The Drosophila daughterless gene autoregulates and is controlled by both positive and negative cis regulation.

As the only class I helix-loop-helix transcription factor in Drosophila, Daughterless (Da) has generally been regarded as a ubiquitously expressed binding partner for other developmentally regulated bHLH transcription factors. From analysis of a novel tissue-specific allele, da(lyh), we show that da expression is not constitutive, but is dynamically regulated. This transcriptional regulation includes somatic ovary-specific activation, autoregulation and negative regulation. Unexpectedly, the diverse functions of da may require that expression levels be tightly controlled in a cell and/or tissue-specific manner. Our analysis of da(lyh) identifies it as the first springer insertion that functions as an insulating element, with its disruptive activity mediated by the product of a fourth chromosome gene, Suppressor of lyh [Su(lyh)].

Type II cAMP-dependent protein kinase-deficient Drosophila are viable but show developmental, circadian, and drug response phenotypes.

We identified a unique type II cAMP-dependent protein kinase regulatory subunit (PKA-RII) gene in Drosophila melanogaster and a severely hypomorphic if not null mutation, pka-RII(EP(2)2162). Extracts from pka- RII(EP(2)2162) flies selectively lack RII-specific autophosphorylation activity and show significantly reduced cAMP binding activity, attributable to the loss of functional PKA-RII. pka-RII(EP(2)2162) shows 2-fold increased basal PKA activity and approximately 40% of normal cAMP-inducible PKA activity. pka-RII(EP(2)2162) is fully viable but displays abnormalities of ovarian development and multiple behavioral phenotypes including arrhythmic circadian locomotor activity, decreased sensitivity to ethanol and cocaine, and a lack of sensitization to repeated cocaine exposures. These findings implicate type II PKA activity in these processes in Drosophila and imply a common role for PKA signaling in regulating responsiveness to cocaine and alcohol.

daughterless is required for Drosophila photoreceptor cell determination, eye morphogenesis, and cell cycle progression.

Initiation of Drosophila peripheral nervous system (PNS) development requires the achaete-scute complex (AS-C) and the atonal (ato) genes. The AS-C and ato encode basic helix-loop-helix (bHLH) transcription factors that dimerize in vitro with another bHLH protein, daughterless (da). da has many functions during Drosophila embryonic development, as it is required for proper sex determination, oogenesis, and neurogenesis. Here, we examine the expression and function of da within the developing Drosophila eye. The use of a monoclonal antibody to the Da protein revealed that Da levels are modulated across the developing eye disc. Within the morphogenetic furrow (MF) and photoreceptor cell R8, there is a cell-by-cell correspondence between high levels of Da protein expression and Ato protein expression. Mosaic analysis of adult tissue demonstrates that da function is cell autonomous and required within R2, R3, R4, R5, and R8. Examination of gene expression in da- imaginal disc clones reveals that da regulates Ato expression in the MF, affects the progression of the MF, and is necessary for the reestablishment of the G2 and M phases of the synchronized cell cycle posterior to the MF.

The daughterless gene functions together with Notch and Delta in the control of ovarian follicle development in Drosophila.

The daughterless (da) gene in Drosophila encodes a broadly expressed transcriptional regulator whose specific functions in the control of sex determination and neurogenesis have been extensively examined. We describe here a third major developmental role for this regulatory gene: follicle formation during oogenesis. A survey of da RNA and protein distribution during oogenesis reveals a multiphasic expression pattern that includes both germline and soma. Whereas the germline expression reflects da's role in progeny sex determination, the somatic ovary expression of da correlates with the gene's role during egg chamber morphogenesis. Severe, but viable, hypomorphic da mutant genotypes exhibit dramatic defects during oogenesis, including aberrantly defined follicles and loss of interfollicular stalks. The follicular defects observed in da mutant ovaries are qualitatively very similar to those described in Notch (N) or Delta (Dl) mutant ovaries. Moreover, in the ovary da- alleles exhibit dominant synergistic interactions with N or Dl mutations. We propose that all three of these genes function in the same regulatory pathway to control follicle formation.

The daughterless gene product in Drosophila is a nuclear protein that is broadly expressed throughout the organism during development.

