Bone morphogenetic protein 2 inhibits FSH responsiveness in hen granulosa cells.

Prior to follicle selection into the preovulatory hierarchy, hen granulosa cells from prehierarchal follicles remain undifferentiated, as defined in part by the virtual absence of LHR mRNA expression and inability to produce progesterone. It has previously been proposed that prior to follicle selection, granulosa cells are actively maintained in an undifferentiated state by epidermal growth factor receptor ligands (EGFRL) signaling via the MAP kinase/extracellular regulated kinase pathway. Moreover, there is recent evidence that EGFRL/MAP kinase signaling modulates FSH receptor (FSHR) transcription, in part, via inhibitor of differentiation/DNA-binding (ID) proteins. In the present studies with undifferentiated granulosa, recombinant human (rh) bone morphogenetic protein 2 (BMP2) induced the phosphorylation of SMAD1/5/8, and blocked transforming growth factor ß and FSH-induced FSHR expression and progesterone production. Significantly, BMP2 rapidly induced mRNAs encoding betacellulin and EGF, plus ID proteins (ID1, ID3, and ID4). Alternatively, the bioactivity of BMPs can be modulated by one or more BMP antagonists, including noggin (NOG). NOG mRNA is expressed by both hen granulosa and theca tissues from prehierarchal follicles. Pretreatment of cultured granulosa with rh NOG reversed both the stimulatory effects of BMP2 on ID1, ID3, and ID4 expression and the inhibitory effects of BMP2 on FSHR mRNA levels and progesterone production. Collectively, these data provide evidence that prior to follicle selection, BMP2 signaling contributes toward maintaining granulosa cells in an undifferentiated state. The actions of BMP2 are, at least in part, mediated indirectly via enhanced EGFRL expression and ERBB receptor-mediated MAP kinase signaling, and can be modulated by the autocrine/paracrine production of NOG.

Role for inhibitor of differentiation/deoxyribonucleic acid-binding (Id) proteins in granulosa cell differentiation.

Recent studies in the hen ovary have linked the initiation of granulosa cell differentiation at follicle selection to the alleviation of inhibitory MAPK signaling. The present studies assessed a role for individual inhibitor of differentiation (Id) protein isoforms as modulators of key transcriptional events occurring within granulosa cells at or immediately subsequent to differentiation. Findings from freshly collected granulosa cells collected at different stages of follicle development demonstrated a negative association between expression levels for Id2 mRNA compared with levels of Id1, Id3, and Id4. Elevated levels of Id2 are related to a differentiating/differentiated phenotype, whereas elevated Id1, Id3, and Id4 are associated with an undifferentiated phenotype. This negative relationship extends to cell signal transduction, because factors that promote inhibitory MAPK signaling (TGF-alpha and betacellulin) block expression of Id2 mRNA but increase levels of Id1, Id3, and Id4. Furthermore, overexpression of Gallus Id2 in cultured granulosa was found to significantly decrease levels of Id1, Id3, and Id4 mRNA but facilitate FSHR mRNA expression and, importantly, initiate LHR mRNA expression plus LH-induced progesterone production. Finally, knockdown studies using small interfering RNA specific for Id2 revealed reduced expression of FSHR and LHR mRNA and attenuated FSH- and LH-induced levels of StAR and p450 cholesterol side-chain cleavage enzyme mRNA plus progesterone production. Collectively, these data demonstrate that Id2 expression is both sufficient and necessary for increasing LHR expression and, as a result, promoting gonadotropin-induced differentiation in hen granulosa cells subsequent to follicle selection.

siRNA-mediated gene targeting in Aedes aegypti embryos reveals that frazzled regulates vector mosquito CNS development.

Although mosquito genome projects uncovered orthologues of many known developmental regulatory genes, extremely little is known about the development of vector mosquitoes. Here, we investigate the role of the Netrin receptor frazzled (fra) during embryonic nerve cord development of two vector mosquito species. Fra expression is detected in neurons just prior to and during axonogenesis in the embryonic ventral nerve cord of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector). Analysis of fra function was investigated through siRNA-mediated knockdown in Ae. aegypti embryos. Confirmation of fra knockdown, which was maintained throughout embryogenesis, indicated that microinjection of siRNA is an effective method for studying gene function in Ae. aegypti embryos. Loss of fra during Ae. aegypti development results in thin and missing commissural axons. These defects are qualitatively similar to those observed in Dr. melanogaster fra null mutants. However, the Aa. aegypti knockdown phenotype is stronger and bears resemblance to the Drosophila commissureless mutant phenotype. The results of this investigation, the first targeted knockdown of a gene during vector mosquito embryogenesis, suggest that although Fra plays a critical role during development of the Ae. aegypti ventral nerve cord, mechanisms regulating embryonic commissural axon guidance have evolved in distantly related insects.

