Variation in mothers' arginine vasopressin receptor 1a and dopamine receptor D4 genes predicts maternal sensitivity via social cognition.

We examined the extent to which the arginine vasopressin receptor 1a (AVPR1a) and dopamine receptor D4 (DRD4) were related to sensitive maternal behavior directly or indirectly via maternal social cognition. Participants were 207 (105 European-American and 102 African-American) mothers and their children (52% females). Sensitive maternal behavior was rated and aggregated across a series of tasks when infants were 6 months, 1 year and 2 years old. At 6 months, mothers were interviewed about their empathy, attributions about infant behavior and beliefs about crying to assess their parenting-related social cognition. Mothers with long alleles for AVPR1a and DRD4 engaged in more mother-oriented social cognition (i.e. negative attributions and beliefs about their infants' crying, ß = 0.13, P < 0.05 and ß = 0.16, P < 0.05, respectively), which in turn predicted less sensitive maternal behavior (ß = -0.23, P < 0.01). Both indirect effects were statistically significant independent of one another and covariates [95% confidence interval (CI): -0.22, -0.03 and ß = -0.03 for AVPR; 95% CI: -0.20, -0.03 and ß = -0.04 for DRD4]. There were no significant direct effects of AVPR1a or DRD4 on maternal sensitivity (ß = 0.02, P = .73 and ß = -0.10, P = .57, respectively). The results did not vary for African-American and European-American mothers (Δχ2 = 18.76, Δdf = 16, P = 0.28). Results support the view that one mechanism by which maternal genes are associated with parental behavior is via social cognition.

A Community's Awareness and Perceptions of Genomic Medicine.

BACKGROUND: Focus groups were conducted in a mid-sized community to explore community members' awareness and perceptions of genomic medicine and identify effective methods to educate the public about this topic. METHODS: Thirteen focus groups were conducted with a demographically representative pool of 121 participants using a semi-structured interview guide. Transcripts were analyzed through a computer assisted approach with Atlas TI consisting of coding, categorizing, comparing, and contrasting relevant data. RESULTS: Identified categories were organized into 6 main themes, which were similar across the groups and included: a lack of awareness, perceived benefits, concerns about genomic medicine, reasons for poor health related behavior, the potential impact of genetic information on health behavior, and the best ways to educate the community. Common concerns included lack of affordability, unanticipated physical harm, mistrust of the government and researchers, downstream effects like overpopulation, playing God/disturbing the natural order, lack of regulations, loss of privacy, genetic discrimination, and moral dilemmas posed by genetic engineering, cloning, choosing traits, and abortions resulting from genetic information. Participants also discussed ways to educate the community. CONCLUSIONS: While individuals recognized that diseases run in families, personal experience was a driving factor in participants' level of knowledge. Many expressed optimism about genomic medicine. However, the lack of depth in responses and their misconceptions reflect a deficiency of knowledge, which along with their personal, moral, and global concerns could impede acceptance and utilization of genomic medicine. Many community members are receptive to learning more about genomic medicine, and many of their concerns and misconceptions can be addressed through a well designed education strategy.

Analysis of transcriptional activity mediated by Drosophila melanogaster ecdysone receptor isoforms in a heterologous cell culture system.

Ecdysteroid regulation of gene transcription in Drosophila melanogaster and other insects is mediated by a heterodimer comprised of Ultraspiracle (USP) and one of three ecdysone receptor (EcR) isoforms (A, B1 and B2). This study revealed that the EcR/USP heterodimer displays isoform-specific capabilities. EcRB1 is normally induced with a form of USP that is missing its DNA-binding domain (DBD), although potentiation by juvenile hormone (JH) III is reduced. The EcRA and B2 isoforms, however, display almost no response to ecdysteroids with the DBD(-) USP. A mutation, K497E, in the shared ligand-binding domain of the EcR isoforms caused elevated EcRB2-specific affinity for a canonical ecdysone response element. The effects of directed modification and mutagenesis offer a strategy for developing hypotheses and considerations for studying in vivo function.