The daughterless (da) gene in Drosophila functions in the regulation of at least three significant developmental pathways: sex determination, neurogenesis and oogenesis. As a member of the helix-loop-helix (HLH) family of DNA binding proteins, the da gene product appears to act as a transcription factor. Based on the genetic and molecular characterization of da, it has been proposed that the da protein (Da) functions as a generic member of this family, serving throughout development as a necessary binding partner for an assortment of other HLH proteins. As a result of temporally and/or spatially restricted expression, these binding partners would provide some regulatory specificity to the functional transcription complex. In order to participate in this way in the regulation of multiple genes, Da must be expressed in numerous times and places during development. Using anti-Da antibodies, we validate two predictions of this scenario of Da function: (1) Da protein is not only nuclear localized, but also associated with chromosomes in vivo; and (2) Da protein is widely distributed, both spatially and temporally, throughout development. With regard to the essential role of maternal da+ in progeny sex determination, little, if any, Da protein is synthesized in the maternal germline. This suggests that the female-specific germline function of da+ is provided to the zygote as maternally synthesized RNA that becomes translated early in embryogenesis.

Molecular characterization of daughterless, a Drosophila sex determination gene with multiple roles in development.

The daughterless (da) gene in Drosophila acts both maternally and zygotically to provide essential functions during development. Maternal da+ expression is required by embryos for the regulation of sex determination and dosage compensation. Zygotic da+ expression is required throughout development--early for the formation of the peripheral nervous system and perhaps for the proper functioning of genes in heterochromatic regions of the genome; during larval stages for growth and differentiation of the future adult epidermis; and in the somatic part of the gonad of adult females for proper egg membrane synthesis. Here we describe the cloning of da by the transposon tagging approach as well as some aspects of the molecular characterization of wild-type and mutant alleles. Despite the multiple developmental roles of da, the organization and expression of this gene appear relatively simple at this level of analysis. The gene codes for two transcripts, present in both sexes and at all stages of development. The nucleotide sequence of a nearly full-length cDNA predicts a protein product of 710 amino acids that shares sequence similarity with the His-Pro repeat of the Drosophila genes bicoid and paired. Two partial-loss-of-function da mutations (one of which is temperature sensitive) appear to be caused by DNA insertions in the 5'-untranslated region of the gene.

The Drosophila sex determination gene daughterless has different functions in the germ line versus the soma.

As a regulator of the female-specific gene Sxl, da+ provides an essential maternal component in the control of sex determination and dosage compensation; nevertheless, neither the maternal nor zygotic phenotypes of the original mutant da allele is sex-specific. Here we clarify the role of da+ in Drosophila development, finding: this sex determination gene is indeed pleiotropic; zygotic functioning of da+ is essential in both sexes for somatic cell development, but not for germ cell development; da female sterility results from a somatic, rather than germ-line, defect; and expression of da+ in the maternal germ line is required only for daughters in the subsequent generation, as expected for a specific regulator of Sxl+. These conclusions follow from the characterization of new da null alleles isolated by a selection for defects in maternally acting positive regulators of Sxl.

The relationship of relative gene dose to the complex phenotype of the daughterless locus in Drosophila.

The daughterless (da) gene provides an essential maternally supplied component for Drosophila sex determination and dosage compensation. In this connection, it is required as a positive regulator of a female-specific master regulatory gene, Sex-lethal (Sxl). In addition, zygotic da gene function is required for male and female viability. Thus, the phenotype da is complex; it includes both maternal and zygotic aspects, as well as both sex-specific and nonsex-specific aspects. Assessment of wild-type da function has relied on the characterization of only a single leaky mutant da allele. In order to better understand the nature of this allele and the relationships between the various aspects of its complex phenotype, tandem duplications of both the mutant and wild-type da alleles were isolated and used in a dose study of this gene's function. Three conclusions were reached: 1) by the most stringent genetic criteria, the mutant da allele is a simple hypomorph, an allele with reduced but non-zero levels of wild-type functions; 2) since increased dose of da+ had no effect on viability or progeny sex ratio, this gene seems not to be a dose-sensitive element of the X/A ratio sex determination signal; and 3) expression of the maternal da+ allele does make a contribution to the nonsex-specific developmental processes that require zygotic da+ function; however, that contribution is clearly minor. In contrast, the zygotic genotype with respect to da appears to have no effect on the expression of Sxl+ in the zygote, the sex-specific process that requires maternal da+ function.

METT-1: a karyotypically normal in vitro line of developmentally totipotent mouse teratocarcinoma cells.

