceeds through repeated firing of one or more DNA replication origins located near the center of each gene cluster. Hypomorphic mutant alleles of the chiffon gene cause thin, fragile chorions and female sterility, and were found to eliminate chorion gene amplification. Null alleles of chiffon had the additional phenotypes of rough eyes and thin thoracic bristles: phenotypes often associated with disruption of normal cell cycle. The chiffon locus was cloned by chromosomal walking from the nearby cactus locus. A 6.5 kb transcript was identified and confirmed to be chiffon by sequencing of mutant alleles and by phenotypic rescue with genomic transformation constructs. The protein predicted by translation of the 5.1 kb chiffon ORF contains two domains related to the S. cerevisiae Dbf4 regulator of DNA replication origin firing and cell cycle progression: a 44 residue domain designated CDDN1 (43% identical) and a 41 residue domain designated CDDN2 (12% identical). The CDDN domains were also found in the S. pombe homolog of Dbf4, Dfp1, as well as in the proteins predicted by translation of the Aspergillus nimO gene and specific human and mouse clones. The data suggest a family of eukaryotic proteins related to Dbf4 and involved in initiation of DNA replication.

Amino Acid Sequence↗
PubMed2000

Anucleate Caenorhabditis elegans sperm can crawl, fertilize oocytes and direct anterior-posterior polarization of the 1-cell embryo.

It has long been appreciated that spermiogenesis, the cellular transformation of sessile spermatids into motile spermatozoa, occurs in the absence of new DNA transcription. However, few studies have addressed whether the physical presence of a sperm nucleus is required either during spermiogenesis or for subsequent sperm functions during egg activation and early zygotic development. To determine the role of the sperm nucleus in these processes, we analyzed two C. elegans mutants whose spermatids lack DNA. Here we show that these anucleate sperm not only differentiate into mature functional spermatozoa, but they also crawl toward and fertilize oocytes. Furthermore, we show that these anucleate sperm induce both normal egg activation and anterior-posterior polarity in the 1-cell C. elegans embryo. The latter finding demonstrates for the first time that although the anterior-posterior embryonic axis in C. elegans is specified by sperm, the sperm pronucleus itself is not required. Also unaffected is the completion of oocyte meiosis, formation of an impermeable eggshell, migration of the oocyte pronucleus, and the separation and expansion of the sperm-contributed centrosomes. Our investigation of these mutants confirms that, in C. elegans, neither the sperm chromatin mass nor a sperm pronucleus is required for spermiogenesis, proper egg activation, or the induction of anterior-posterior polarity.

Animals↗
PubMed2000

Drosophila bunched integrates opposing DPP and EGF signals to set the operculum boundary.

The Drosophila BMP homolog DPP can function as a morphogen, inducing multiple cell fates across a developmental field. However, it is unknown how graded levels of extracellular DPP are interpreted to organize a sharp boundary between different fates. Here we show that opposing DPP and EGF signals set the boundary for an ovarian follicle cell fate. First, DPP regulates gene expression in the follicle cells that will create the operculum of the eggshell. DPP induces expression of the enhancer trap reporter A359 and represses expression of bunched, which encodes a protein similar to the mammalian transcription factor TSC-22. Second, DPP signaling indirectly regulates A359 expression in these cells by downregulating expression of bunched. Reduced bunched function restores A359 expression in cells that lack the Smad protein MAD; ectopic expression of BUNCHED suppresses A359 expression in this region. Importantly, reduction of bunched function leads to an expansion of the operculum and loss of the collar at its boundary. Third, EGF signaling upregulates expression of bunched. We previously demonstrated that the bunched expression pattern requires the EGF receptor ligand GURKEN. Here we show that activated EGF receptor is sufficient to induce ectopic bunched expression. Thus, the balance of DPP and EGF signals sets the boundary of bunched expression. We propose that the juxtaposition of cells with high and low BUNCHED activity organizes a sharp boundary for the operculum fate.

Amino Acid Sequence↗
PubMed2001

Establishment of dorsal-ventral polarity of the Drosophila egg requires capicua action in ovarian follicle cells.