Differential control of gene activity by isoforms A, B1 and B2 of the Drosophila ecdysone receptor.

The steroid hormone ecdysone initiates molting and metamorphosis in Drosophila via a heterodimeric receptor consisting of EcR that binds hormone, and USP, a homolog of the vertebrate RXR receptor. EcR exists in three isoforms EcRA, EcRB1 and EcRB2 that are thought to direct specific physiological responses to ecdysone. These three isoforms differ only in their N-terminal A/B domain that implies that sequences responsible for the differential physiological effects lie within the A/B domains of the EcR isoforms. In the present study, we set out to determine the capability of the three isoforms and their A/B domains to control gene transcription. When full-length EcR plasmids were cotransfected into mammalian cells with a USP expressing and a cognate reporter plasmid, the three EcR isoforms showed striking differences in their ability to control gene transcription, both in the presence and in the absence of hormone. Furthermore, the A/B domains of EcRB1 and of EcRB2 when fused to the GAL4 DNA binding domain are sufficient to activate transcription of a reporter gene, in yeast as well as in mammalian cells. In contrast, a fusion construct containing the A/B domain of EcRA represses basal transcription of the reporter gene. All these findings emphasize the importance of the A/B domains of the three EcR isoforms for differentially controlling gene transcription. Furthermore, they provide evidence for the existence of an autonomous ligand-independent activation function (AF1) in the A/B domains of EcRB1 and EcRB2 and of an inhibitory function (IF) in the A/B domain of EcRA.

Developmental effects of a chimeric ultraspiracle gene derived from Drosophila and Chironomus.

The ultraspiracle (usp) gene encodes a nuclear receptor that forms a heterodimer with the ecdysone receptor (EcR) to mediate transcriptional responses to the insect steroid hormone, 20-hydroxyecdysone (20HE). The responses ultimately elicit changes associated with molting and metamorphosis. Although Ultraspiracle (USP) is required at several developmental times, it is unclear whether USP plays stage-specific roles in Drosophila. A chimeric transgene (d/cusp), produced by replacing the ligand-binding domain (LBD) of Drosophila USP with the equivalent domain from another Diptera, Chironomus tentans, was tested for its ability to rescue Drosophila usp mutants from early larval lethality. A single copy of the d/cusp was sufficient to rescue transformants from several lines through larval development but they died suddenly during the late third instar. Additional doses of d/cusp were required to allow survival through the adult stage, but they did not restore a normal prepupal contraction. Thus, the arrest at the onset of metamorphosis apparently is caused by the impaired ability of the chimeric USP to mediate a stage-specific function associated with the LBD.

Functional characterization of two Ultraspiracle forms (CtUSP-1 and CtUSP-2) from Chironomus tentans.

Two forms, CtUSP-1 and CtUSP-2, of the Chironomus tentans homolog of Ultraspiracle (new nomenclature: Chironomus NR2B4) were described and verified as components of the functional ecdysteroid receptor. The two forms differed from each other in the most N-terminal regions of the A/B domain and were tested for several properties. Both forms showed the ability to heterodimerize with CtEcR and interact with a variety of direct repeat and palindromic EcREs, and both conferred specific ligand binding when heterodimerized with EcR. CtUSP-2 showed a twofold higher ponasterone-binding potential than CtUSP-1. Both USP forms demonstrated the ability to activate ecdysteroid-inducible transcription in HeLa cells and the variations in the A/B domain of these forms were not associated with detectable differences in transcriptional activation. Thus, the two forms function similarly. Among species for which USP forms have been reported, Chironomus is the most closely related one evolutionarily to Drosophila. Despite this proximity, a variety of structural differences were noted in both the A/B and E domains of USP between the two species. The Chironomus USP forms lack many of the amino acid residues associated with the ligand-dependent AF2 transactivation function found in all other RXRs and USPs reported so far.