Semaphorin-1a is required for Aedes aegypti embryonic nerve cord development.

Although mosquito genome projects have uncovered orthologues of many known developmental regulatory genes, extremely little is known about mosquito development. In this study, the role of semaphorin-1a (sema1a) was investigated during vector mosquito embryonic ventral nerve cord development. Expression of sema1a and the plexin A (plexA) receptor are detected in the embryonic ventral nerve cords of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector), suggesting that Sema1a signaling may regulate mosquito nervous system development. Analysis of sema1a function was investigated through siRNA-mediated knockdown in A. aegypti embryos. Knockdown of sema1a during A. aegypti development results in a number of nerve cord phenotypes, including thinning, breakage, and occasional fusion of the longitudinal connectives, thin or absent commissures, and general distortion of the nerve cord. Although analysis of Drosophila melanogaster sema1a loss-of-function mutants uncovered many similar phenotypes, aspects of the longitudinal phenotypes differed between D. melanogaster and A. aegypti. The results of this investigation suggest that Sema1a is required for development of the insect ventral nerve cord, but that the developmental roles of this guidance molecule have diverged in dipteran insects.

Comparative genomic analysis of Drosophila melanogaster and vector mosquito developmental genes.

Genome sequencing projects have presented the opportunity for analysis of developmental genes in three vector mosquito species: Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae. A comparative genomic analysis of developmental genes in Drosophila melanogaster and these three important vectors of human disease was performed in this investigation. While the study was comprehensive, special emphasis centered on genes that 1) are components of developmental signaling pathways, 2) regulate fundamental developmental processes, 3) are critical for the development of tissues of vector importance, 4) function in developmental processes known to have diverged within insects, and 5) encode microRNAs (miRNAs) that regulate developmental transcripts in Drosophila. While most fruit fly developmental genes are conserved in the three vector mosquito species, several genes known to be critical for Drosophila development were not identified in one or more mosquito genomes. In other cases, mosquito lineage-specific gene gains with respect to D. melanogaster were noted. Sequence analyses also revealed that numerous repetitive sequences are a common structural feature of Drosophila and mosquito developmental genes. Finally, analysis of predicted miRNA binding sites in fruit fly and mosquito developmental genes suggests that the repertoire of developmental genes targeted by miRNAs is species-specific. The results of this study provide insight into the evolution of developmental genes and processes in dipterans and other arthropods, serve as a resource for those pursuing analysis of mosquito development, and will promote the design and refinement of functional analysis experiments.

Functional analysis of genes in Aedes aegypti embryos.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. In recent years, RNA interference (RNAi) has proven to be an effective strategy for inhibiting gene function in many organisms. This protocol describes a method for knockdown of embryonic genes in Ae. aegypti embryos by microinjection of small interfering RNA (siRNA) designed to target a specific gene of interest. The procedure includes a strategy for siRNA design, microinjection, and measurement of knockdown effectiveness.

Culturing and egg collection of Aedes aegypti.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol describes methods for culturing Ae. aegypti and includes a procedure for egg collection that can be used in conjunction with fixation, immunohistochemistry, and in situ protocols.

Fixation and preparation of developing tissues from Aedes aegypti.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol describes a method for fixation and dissection of Ae. aegypti embryos, larvae, and pupae. Tissue processed in this manner can be used subsequently for in situ hybridization detection of mRNA or immunohistochemical analysis of protein expression.

Whole-mount in situ hybridization for analysis of gene expression during Aedes aegypti development.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol for whole-mount in situ hybridization can be used to analyze gene expression in Ae. aegypti embryos and larvae, a critical aspect of understanding developmental gene function in this vector mosquito.

Aedes aegypti: an emerging model for vector mosquito development.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects. Targets of particular interest include genes that regulate development. However, although the Ae. aegypti genome project uncovered homologs of many known developmental regulatory genes, little is known of the genetic regulation of development in Ae. aegypti or other vector mosquitoes. This article provides an overview of the background, husbandry, and potential uses of Ae. aegypti as a model species. Methods for culturing, collecting and fixing developing tissues, analyzing gene and protein expression, and knocking down genes are permitting detailed analyses of the functions of developmental regulatory genes and the selective inhibition of such genes during Ae. aegypti development. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.