The dorsal-ventral pattern of the Drosophila egg is established during oogenesis. Epidermal growth factor receptor (Egfr) signaling within the follicular epithelium is spatially regulated by the dorsally restricted distribution of its presumptive ligand, Gurken. As a consequence, pipe is transcribed in a broad ventral domain to initiate the Toll signaling pathway in the embryo, resulting in a gradient of Dorsal nuclear translocation. We show that expression of pipe RNA requires the action of fettucine (fet) in ovarian follicle cells. Loss of maternal fet activity produces a dorsalized eggshell and embryo. Although similar mutant phenotypes are observed with regulators of Egfr signaling, genetic analysis suggests that fet acts downstream of this event. The fet mutant phenotype is rescued by a transgene of capicua (cic), which encodes an HMG-box transcription factor. We show that Cic protein is initially expressed uniformly in ovarian follicle cell nuclei, and is subsequently downregulated on the dorsal side. Earlier studies described a requirement for cic in repressing zygotic target genes of both the torso and Toll pathways in the embryo. Our experiments reveal that cic controls dorsal-ventral patterning by regulating pipe expression in ovarian follicle cells, before its previously described role in interpreting the Dorsal gradient.

Animals↗
PubMed2001

Drosophila E2f2 promotes the conversion from genomic DNA replication to gene amplification in ovarian follicle cells.

Drosophila contains two members of the E2F transcription factor family (E2f and E2f2), which controls the expression of genes that regulate the G1-S transition of the cell cycle. Previous genetic analyses have indicated that E2f is an essential gene that stimulates DNA replication. We show that loss of E2f2 is viable, but causes partial female sterility associated with changes in the mode of DNA replication in the follicle cells that surround the developing oocyte. Late in wild-type oogenesis, polyploid follicle cells terminate a program of asynchronous endocycles in which the euchromatin is entirely replicated, and then confine DNA synthesis to the synchronous amplification of specific loci, including two clusters of chorion genes that encode eggshell proteins. E2f2 mutant follicle cells terminate endocycles on schedule, but then fail to confine DNA synthesis to sites of gene amplification and inappropriately begin genomic DNA replication. This ectopic DNA synthesis does not represent a continuation of the endocycle program, as the cells do not complete an entire additional S phase. E2f2 mutant females display a 50% reduction in chorion gene amplification, and lay poorly viable eggs with a defective chorion. The replication proteins ORC2, CDC45L and ORC5, which in wild-type follicle cell nuclei localize to sites of gene amplification, are distributed throughout the entire follicle cell nucleus in E2f2 mutants, consistent with their use at many genomic replication origins rather than only at sites of gene amplification. RT-PCR analyses of RNA purified from E2f2 mutant follicle cells indicate an increase in the level of Orc5 mRNA relative to wild type. These data indicate that E2f2 functions to inhibit widespread genomic DNA synthesis in late stage follicle cells, and may do so by repressing the expression of specific components of the replication machinery.

Animals↗
PubMed2002

LET-99 determines spindle position and is asymmetrically enriched in response to PAR polarity cues in C. elegans embryos.

Asymmetric cell division depends on coordinating the position of the mitotic spindle with the axis of cellular polarity. We provide evidence that LET-99 is a link between polarity cues and the downstream machinery that determines spindle positioning in C. elegans embryos. In let-99 one-cell embryos, the nuclear-centrosome complex exhibits a hyperactive oscillation that is dynein dependent, instead of the normal anteriorly directed migration and rotation of the nuclear-centrosome complex. Furthermore, at anaphase in let-99 embryos the spindle poles do not show the characteristic asymmetric movements typical of wild type animals. LET-99 is a DEP domain protein that is asymmetrically enriched in a band that encircles P lineage cells. The LET-99 localization pattern is dependent on PAR polarity cues and correlates with nuclear rotation and anaphase spindle pole movements in wild-type embryos, as well as with changes in these movements in par mutant embryos. In particular, LET-99 is uniformly localized in one-cell par-3 embryos at the time of nuclear rotation. Rotation fails in spherical par-3 embryos in which the eggshell has been removed, but rotation occurs normally in spherical wild-type embryos. The latter results indicate that nuclear rotation in intact par-3 embryos is dictated by the geometry of the oblong egg and are consistent with the model that the LET-99 band is important for rotation in wild-type embryos. Together, the data indicate that LET-99 acts downstream of PAR-3 and PAR-2 to determine spindle positioning, potentially through the asymmetric regulation of forces on the spindle.

Anaphase↗
PubMed2006

Cadherin Cad99C is required for normal microvilli morphology in Drosophila follicle cells.

Microvilli are actin-filled membranous extensions common to epithelial cells. Several proteins have been identified that localize to microvilli. However, most of these proteins are dispensable for the normal morphogenesis of microvilli. Here, we show by immunoelectron microscopy that the non-classical cadherin Cad99C localizes to microvilli of Drosophila ovarian follicle cells. Loss of Cad99C function leads to disorganized and abnormal follicle cell microvilli. Conversely, overexpression of Cad99C in follicle cells results in large bundles of microvilli. Furthermore, altered microvilli morphology correlates with defects in the assembly of the vitelline membrane, an extracellular layer secreted by follicle cells that is part of the eggshell. Finally, we provide evidence that Cad99C is the homolog of vertebrate protocadherin 15. Mutations in the gene encoding protocadherin 15 lead to the disorganization of stereocilia, which are microvilli-derived extensions of cochlear hair cells, and deafness (Usher syndrome type 1F). Our data suggest an essential role for Cad99C in microvilli morphogenesis that is important for follicle cell function. Furthermore, these results indicate that insects and vertebrates use related cadherins to organize microvilli-like cellular extensions.

Animals↗
PubMed1993

Transepithelial calcium transport in the chick chorioallantoic membrane. II. Compartmentalization of calcium during uptake.

Calcium transport from the eggshell to the developing chick embryo is carried out by the ectoderm cells of the chick chorioallantoic membrane. Primary cells isolated from chick chorioallantoic membrane ectoderm were used to analyze the subcellular distribution of 45Ca2+ accumulated from the extracellular medium. We present evidence suggesting that calcium may be sequestered into endosome-like vesicles during the initial phase of uptake. A combination of techniques were utilized to monitor calcium fluxes and calcium compartmentalization in the cultured chorioallantoic membrane cells: (1) fura-2 fluorescence was used to indicate cytosolic free calcium concentrations, (2) 45Ca2+ tracer was used to follow calcium accumulation in all cellular compartments, and (3) digitonin was used to differentially permeabilize subcellular membranes in order to localize 45Ca2+ by following tracer release profiles. Differences between cytosolic calcium flux and whole cell calcium accumulation suggested that the pathway of calcium uptake from the medium involves sequestration into an internal compartment separate from the cytosol. Kinetic analysis of the digitonin-mediated release of specific subcellular markers (lactate dehydrogenase, NAD-dependent isocitrate dehydrogenase, [3H]inulin, and [3H]-2-deoxyglucose) and preloaded 45Ca2+ indicated that calcium was localized in a compartment similar to endosomal vesicles. Our results are consistent with a transcytotic mechanism for chorioallantoic membrane calcium transport.

Allantois↗
PubMed1989

Respiratory gases and acid-base balance in shell-less avian embryos.

1. The PCO2 and PO2 levels of oxygenated and deoxygenated embryonic blood were measured in normal and shell-less cultured embryos. 2. Embryos in shell-less culture were both hypoxic and hypocapnic, suggesting that wetting the respiratory membranes and the reduction in their functional area is distorting their normal function. 3. Despite this hypocapnia, there was evidence for an increase in base excess during incubation. This suggests that plasma bicarbonate is derived from renal sources rather than by resorbing the eggshell calcium carbonate, which is, of course, absent from shell-less cultures.

Acid-Base Equilibrium↗
PubMed1994

Mobilization of shell calcium by the chick chorioallantoic membrane in vitro.

Two explants of shell were removed from each of several fertile eggs of domestic fowl at different times during incubation. The chorioallantoic membrane (CAM) was removed from one of the explants (SHELL ONLY) and was left in situ on the other (SHELL+CAM). Explants were cultured for 24, 48 or 96 h at 37 degrees C and 5% CO2 in air in individual Petri dishes containing Dulbecco's modified Eagle's medium, bovine serum albumin, penicillin and streptomycin. Both SHELL+CAM and SHELL ONLY explants released calcium into the culture medium, but the former released considerably more calcium than the latter. More calcium was released by SHELL+CAM explants taken from older eggs than from younger ones, but the age of the donor eggs did not affect release of calcium by SHELL ONLY explants. In addition, release of calcium by SHELL+CAM explants exceeded that shown by SHELL ONLY explants for multiple 24 h intervals. However, the capacity for sustained release of calcium by SHELL+CAM explants declined with age and maturity of the CAM. Manipulations that lead to the death of the CAM abolish the capacity for SHELL+CAM explants to release more calcium than SHELL ONLY explants. Differential release of calcium by SHELL+CAM explants was not attributable to calcium present in the CAM at the onset of culture or to non-specific degradation of the shell by intracellular constituents released as a result of the death of the CAM. Taken in concert, these results indicate that the CAM mobilizes calcium from the eggshell during in vitro culture.

Age Factors↗
PubMed2002

Respiration and energetics of embryonic development in a large altricial bird, the Australian pelican (Pelecanus conspicillatus).

We examined whether the previously reported low cost of embryonic development in pelicans could be attributed to a more efficient conversion of egg energy to hatchling tissues as a result of high initial egg water content, low embryonic metabolic rate and growth later in incubation than in more precocious species. We therefore determined egg and hatchling composition and the development of embryonic respiration in the Australian pelican Pelecanus conspicillatus, which lays one of the largest eggs (140-210 g) with an altricial developmental mode. The small yolk fraction (21%) is typical of all pelecaniforms; however, we found that intraspecific variability in fresh egg mass was related to water content (principally in the albumen), but independent of yolk mass (mean 13 g dry mass). P. conspicillatus eggs have, on average, 635 kJ of energy, irrespective of egg mass across the whole range of egg mass. The embryonic developmental pattern of O(2) consumption and CO(2) production showed clear plateaus lasting 2-3 days immediately prior to internal pipping, resembling the typical precocial pattern. However, the rate of pre-internal pipping O(2) consumption was low in comparison with that of precocial species of similar egg mass. There is no evidence to support the hypothesis that the observed plateau in rates of O(2) uptake is due to a diffusion limitation of the eggshell gas conductance in this species. Embryonic metabolic rate nearly doubled during the pipping period, but the mass-independent metabolic rate of the hatchling was low in comparison with that of the resting adult. The total O(2) consumed (11 063 ml) is equivalent to 217.3 kJ (or 34% of egg energy) based on indirect calorimetry and the observed respiratory exchange ratio of 0.71. Thus, the cost of development (direct calorimetry) was 0.29 kJ J(-1) in the egg (mean egg mass 168 g), which is one of lowest reported values. As a result, the production efficiency of pelican embryonic development was 61.6%, higher than the average for birds in general (56.9%) and, in particular, of seabirds that have prolonged incubation periods on the basis of egg mass. High efficiency in embryonic development in this species was attained as a result of rapid embryonic growth later in incubation, low hatchling energy density (23.6 kJ g(-1) dry matter) and dry matter content, low embryonic metabolic rate throughout incubation and a shorter than expected incubation period of 33 days (predicted 36 days).

Animals↗
PubMed1995

A review of factors affecting productivity of bald eagles in the Great Lakes region: implications for recovery.

The bald eagle (Haliaeetus leucocephalus) population in North America declined greatly after World War II due primarily to the eggshell thinning effects of p,p'-DDE, a biodegradation product of DDT. After the banning of DDT in the United States and Canada during the early 1970s, the bald eagle population started to increase. However, this population recovery has not been uniform. Eagles nesting along the shorelines of the North American Great Lakes and rivers open to spawning runs of anadromous fishes from the Great Lakes still exhibit impaired reproduction. We have explored both ecological and toxicological factors that would limit reproduction of bald eagles in the Great Lakes region. Based on our studies, the most critical factors influencing eagle populations are concentrations of environmental toxicants. While there might be some continuing effects of DDE, total PCBs and most importantly 2,3,7,8-tetrachlordibenzo-p-dioxin equivalents (TCDD-EQ) in fishes from the Great Lakes and rivers open to spawning runs of anadromous fishes from the Great Lakes currently represent a significant hazard to bald eagles living along these shorelines or near these rivers and are most likely related to the impaired reproduction in bald eagles living there.

Animals↗