Insect nuclear receptors: a developmental and comparative perspective.

The appearance of puffs on the polytene chromosomes of insect salivary glands incubated with 20-hydroxyecdysone provided the first demonstration that steroids act directly at the gene transcriptional level to bring about subsequent cellular changes (Becker, 1959; Clever and Karlson, 1960). Despite that auspicious beginning, learning about the molecular mechanisms that underlie the hormonal regulation of insect development was impeded for many years by the difficulty associated with isolating and identifying rare regulatory factors from limited tissue sources. The advent of recombinant DNA methodology and powerful techniques such as the polymerase chain reaction (PCR) along with the recognition that many important endocrine factors are structurally conserved across a wide range of species has, however, all but eliminated the technical obstacles once facing the insect endocrinologist trying to isolate and study these regulatory molecules. This review will discuss recent progress and recall some earlier experiments concerning the molecular basis of hormonal action in insects focusing primarily on the members of the nuclear hormone receptor superfamily in Drosophila melanogaster. Two members of this family comprise the functional ecdysteroid receptor and at least a dozen other "orphans" have been identified in Drosophila for which no cognate ligand has yet been found. Many of these orphans are regulated by ecdysteroids. A discussion of juvenile hormone binding proteins that are not family members has been included because of their potential impact on nuclear receptor function. As receptor homologues have been identified in other insects, several general ideas concerning insect hormonal regulation have begun to emerge and these will be examined from a comparative point of view.

Comparison of ecdysteroid production in Drosophila and Manduca: pharmacology and cross-species neural reactivity.

In both Manduca sexta and Drosophila melanogaster, metamorphic events are driven by ecdysteroids whose production in prothoracic gland (PGs) is stimulated periodically by neural factors. Differences in the life cycle of moths and flies have made it difficult to compare the regulation of ecdysteroid biosynthesis in these two species. As in Manduca, at least two neural factors in the larval Drosophila BVG complex were separable by molecular weight, and they stimulated increased ecdysteroid biosynthesis from the ring gland, a composite organ that includes PG cells. Drosophila neural extracts accelerated ecdysteroid biosynthesis in Manduca PGs and, conversely, partially purified Manduca PTTH preparations elevated ecdysteroid biosynthesis in Drosophila ring glands, suggesting that the two species may share structurally similar prothoracicotropic factors. Drosophila ring glands required the presence of calcium ions to respond to neural extracts, but the phosphodiesterase inhibitor MIX and cAMP analogues exerted little, if any, positive effect on production. Mean ecdysteroid production rates of BVG-ring gland complexes taken from Drosophila larvae during various phases of the wandering period were often submaximal and highly variable, suggesting that they fluctuate widely prior to pupariation. Based on available data in Drosophila and the Manduca model for the control of ecdysteroid biosynthesis, a developmental scheme for neuroendocrine control in Drosophila is proposed.

Expression and function of the ultraspiracle (usp) gene during development of Drosophila melanogaster.

The usp locus encodes a member of the nuclear hormone receptor superfamily in Drosophila melanogaster that interacts with EcR (ecdysone receptor) to mediate ecdysteroid-induced gene expression. A 2.7-kb usp mRNA was detected at all developmental times tested, although its abundance varied. Among premetamorphic stages, both the 2.7-kb transcript and Usp protein attained their highest levels in the late third larval instar. The 2.7-kb usp transcript was also found in adult stages and a 1.2-kb transcript was detected in the polyadenylated RNA fraction of both mature adult females and early embryos. Aneuploids carrying two usp mutant alleles and a putative variegating usp+ allele often developed deformities of the adult wing disc that apparently resulted from mutational disruption of usp activity before metamorphosis and whose frequency was affected by maternal genotype. Both of the recessive lethal usp mutations associated with this "cleft thorax" phenotype involved substitutions of conserved arginine residues in the DNA-binding domain, although the frequency of the phenotype was not the same for the two alleles. Both mutant proteins retained the ability to form heterodimers with EcR in vitro but showed reduced affinity for an ecdysone response element.