Actions of epidermal growth factor receptor/mitogen-activated protein kinase and protein kinase C signaling in granulosa cells from Gallus gallus are dependent upon stage of differentiation.

Studies in both mammalian and nonmammalian ovarian model systems have demonstrated that activation of the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways modulates steroid biosynthesis during follicle development, yet the collective evidence for facilitory versus inhibitory roles of these pathways is inconsistent. The present studies in the hen ovary describe the changing role of MAPK and PKC signaling in the regulation of steroidogenic acute regulatory protein (STAR) expression and progesterone production in undifferentiated granulosa cells collected from prehierarchal follicles prior to follicle selection versus differentiated granulosa from preovulatory follicles subsequent to selection. Treatment of undifferentiated granulosa cells with a selective epidermal growth factor receptor (EGFR) and ERBB4 receptor tyrosine kinase inhibitor (AG1478) both augments FSH receptor (Fshr) mRNA expression and initiates progesterone production. Conversely, selective inhibitors of both EGFR/ERBB4 and MAPK activity attenuate steroidogenesis in differentiated granulosa cells subsequent to follicle selection. In addition, inhibition of PKC signaling with GF109203X augments FSH-induced Fshr mRNA plus STAR protein expression and initiates progesterone synthesis in undifferentiated granulosa cells, but inhibits both gonadotropin-induced STAR expression and progesterone production in differentiated granulosa. Granulosa cells from the most recently selected (9- to 12-mm) follicle represent a stage of transition as inhibition of MAPK signaling promotes, while inhibition of PKC signaling blocks gonadotropin-induced progesterone production. Collectively, these data describe stage-of-development-related changes in cell signaling whereby the differentiation-inhibiting actions of MAPK and PKC signaling in prehierarchal follicle granulosa cells undergo a transition at the time of follicle selection to become obligatory for gonadotropin-stimulated progesterone production in differentiated granulosa from preovulatory follicles.

Tumor necrosis factor-related apoptosis inducing ligand expression and activity in hen granulosa cells.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) represents one of several cytokine members of the tumor necrosis factor superfamily reported to initiate apoptosis in a wide range of transformed, but not most normal, cell types. The present studies were conducted to evaluate the potential for TRAIL to promote apoptotic cell death in differentiated granulosa cells collected from hen preovulatory follicles. While mRNA encoding critical components (including TRAIL) required for a functional extrinsic cell death pathway are expressed in granulosa cells, TRAIL treatment by itself fails to induce either caspase-3 activity or a decrease in cell viability. On the other hand, preculture of cells with the conventional chemotherapeutic, cisplatin, or the 20S proteosome inhibitor, Z-LLF-CHO, sensitizes granulosa cells to TRAIL as evidenced by enhanced caspase-3 activity after 4 h of culture and loss of cell viability after 24 h when compared with either cisplatin or Z-LLF-CHO treatment alone. Moreover, the sensitizing effect of Z-LLF-CHO on TRAIL-induced loss of cell viability is prevented by the selective caspase-8 inhibitor, Z-IETD-FMK. Interestingly, TRAIL mRNA expression is elevated both in prehierarchal follicles undergoing spontaneous atresia and in prehierarchal follicles induced to undergo atresia for 6 h in vitro. In summary, the data demonstrate the presence of a functional TRAIL signaling pathway in hen granulosa cells, and are consistent with the possibility that TRAIL signaling may directly or indirectly participate in the process of follicle atresia in vivo.

Opposing actions of TGFbeta and MAP kinase signaling in undifferentiated hen granulosa cells.

The present studies were conducted to establish interactions between transforming growth factor (TGF)-beta and the epidermal growth factor (EGF) family members, TGFalpha and betacellulin (BTC), relative to proliferation and differentiation of granulosa cells in hen ovarian follicles. Results presented demonstrate expression of TGFbeta isoforms, plus TGFalpha, BTC, and ErbB receptors in prehierarchal follicles, thus establishing the potential for autocrine/paracrine signaling and cross-talk within granulosa cells at the onset of differentiation. Treatment with TGFalpha or BTC increases levels of TGFbeta1 mRNA in undifferentiated granulosa cells, while the selective inhibitor of mitogen activated protein kinase signaling, U0126, reverses these effects. Moreover, TGFbeta1 attenuates c-myc mRNA expression and granulosa cell proliferation, while TGFalpha blocks both these inhibitory effects. Collectively, these data provide evidence that EGF family ligands regulate both the expression and biological actions of TGFbeta1 in hen granulosa cells, and indicate that the timely interaction of these opposing factors is an important modulator of both granulosa cell proliferation and differentiation.