Two Drosophila learning mutants, dunce and rutabaga, provide evidence of a maternal role for cAMP on embryogenesis

The dunce gene of Drosophila melanogaster encodes a cAMP-specific phosphodiesterase (form II). Mutant dunce flies have elevated levels of cAMP and exhibit a number of defects including learning deficiencies and female sterility. Two partial suppressors of the female sterility phenotype have been selected in an X chromosome containing a dunce null mutation. Both suppressors are associated with reduced AC2 activity. Complementation analyses suggest that both are alleles of the learning mutant rutabaga. Females homozygous for dunce null mutations that abolish PDE activity do not deposit eggs. The suppressors exhibit differential effects on egg deposition and production of progeny; double-mutant females deposit many eggs that fail to hatch, but some develop to adults. These adult progeny exhibit morphological defects that are confined mostly to the second and third thoracic segments or to the first five abdominal segments. These observations demonstrate that the dunce gene is required in adult females for egg laying and that the dunce gene provides an essential maternal function required for normal development of the zygote. Clonal analysis, employing the dominant female-sterile mutation ovoD1, demonstrates that the former requirement for PDE activity resides in somatic cells and that the latter requirement resides in germ line cells. Female germ line cells homozygous for a dunce null mutation produce oocytes that fail to develop. Thus, homozygous dunce null-mutant zygotes develop to adults solely because of the enzyme or mRNA present in the oocytes of heterozygous mothers. Mutant alleles of rutabaga act in the germ line cells to partially suppress the developmental defects caused by dunce mutations. Thus the rutabaga gene, as well as the dunce gene, functions in both somatic and germ line cells.     

Genetic Characterization of Small Ovaries, a Gene Required in the Soma for the Development of the Drosophila Ovary and the Female Germline

The small ovary gene (sov) is required for the development of the Drosophila ovary. Six EMS-induced recessive alleles have been identified. Hypomorphic alleles are female sterile and have no effect on male fertility, whereas more severe mutations result in lethality. The female-sterile alleles produce a range of mutant phenotypes that affect the differentiation of both somatic and germline tissues. These mutations generally produce small ovaries that contain few egg cysts and disorganized ovarioles, and in the most extreme case no ovarian tissue is present. The mutant egg cysts that develop have aberrant morphology, including abnormal numbers of nurse cells and patches of necrotic cells. We demonstrate that sov gene expression is not required in the germline for the development of functional egg cysts. This indicates that the sov function is somatic dependent. We present evidence using loss-of-function and constitutive forms of the somatic sex regulatory genes that sov activity is essential for the development of the somatic ovary regardless of the chromosomal sex of the fly. In addition, the genetic mapping of the sov locus is presented, including the characterization of two lethal sov alleles and complementation mapping with existing rearrangements.     

Segmental determination in Drosophila central nervous system: analysis of the abdominal-A region of the bithorax complex.

The bithorax complex (BX-C) comprises several genes required for the diversification of posterior segments in Drosophila. The BX-C genes control segment differences not only in the epidermis but in other tissues as well, especially in the central nervous system. We have examined the control of one segment-specific neural structure: the lateral dots, a paired structure present in the first abdominal segment of the larval CNS and absent in all following abdominal segments. Our results show that the suppression of lateral dots in segments A3 and A4 requires the presence of two active copies of one of the BX-C genes, abdominal-A (abd-A). We also show that the adjacent BX-C regions, iab-3 and iab-4, can act in trans on abd-A not only when the two copies of BX-C are paired but also, at least to some extent, when pairing is disturbed.     

Genetic and developmental evidence for a repressed genital primordium in Drosophila melanogaster

Agametic, a maternal-effect mutation, causes the absence of germ cells in approximately 40% of the gonads of flies derived from homozygous females. The nature of the deficiency in the eggs produced by these flies was examined. Ultrastructural abnormalities were seen in the polar granules of some eggs shortly after fertilization. Although a normal number of pole cells form, some are abnormal with degenerating polar granules and nuclear bodies and they contain myeloid bodies. The pole cells reach the gonads and at 14 hr of development all the gonads contain germ cells. However, in 40% of the gonads the germ cells become necrotic and disappear. Thus, the source of agametic gonads in the adult is embryonic death of pole cells in some gonads. To test whether this gonadal death is an autonomous deficiency of the mutant pole cells, mosaic pole cell populations were produced by reciprocal pole cell transplantation. In both types of transplants, the mutant pole cells died autonomously. In eight instances gonads containing only donor pole cells were obtained. Since mutant pole cells die when wild-type pole cells normally begin dividing, we suggest that the lesion affects the ability of these mutant pole cells to reenter the cell cycle.     