Developmental requirements for the ecdysoneless (ecd) locus in Drosophila melanogaster.

The ecdysoneless locus in Drosophila melanogaster has been defined previously by a single conditional mutation, I(3)ecd1, that causes an ecdysteroid deficit and larval death at the restrictive temperature, 29 degrees C, although the primary role of the mutation in developmental processes has been unclear. Gene dosage and complementation studies reported here for ecd1 and five nonconditional lethal alleles indicate that the ecd locus plays prezygotic and postzygotic roles essential for normal embryonic development, the successful completion of each larval molt, adult eclosion, and female fertility. The ecd locus is also required for normal macrochaete differentiation. For each observed phenotype, the severity of mutational effects was correlated with ecd mutant genotypes. In all cases, ecd1 homozygotes were least affected. Mutants heteroallelic for ecd1 and any one of four nonconditional recessive mutations were more severely affected than ecd1 homozygotes, revealing these as hypomorphic alleles. For all phenotypic effects, mutants heteroallelic for ecd1 and a dominant mutation (ecd3D) were most severely affected. These individuals died during embryogenesis at 29 degrees C and developed no macrochaetes on the dorsal thorax when transferred to 29 degrees C during the white prepupal stage. The ecd3D mutation also caused female semisterility in heterozygotes. Ecdysteroid regulation has been implicated previously in all the developmental processes disrupted by these ecd mutations except for macrochaete differentiation.

An ultrastructural analysis of the ecdysoneless (l(3)ecd1ts) ring gland during the third larval instar of Drosophila melanogaster.

In the late third larval instar of Drosophila melanogaster, the prothoracic gland, an endocrine portion of the ring gland, synthesizes ecdysteroids at an accelerated rate. The resultant ecdysteroid titer peak initiates the events associated with metamorphosis. The normal prothoracic gland displays several ultrastructural features at this developmental stage that reflect increased steroidogenic activity, including extensive infoldings of the plasma membrane (membrane invaginations) and an increase in both the concentration of smooth endoplasmic reticulum (SER) (or transitional ER) and elongated mitochondria. By contrast, the prothoracic glands of larvae homozygous for a conditional larval lethal mutation, l(3)ecd1ts, not only fail to produce ecdysteroids at normal levels at the restrictive temperature (29 degrees C), but also acquire abnormal morphological features that reflect the disruptive effects of the mutation. These abnormalities include an accumulation of lipid droplets presumed to contain sterol precursors of ecdysteroids, a disappearance of SER and a drastic reduction of membrane invaginations in the peripheral area of the cell. These morphological defects are observed in prothoracic glands dissected from larvae transferred from 18 degrees C to 29 degrees C approximately 24 h before observation and also within 4 h of an in vitro transfer to 29 degrees C following dissection from wandering third instar larvae reared at 18 degrees C. No ultrastructural abnormalities were noted in the corpus allatum portion of mutant ring glands. These observations further indicate the direct involvement of the ecd gene product in ecdysteroid synthesis and suggest a role for the gene in the proper transport of precursors to the site where they can be utilized in ecdysteroid biosynthesis.

A steroid/thyroid hormone receptor superfamily member in Drosophila melanogaster that shares extensive sequence similarity with a mammalian homologue.