A karyotypically normal, chromosomally female (X/X) in vitro line of mouse teratocarcinoma stem cells was established from a malignant mouse teratocarcinoma of the 129/Sv Sl C P inbred strain. The tumor of origin was experimentally induced by ectopic transplantation of a 6-day embryo. The normal number of chromosomes was observed in 92% of metaphases of the cultured cells. This high frequency of euploidy, as well as karyotypic normalcy, were maintained during numerous passages in culture without a feeder-cell layer and after freezing and thawing of the cells. The line has been designated METT-1 (Mouse Euploid Totipotent Teratocarcinoma), signifying that it is the first such in vitro line that has proved (in tests by T. Stewart and B. Mintz, manuscript in preparation) to be developmentally totipotent, i.e., capable of both somatic and germinal differentiation when injected into blastocysts, even after freezing and thawing and prolonged culture. This unique ensemble of properties renders the cell line suitable for selection of specific mutant genes and for gene-transfer experiments in culture, for the purpose of producing from the mutant cells new strains of mice with predetermined genetic changes.

Normal blood cells of anemic genotype in teratocarcinoma-derived mosaic mice.

In allophenic (mosaic) mice produced from blastocysts injected with teratocarcinoma stem cells of the OTT 6050 transplant line, an unexpected coat phenotype led to the discovery that the tumor-lineage cells carried the steel gene (Sl(J)/+). Because steel also causes a macrocytic anemia, mosaics comprising both genetically anemic and normal (+/+) cells fortuitously provided a unique opportunity to examine in vivo the etiology of this anemia in light of previous results indicating that the lesion is extrinsic to the erythroid cells. The experiment differs from previous ones, which involved postnatal grafting, in that here hematopoietic stem cells of anemic and normal genotypes coexist throughout all developmental stages, confronted by tissues of the hematopoietic microenvironment that consist partly or solely of genetically normal cells. Therefore, the possibility exists that the anemia might be completely prevented rather than secondarily ameliorated. Moreover, variation in proportion of normal-strain cells in the hematopoietic supporting tissues could serve to "titrate" minimal requirements to promote normal erythropoiesis. Mice with mixed populations of steel- and normal-genotype cells in blood and other tissues were identified by means of independent markers specific for tumor vs. blastocyst strains of origin. The clinical blood picture of these mosaics proved to be indistinguishable from that of normal controls, even when only a small minority of cells in all tissues of one of the animals were genetically normal. Phenotypic blood normalcy was shown, by occurrence of the typical steel anemia among F(1) germ-line progeny of mosaics, not to be due to any change in the capacity of the mutant gene to elicit the anemia. The results from the mosaics thus demonstrate that the primary expression of the steel lesion is indeed in the hematopoietic microenvironment. However, they also reveal that a surprisingly small complement of normal cells there appears to be adequate to prevent this anemia permanently. The hypothesis is advanced that relatively short-range diffusible substances, produced by cells in the microenvironment and required for normal erythropoiesis, may account for the inductive effectiveness of small cell numbers.

Somatic cell origin of teratocarcinomas.

Malignant teratocarcinomas arise from developmentally totipotent normal stem cells. Whether the targets are embryonal somatic cells or germinal cells has long been a matter of controversy. Past experiments on teratocarcinoma induction by ectopic grafting of early rodent embryos or fetal germinal ridges have remained ambiguous because embryos ordinarily soon form germ cells, and parthenogenetic germ cells form "embryos." In order to interrupt the developmental cycle at its most telling point, day 6 (egg-cylinder stage) mouse embryos of genetically sterile types were grafted; in such grafts, only a terminal residue of totipotent embryonal somatic ("ectoderm") cells is available, and subsequent germ cell development is severely impaired. One graft series, from S1(J)/+ matings, comprised 25% S1(J)/S1(J) presumptive sterile embryos; these grafts formed tumors containing embryonal carcinoma cells as often (47%) as did control +/+ grafts (41%) on the same genetic background. In another series, from W/+ matings, tumors of the sterile W/W genotype were individually identified by means of a closely linked marker, phosphoglucomutase (PGM, EC 2.7.5.1; Pgm-1 locus), coding for electrophoretic enzyme variants and incorporated into the stock. Four tumors were obtained (out of 16) that had the PGM-1D phenotype diagnostic for W/W, and that also contained embryonal carcinoma cells. Therefore, the malignancy arises here in susceptible somatic embryonal stem cells at the terminal stage of their capacity for totipotency. Other teratocarcinomas-whether induced or spontaneous-of ostensible germ-cell origin by parthenogenesis may also depend upon development of the same somatic target cells before neoplastic conversion can occur. A general model based on these experiments is proposed for all malignancies: Malignant transformation of a particular kind of normal stem cell may be possible only when that stem cell has progressed to the threshold of further differentiation.