Female-specific myoinhibitory peptide neurons regulate mating receptivity in Drosophila melanogaster.

Upon mating, fruit fly females become refractory to further mating for several days. An ejaculate protein called sex peptide (SP) acts on uterine neurons to trigger this behavioural change, but it is still unclear how the SP signal modifies the mating decision. Here we describe two groups of female-specific local interneurons that are important for this process-the ventral abdominal lateral (vAL) and ventral abdominal medial (vAM) interneurons. Both vAL and vAM express myoinhibitory peptide (Mip)-GAL4. vAL is positive for Mip neuropeptides and the sex-determining transcriptional factor doublesex. Silencing the Mip neurons in females induces active rejection of male courtship attempts, whereas activation of the Mip neurons makes even mated females receptive to re-mating. vAL and vAM are located in the abdominal ganglion (AG) where they relay the SP signal to other AG neurons that project to the brain. Mip neuropeptides appear to promote mating receptivity both in virgins and mated females, although it is dispensable for normal mating in virgin females.

Characterization of a new Gsx2-cre line in the developing mouse telencephalon.

In this study, we generated a transgenic mouse line driving Cre and EGFP expression with two putative cis-regulatory modules (CRMs) (i.e., hs687 and hs678) upstream of the homeobox gene Gsx2 (formerly Gsh2), a critical gene for establishing lateral ganglionic eminence (LGE) identity. The combination of these two CRMs drives transgene expression within the endogenous Gsx2 expression domains along the anterior-posterior neuraxis. By crossing this transgenic line with the RosatdTomato (Ai14) reporter mouse line, we observed a unique recombination pattern in the lateral ventral telencephalon, namely the LGE and the dorsal half of the medial GE (MGE), but not in the septum. We found robust recombination in many cell types derived from these embryonic regions, including olfactory bulb and amygdala interneurons and striatal projection neurons from the LGE, as well as cortical interneurons from the MGE and caudal GE (CGE). In summary, this transgenic mouse line represents a new tool for genetic manipulation in the LGE/CGE and the dorsal half of MGE.

Retinal homeobox promotes cell growth, proliferation and survival of mushroom body neuroblasts in the Drosophila brain.

The Drosophila mushroom bodies, centers of olfactory learning and memory in the fly 'forebrain', develop from a set of neural stem cells (neuroblasts) that generate a large number of Kenyon cells (KCs) during sustained cell divisions from embryonic to late pupal stage. We show that retinal homeobox (rx), encoding for an evolutionarily conserved transcription factor, is required for proper development of the mushroom bodies. Throughout development rx is expressed in mushroom body neuroblasts (MBNBs), their ganglion mother cells (MB-GMCs) and young KCs. In the absence of rx function, MBNBs form correctly but exhibit a reduction in cell size and mitotic activity, whereas overexpression of rx increases growth of MBNBs. These data suggest that Rx is involved in the control of MBNB growth and proliferation. Rx also promotes cell cycling of MB-GMCs. Moreover, we show that Rx is important for the survival of MBNBs and Kenyon cells which undergo premature cell death in the absence of rx function. Simultaneous blocking of cell death restores the normal set of MBNBs and part of the KCs, demonstrating that both, impaired proliferation and premature cell death (of MBNBs and KCs) account for the observed defects in mushroom body development. We then show that Rx controls proliferation within the MBNB clones independently of Tailless (Tll) and Prospero (Pros), and does not regulate the expression of other key regulators of MB development, Eyeless (Ey) and Dachshund (Dac). Our data support that the role of Rx in forebrain development is conserved between vertebrates and fly.

Prediction of G protein-coupled receptor encoding sequences from the synganglion transcriptome of the cattle tick, Rhipicephalus microplus.

The cattle tick, Rhipicephalus (Boophilus) microplus, is a pest which causes multiple health complications in cattle. The G protein-coupled receptor (GPCR) super-family presents a candidate target for developing novel tick control methods. However, GPCRs share limited sequence similarity among orthologous family members, and there is no reference genome available for R. microplus. This limits the effectiveness of alignment-dependent methods such as BLAST and Pfam for identifying GPCRs from R. microplus. However, GPCRs share a common structure consisting of seven transmembrane helices. We present an analysis of the R. microplus synganglion transcriptome using a combination of structurally-based and alignment-free methods which supplement the identification of GPCRs by sequence similarity. TMHMM predicts the number of transmembrane helices in a protein sequence. GPCRpred is a support vector machine-based method developed to predict and classify GPCRs using the dipeptide composition of a query amino acid sequence. These two bioinformatic tools were applied to our transcriptome assembly of the cattle tick synganglion. Together, BLAST and Pfam identified 85 unique contigs as encoding partial or full length candidate cattle tick GPCRs. Collectively, TMHMM and GPCRpred identified 27 additional GPCR candidates that BLAST and Pfam missed. This demonstrates that the addition of structurally-based and alignment-free bioinformatic approaches to transcriptome annotation and analysis produces a greater collection of prospective GPCRs than an analysis based solely upon methodologies dependent upon sequence alignment and similarity.

Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo.