The sys-1 gene and sexual dimorphism during gonadogenesis in Caenorhabditis elegans

In wild-type Caenorhabditis elegans, the hermaphrodite gonad is a symmetrical structure, whereas the male gonad is asymmetric. Two cellular processes are critical for the generation of these sexually dimorphic gonadal shapes during early larval development. First, regulatory "leader" cells that control tube extension and gonadal shape are generated. Second, the somatic gonadal precursor cells migrate and become rearranged to establish the adult pattern. In this paper, we introduce sys-1, a gene required for early organization of the hermaphrodite, but not the male, gonad. The sys-1(q544) allele behaves genetically as a strong loss-of-function mutant and putative null. All hermaphrodites that are homozygous for sys-1(q544) possess a grossly malformed gonad and are sterile; in contrast, sys-1(q544) males exhibit much later and only partially penetrant gonadal defects. The sys-1(q544) hermaphrodites exhibit two striking early gonadal defects. First, the cell lineages of Z1 and Z4, the somatic gonadal progenitor cells, produce extra cells during L2, but the regulatory cells that control gonadal shape are not generated. Second, somatic gonadal precursor cells do not cluster centrally during late L2, and the somatic gonadal primordium typical of hermaphrodites is not established. In contrast, the early male gonadal lineage is asymmetric as normal, the somatic gonadal primordium typical of males is established correctly, and the male adult gonadal structures can be normal. We conclude that the primary role of sys-1 is to establish the shape and polarity of the hermaphrodite gonad.     

Ornithine decarboxylase activity during the development of Anastrepha fraterculus (Diptera, Tephritidae)

Ornithine decarboxylase (ODC) (EC 4.1.1.17) is very important for polyamine biosynthesis, which is required for main biological events. In the present study, ODC activity was measured in samples of Anastrepha fraterculus's egg, larva, pupa body and abdomen, adult body, ovaries, and fat body of young females, and in ovaries of mature flies. The kinetic parameters (Km app and Vmax) for ODC activity were determined for pupa, larva, and young ovary. ODC activity showed fluctuations during A. fraterculus's life development. In its earlier stages, prior to emergence, the egg has high ODC-specific activity probably due to embryogenesis, which is characterized by a high rate of cell division. This enzyme activity is also significantly high in the ovary and fat body of young females possibly related to the increased oogenesis and vitellogenesis. The kinetic parameters (Km app and Vmax) had great variation. Our results using GTP showed that the great variation in kinetic parameters can be accounted for by post-translational modifications.     

Defects in the germ line and gonads of mice lacking connexin43

The connexins are a family of at least 15 proteins that form the intercellular membrane channels of gap junctions. Numerous connexins, including connexin43 (Cx43), have been implicated in reproductive processes by virtue of their expression in adult gonads. In the present study, we examined the gonads of fetal and neonatal mice homozygous for a null mutation in the Gja1 gene encoding Cx43 to determine whether the absence of this connexin has any consequences for gonadal development. We found that in both sexes at the time of birth, the gonads of homozygous mutants were unusually small. This appears to be caused, at least in part, by a deficiency of germ cells. The germ cell deficiency was traced back as far as Day 11.5 of gestation, implying that it arises during early stages of germ line development. We also used an organ culture technique to examine postnatal folliculogenesis in the mutant ovaries, an approach necessitated by the fact that Gja1 null mutant offspring die soon after birth because of a heart abnormality. The results demonstrated that folliculogenesis can proceed to the primary (unilaminar) follicle stage in the absence of Cx43 but that subsequent development is impaired. In neonatal ovaries of normal mice, Cx43 could be detected in the somatic cells as early as Day 1, when primordial follicles begin to appear, supporting the conclusion that this connexin is required for the earliest stages of folliculogenesis. These results imply that gap junctional coupling mediated by Cx43 channels plays indispensable roles in both germ line development and postnatal folliculogenesis.     