A gene in Drosophila melanogaster that maps cytologically to 2C1-3 on the distal portion of the X-chromosome encodes a member of the steroid/thyroid hormone receptor superfamily. The gene was isolated from an embryonic cDNA library using an oligonucleotide probe that specifies the consensus amino acid sequence in the DNA-binding domain of several human receptors. The conceptual amino acid sequence of 2C reveals at least four regions of homology that are shared with all identified vertebrate receptors. Region I includes the two cysteine-cysteine zinc fingers that comprise a DNA-binding domain which typifies all members of the superfamily. In addition, three regions (Regions II-IV) in the carboxy-terminal portion of the protein that encode the putative hormone-binding domain of the 2C gene product resemble similar sequences in vertebrate steroid/thyroid hormone receptors. The similarity suggests that this Drosophila receptor possesses many of the regulatory functions attributed to these regions in vertebrate counterparts. A portion of Region II also resembles part of the human c-jun oncoprotein's leucine zipper, which in turn, has been demonstrated to be the heterodimerization site between the jun and fos oncoproteins. The 2C receptor-like protein most resembles the mouse H2RII binding protein, a member of the superfamily which has been implicated in the regulation of major histocompatibility complex (MHC) class I gene expression. These two gene products are 83% identical in the DNA-binding domain and 50% identical in the putative hormone-binding domain, although no ligand has been identified for either protein. The high degree of similarity in the hormone-binding domain between the 2C protein and the H2RII binding protein outside regions II-IV suggests specific functional roles which are not shared by other members of the superfamily.

The genetics of the Dras3-Roughened-ecdysoneless chromosomal region (62B3-4 to 62D3-4) in Drosophila melanogaster: analysis of recessive lethal mutations.

The genetic organization of interval 62B3-4 to 62D3-4 on the Drosophila third chromosome was investigated. The region (designated DRE) includes four known loci: Roughened (R; 3-1.4), defined by a dominant mutation disrupting eye morphology; the nonvital locus Aprt, structural gene for adenine phosphoribosyltransferase; Dras3, a homolog of the vertebrate ras oncogene; and 1(3)ecdysoneless (1(3)ecd), a gene that has been implicated in the regulation of larval molting hormone (ecdysteroid) synthesis. Overlapping chromosomal deletions of the region were generated by gamma-ray-induced reversion of the R mutation. Recessive lethal mutations were isolated based upon failure to complement the recessive lethality of Df(3L)RR2, a deletion of the DRE region that removes 16-18 polytene chromosome bands. A total of 117 mutations were isolated following ethyl methanesulfonate and gamma-ray mutagenesis. These and two additional define 13 lethal complementation groups. Mutations at two loci were recovered at disproportionately high rates. One of these loci is preferentially sensitive to radiation-induced mutational alterations. Additionally, an unusually low recovery rate for cytologically detectable rearrangement breakpoints within the gamma-ray-sensitive locus suggests that an interval of the DRE region closely linked to the R locus may be dominantly sensitive to position effects. Lethal phase analysis of mutant hemizygotes indicates that a high proportion of DRE-region loci (11 of 13) are necessary for larval development. Mutations in five loci cause predominantly first-instar larval lethality, while mutations in four other loci cause predominantly second-instar lethality. Mutations in two loci cause late-larval lethality associated with abnormal imaginal disc development. A temperature-sensitive allele of one newly identified complementation group blocks ecdysteroid-induced pupariation. This developmental block is overcome by dietary 20-hydroxyecdysone, suggesting that a second locus in the region in addition to l(3)ecd may play a role in the regulation of late larval ecdysteroid levels.

Induction of diapause in Drosophila melanogaster: photoperiodic regulation and the impact of arrhythmic clock mutations on time measurement.

The fruit fly Drosophila melanogaster displays an ovarian diapause that is regulated by photoperiod. Newly eclosed female flies (Canton-S wild type) exposed to short days (less than 14 hr of light per day) at 12 degrees C (or 10 degrees C) enter a fairly shallow reproductive diapause. Females exposed to long days (16 hr of light per day) at the same low temperature undergo ovarian maturation. The short day induced diapause continues for 6-7 weeks under a 10:14 light/dark cycle at 12 degrees C but is terminated rapidly after a transfer to higher temperature (18 or 25 degrees C) or to long days (18:6 light/dark cycle). Females from three strains homozygous for alleles of the period (per) locus, reportedly arrhythmic for behavioral circadian rhythms, and females that possessed two overlapping deletions of per were also capable of discriminating between long and short days, although, when compared with the wild-type flies, the critical day length was shifted to shorter values by approximately 2 hr. It is concluded that the period locus is not causally involved in photoperiod time measurement.