Pigment epithelium-derived factor (PEDF) is a potent multifunctional protein that inhibits angiogenesis and has neurogenic and neuroprotective properties. Since the wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV), PEDF would be an ideal candidate to inhibit CNV and support retinal pigment epithelial (RPE) cells. However, its short half-life has precluded its clinical use. To deliver PEDF to the subretinal space, we transfected RPE cells with the PEDF gene using the Sleeping Beauty transposon system. Transfected cells expressed and secreted biologically active recombinant PEDF (rPEDF). In cultures of human umbilical vein endothelial cells, rPEDF reduced VEGF-induced cumulative sprouting by ≥47%, decreased migration by 77%, and increased rate of apoptosis at least 3.4 times. rPEDF induced neurite outgrowth in neuroblastoma cells and protected ganglion and photoreceptor cells in organotypic retinal cultures. In a rat model of CNV, subretinal transplantation of PEDF-transfected cells led to a reduction of the CNV area by 48% 14 days after transplantation and decreased clinical significant lesions by 55% and 40% after 7 and 14 days, respectively. We showed that transplantation of pigment epithelial cells overexpressing PEDF can restore a permissive subretinal environment for RPE and photoreceptor maintenance, while inhibiting choroidal blood vessel growth.

Tfap2a promotes specification and maturation of neurons in the inner ear through modulation of Bmp, Fgf and notch signaling.

Neurons of the statoacoustic ganglion (SAG) transmit auditory and vestibular information from the inner ear to the hindbrain. SAG neuroblasts originate in the floor of the otic vesicle. New neuroblasts soon delaminate and migrate towards the hindbrain while continuing to proliferate, a phase known as transit amplification. SAG cells eventually come to rest between the ear and hindbrain before terminally differentiating. Regulation of these events is only partially understood. Fgf initiates neuroblast specification within the ear. Subsequently, Fgf secreted by mature SAG neurons exceeds a maximum threshold, serving to terminate specification and delay maturation of transit-amplifying cells. Notch signaling also limits SAG development, but how it is coordinated with Fgf is unknown. Here we show that transcription factor Tfap2a coordinates multiple signaling pathways to promote neurogenesis in the zebrafish inner ear. In both zebrafish and chick, Tfap2a is expressed in a ventrolateral domain of the otic vesicle that includes neurogenic precursors. Functional studies were conducted in zebrafish. Loss of Tfap2a elevated Fgf and Notch signaling, thereby inhibiting SAG specification and slowing maturation of transit-amplifying cells. Conversely, overexpression of Tfap2a inhibited Fgf and Notch signaling, leading to excess and accelerated SAG production. However, most SAG neurons produced by Tfap2a overexpression died soon after maturation. Directly blocking either Fgf or Notch caused less dramatic acceleration of SAG development without neuronal death, whereas blocking both pathways mimicked all observed effects of Tfap2a overexpression, including apoptosis of mature neurons. Analysis of genetic mosaics showed that Tfap2a acts non-autonomously to inhibit Fgf. This led to the discovery that Tfap2a activates expression of Bmp7a, which in turn inhibits both Fgf and Notch signaling. Blocking Bmp signaling reversed the effects of overexpressing Tfap2a. Together, these data support a model in which Tfap2a, acting through Bmp7a, modulates Fgf and Notch signaling to control the duration, amount and speed of SAG neural development.

Three independent techniques localize expression of transcript afp-11 and its bioactive peptide products to the paired AVK neurons in Ascaris suum: in situ hybridization, immunocytochemistry, and single cell mass spectrometry.

We utilized three independent techniques, immunocytochemistry (ICC), single cell mass spectrometry (MS), and in situ hybridization (ISH), to localize neuropeptides and their transcripts in the nervous system of the nematode Ascaris suum . AF11 (SDIGISEPNFLRFa) is an endogenous peptide with potent paralytic effects on A. suum locomotory behavior. A highly specific antibody to AF11 showed robust immunostaining for AF11 in the paired AVK neurons in the ventral ganglion. We traced the processes from the AVK neurons into the ventral nerve cord and identified them as ventral cord interneurons. MS and MS/MS of single dissected AVKs detected AF11, two previously characterized peptides (AF25 and AF26), seven novel sequence-related peptides, including several sharing a PNFLRFamide C-terminus, and peptide NY, a peptide with an unrelated sequence. Also present in a subset of AVKs was AF2, a peptide encoded by the afp-4 transcript. By sequencing the afp-11 transcript, we discovered that it encodes AF11, all the AF11-related peptides detected by MS in AVK, and peptide NY. ISH detected the afp-11 transcript in AVK neurons, consistent with other techniques. ISH did not detect afp-11 in the ALA neuron, although both ICC and MS found AF11 in ca. 30% of ALAs. All 10 AF11-related peptides reduced acetylcholine-induced muscle contraction, but they differed in their rate of reversal of inhibition after removal of the peptide.

The role of Pax2 in mouse inner ear development.

The paired box transcription factor, Pax2, is important for cochlear development in the mouse inner ear. Two mutant alleles of Pax2, a knockout and a frameshift mutation (Pax21Neu), show either agenesis or severe malformation of the cochlea, respectively. In humans, mutations in the PAX2 gene cause renal coloboma syndrome that is characterized by kidney abnormalities, optic nerve colobomas and mild sensorineural deafness. To better understand the role of Pax2 in inner ear development, we examined the inner ear phenotype in the Pax2 knockout mice using paint-fill and gene expression analyses. We show that Pax2-/- ears often lack a distinct saccule, and the endolymphatic duct and common crus are invariably fused. However, a rudimentary cochlea is always present in all Pax2 knockout inner ears. Cochlear outgrowth in the mutants is arrested at an early stage due to apoptosis of cells that normally express Pax2 in the cochlear anlage. Lack of Pax2 affects tissue specification within the cochlear duct, particularly regions between the sensory tissue and the stria vascularis. Because the cochlear phenotypes observed in Pax2 mutants are more severe than those observed in mice lacking Otx1 and Otx2, we postulate that Pax2 plays a key role in regulating the differential growth within the cochlear duct and thus, its proper outgrowth and coiling.