Analysis of the Drosophila female sterile mutation fs(1) 1304 by pole cell transplantation experiments

The Drosophila melanogaster mutant fs(1)1304 is an ovary autonomous female sterile mutant that causes abnormal morphology of the egg. Vitellogenesis proceeds at an abnormally slow rate in homozygous females. We have used pole cell transplantation to construct germ line mosaics in order to determine whether the 1304 defect depends upon the genotype of the germ line cells (oocyte or nurse cells) or the somatic line (follicle cells). We have found that the germ line is the primary target tissue where the mutant gene is expressed.     

Transvection in the Iab-5,6,7 Region of the Bithorax Complex of Drosophila: Homology Independent Interactions in Trans

The Abdominal-B (Abd-B) gene of the bithorax complex (BX-C) of Drosophila controls the identities of the fifth through seventh abdominal segments and segments in the genitalia (more precisely, parasegments 10-14). Here we focus on iab-5, iab-6 and iab-7, regulatory regions of Abd-B that control expression in the fifth, sixth and seventh abdominal segments (parasegments 10-12). By analysis of partial BX-C deficiencies, we show that these regions are able to promote fifth and sixth abdominal segment identities in the absence of an Abd-B gene in cis. We establish that this ability does not result from cis-regulation of the adjacent abd-A or Ubx genes of the BX-C but rather occurs because the iab-5,6,7 region is able to interact with Abd-B in trans. We demonstrate that this interaction is proximity dependent and is, therefore, a case of what E. B. LEWIS has called transvection. Interactions of this type are presumably facilitated by the synapsis of homologues that occurs in somatic cells of Dipterans. Although transvection has been detected in a number of Drosophila genes, transvection of the iab-5,6,7 region is exceptional in two ways. First, interaction in trans with Abd-B does not require that homologues share homologous sequences within, or for some distance to either side of, the BX-C. This is the first case of transvection shown to be independent of local synapsis. A second unusual feature of iab-5,6,7 transvection is that it is remarkably difficult to disrupt by heterozygosity for chromosome rearrangements. The lack of requirement for local synapsis and the tenacity of trans-interaction argue that the iab-5,6,7 region can locate and interact with Abd-B over considerable distance. This is consistent with the normal role of iab-5,6,7, which must act over some 20-60 kb to influence its regulatory target in cis at the Abd-B promoter. Evidence is presented that trans-action of iab-5,6,7 requires, and may be mediated by, the region between distal iab-7 and Abd-B. Also, we show that iab-5,6,7 transvection is independent of the allelic state of zeste, a gene that influences several other cases of transvection. The long-range nature of interactions in trans between iab-5,6,7 and Abd-B suggests that similar interactions could operate effectively in organisms lacking extensive somatic pairing. Transvection may, therefore, be of more general significance than previously suspected.     

The molecular genetics of the bithorax complex of Drosophila: cis-regulation in the Abdominal-B domain.

In Drosophila the Abdominal-B (Abd-B) domain of the bithorax complex (BX-C) spans over 100 kb and is responsible for specifying the identities of adult abdominal segments five (A5) to nine (A9), inclusive, and correspondingly, neuromeres 10-14 of the embryonic central nervous system. The domain consists of a region coding for two proteins, ABD-BI (54 kd) and ABD-BII (36 kd) and cis-regulatory regions extending from infra-abdominal-5 (iab-5) to iab-9, inclusive. We have used a monoclonal anti-ABD-B antibody to infer that mutants in iab-8 eliminate the expression of ABD-BI in neuromeres 10-13, inclusive, and that mutants in iab-9 eliminate expression of ABD-BII in neuromere 14. ABD-B expression is also analyzed in homozygotes for (i) loss-of-function mutants involving the iab-5, iab-6 and iab-7 regions, (ii) gain-of-function mutants Miscadastral pigmentation (Mcp) and Superabdominal (Sab), and (iii) a trans-regulator, Polycomb (Pc). ABD-B expression along the antero-posterior axis is colinear with the chromosomal order of the cis-regulatory regions. The behavior of rearrangement-associated iab-6 and iab-7 mutants suggests that the enhancer-like region, iab-5, and possibly also iab-6, may be shared between the abd-A and Abd-B domains. Such sharing is proposed as a factor that tends to keep gene complexes intact during evolution.     