Juvenile hormone bisepoxide biosynthesis in vitro by the ring gland of Drosophila melanogaster: a putative juvenile hormone in the higher Diptera.

The in vitro production of juvenile hormone (JH) was investigated by using isolated ring glands from third instar Drosophila melanogaster. A JH-like molecule is secreted that comigrates with a synthetic sample of methyl 6,7;10,11-bisepoxy-3,7,11-trimethyl-(2E)-dodecenoate (JHB3) during TLC, liquid chromatography, and GC analysis. Purified product from farnesoic acid-stimulated ring glands was analyzed by electron impact GC/MS and gave a mass spectrum identical to synthetic JHB3. Additional structure confirmation was obtained following conversion of product from unstimulated biosynthesis to a derivative that comigrated on liquid chromatography with the derivative prepared from synthetic JHB3. Physiological studies revealed that JHB3 is produced solely by the corpus allatum portion of the ring gland in vitro. Isolated ring glands from other cyclorrhaphous dipteran larvae also produce JHB3 almost exclusively in vitro. Corpora allata from mosquito larvae, however, produce only JH III, indicating that JHB3 production may be restricted to the higher Diptera. Topically applied synthetic JHB3 caused developmental responses in newly formed D. melanogaster white puparia similar to those obtained with JH III. The data suggest that JHB3 is a fly juvenile hormone.

The ecdysoneless (ecd1ts) mutation disrupts ecdysteroid synthesis autonomously in the ring gland of Drosophila melanogaster.

Ring glands dissected from homozygous l(3)ecd1ts wandering larvae and upshifted in vitro to the restrictive temperature, 29 degrees C, synthesize abnormally low quantities of ecdysteroid. Nevertheless, ecd1 ring glands retain the ability to respond at 29 degrees C to an extract prepared from wild-type larval neural tissues that presumably contain prothoracicotropic hormone (PTTH), although both basal and stimulated levels of synthesis are lower than those in wild-type ring glands. Extracts prepared from ecd1 neural tissue exhibit an unusually high level of PTTH activity. Mutant ring glands downshifted in vitro to the permissive temperature after removal from larvae maintained at 29 degrees C regain the ability to produce normal basal and stimulated ecdysteroid levels. Collectively, these experiments demonstrate that the ecd1 mutation disrupts the physiology of the ring gland at 29 degrees C autonomously and may also interfere with PTTH release.

Neural factors that stimulate ecdysteroid synthesis by the larval ring gland of Drosophila melanogaster.

The larval ring gland of Drosophila melanogaster is the source of ecdysteroids responsible for larval-larval and larval-pupal molting. An extract prepared from the Drosophila larval central nervous system, that presumably contains prothoracicotropic hormone, elicits a significant and dose-dependent in vitro increase in ecdysteroid synthesis by ring glands from wandering third instar larvae. The synthesis of all three ecdysteroids previously identified as ring gland products is elevated by more than two-fold in the presence of neural extract. The maximum response occurs within 30 min and can be sustained for at least two hours after a 30 min exposure to neural extract. No non-neural tissue extracts evoke a response and most of the prothoracicotropic activity originates in the ventral ganglion. However, while extract prepared from larval brains elicits only a slight increase in ecdysteroid synthesis, it enhances the activity of a submaximal dose of ventral ganglion extract. This suggests that two or more neural factors, at least one from the brain lobes and another from the ventral ganglion, interact to stimulate ecdysteroid synthesis by the larval ring gland.