Ontogenesis of the ovary in a moth midge,Tinearia alternata Say (Diptera: Psychodidae)

In a psychodid, Tinearia alternata, the initial differentiation of the polytrophic ovary occurs during the early larval stages. Early in development, each ovary anlage is a solid organ subdivided into three distinct zones: the cortex houses germ cells and somatic interstitial tissue, while two other somatic regions will give rise to the oviduct calyx and anterior part of the lateral oviduct. Germ cell cluster formation precedes the development of ovarioles. Each ovariole houses only one functional egg chamber. All ovarioles within paired ovaries are developmentally synchronized. In the larval ovaries, the newly formed egg chambers and then the ovarioles are intermingeled with and surrounded by the somatic interstitial tissue of the ovary cortex. The interstitial cells give rise to all the somatic elements of the ovarioles. In the pupal ovaries, the remaining interstitial tissue degenerates; thus, the ovarioles protrude into the body cavity. The ovaries in psychodids develop relatively large and swollen oviduct calyxes that are equivalent to receptaculum seminis (spermatheca). The morphological differentiation of germ cells within the egg chambers starts during late larval/early pupal stages. Nurse cell nuclei contain prominent nucleoli and polytene chromosomes. Oocyte growth results from accumulation of yolk and then, in the final stages of oogenesis, from an inflow of cytoplasm from the nurse cells. J. Morphol. 236:167–177, 1998. © 1998 Wiley-Liss, Inc.     

Developmental Analysis of the Ovarian Tumor Gene during Drosophila Oogenesis

Severe alleles of the ovarian tumor (otu) and ovo genes result in female sterility in Drosophila melanogaster, producing adult ovaries that completely lack egg chambers. We examined the developmental stage in which the agametic phenotype first becomes apparent. Germ cell development in embryos was studied using a strategy that allowed simultaneous labeling of pole cells with the determination of embryonic genotype. We found that ovo(-) or otu(-) XX embryonic germ cells were indistinguishable in number and morphology from those present in wild-type siblings. The effects of the mutations were not consistently manifested in the female germline until pupariation, and there was no evidence that either gene was required for germ cell viability at earlier stages of development. The requirement for otu function in the pupal and adult ovary is supported by temperature-shift experiments using a heat-inducible otu gene construct. We demonstrate that otu activity limited to prepupal stages was not sufficient to support oogenesis, while induction during the pupal and adult periods caused suppression of the otu mutant phenotype.     

The chorion defect of the ocelliless mutation caused by abnormal follicle cell function

Homozygous Drosophila females bearing the ocelliless mutation are sterile and produce oocytes with abnormal chorions. It has been possible to determine in which tissues these defects reside by generating ovarian chimeras. Pole cells from ocelliless female embryos can give rise to functional oocytes surrounded by normal chorions when placed in a wild-type environment. Conversely, when wild-type pole cells are placed in homozygous ocelliless females, the oocytes that form from them have abnormal chorions and never give rise to progeny. Thus the chorion defect and sterility of the ocelliless mutation are not germ-line autonomous. Homozygous ocelliless ovaries will attach to the uterus when placed in a wild-type third instar larva, but few eggs are ever laid, and the chorions of stage 14 oocytes remain ocelliless in morphology. Wild-type ovaries continue to produce oocytes with normal chorion morphology when placed into ocelliless hosts, indicating that the ocelliless chorion defect is ovary autonomous. Thus the chorion defect of the ocelliless mutation resides in the ovarian somatic tissue, presumably the follicle cells.     

Marker genes identify three somatic cell types in the fetal mouse ovary

The two main functions of the ovary are the production of oocytes, which allows the continuation of the species, and secretion of female sex hormones, which control many aspects of female development and physiology. Normal development of the ovaries during embryogenesis is critical for their function and the health of the individual in later life. Although the adult ovary has been investigated in great detail, we are only starting to understand the cellular and molecular biology of early ovarian development. Here we show that the adult stem cell marker Lgr5 is expressed in the cortical region of the fetal ovary and this expression is mutually exclusive to FOXL2. Strikingly, a third somatic cell population can be identified, marked by the expression of NR2F2, which is expressed in LGR5- and FOXL2 double-negative ovarian somatic cells. Together, these three marker genes label distinct ovarian somatic cell types. Using lineage tracing in mice, we show that Lgr5-positive cells give rise to adult cortical granulosa cells, which form the follicles of the definitive reserve. Moreover, LGR5 is required for correct timing of germ cell differentiation as evidenced by a delay of entry into meiosis in Lgr5 loss-of-function mutants, demonstrating a key role for LGR5 in the differentiation of pre-granulosa cells, which ensure the differentiation of oogonia, the formation of the definitive follicle reserve, and long-term female fertility.     

Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila

Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. We have identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. We show that inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion.