Genomic Identification and Functional Analysis of JHAMTs in the Pond Wolf Spider, Pardosa pseudoannulata.

Juvenile hormone (JH) plays a critical role in many physiological activities of Arthropoda. Juvenile hormone acid methyltransferase (JHAMT) is involved in the last steps of JH biosynthesis as an important rate-limiting enzyme. In recent studies, an increasing number of JHAMTs were identified in arthropods, but no JHAMT was reported in spiders. Herein, eight JHAMTs were identified in the pond wolf spider, Pardosa pseudoannulata, all containing the well conserved S-adenosyl-L-methionine binding motif. JHAMT-1 and the other seven JHAMTs were located at chromosome 13 and chromosome 1, respectively. Multiple alignment and phylogenetic analysis showed that JHAMT-1 was grouped together with insect JHAMTs independently and shared high similarities with insect JHAMTs compared to the other seven JHAMTs. In addition, JHAMT-1, JHAMT-2, and JHAMT-3 were highly expressed in the abdomen of spiderlings and could respond to the stimulation of exogenous farnesoic acid. Meanwhile, knockdown of these three JHAMTs caused the overweight and accelerated molting of spiderlings. These results demonstrated the cooperation of multi-JHAMTs in spider development and provided a new evolutionary perspective of the expansion of JHAMT in Arachnida.

Structural basis for juvenile hormone biosynthesis by the juvenile hormone acid methyltransferase.

Juvenile hormone (JH) acid methyltransferase (JHAMT) is a rate-limiting enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis in insects and thus presents an excellent target for the development of insect growth regulators or insecticides. However, the three-dimensional properties and catalytic mechanism of this enzyme are not known. Herein, we report the crystal structure of the JHAMT apoenzyme, the three-dimensional holoprotein in binary complex with its cofactor S-adenosyl-l-homocysteine, and the ternary complex with S-adenosyl-l-homocysteine and its substrate methyl farnesoate. These structures reveal the ultrafine definition of the binding patterns for JHAMT with its substrate/cofactor. Comparative structural analyses led to novel findings concerning the structural specificity of the progressive conformational changes required for binding interactions that are induced in the presence of cofactor and substrate. Importantly, structural and biochemical analyses enabled identification of one strictly conserved catalytic Gln/His pair within JHAMTs required for catalysis and further provide a molecular basis for substrate recognition and the catalytic mechanism of JHAMTs. These findings lay the foundation for the mechanistic understanding of JH biosynthesis by JHAMTs and provide a rational framework for the discovery and development of specific JHAMT inhibitors as insect growth regulators or insecticides.

A haemocyte-expressed Methyltransf_FA domain containing protein (MFCP) exhibiting microbe binding activity in oyster Crassostrea gigas.

The Methyltransf_FA domain is well-known as a key protein domain of enzyme synthesizing juvenile hormone, and Methyltransf_FA domain containing proteins (MFCPs) are widely existed in vertebrates and invertebrates. In the present study, a CgMFCP with a single Methyltransf_FA domain was screened from oyster Crassostrea gigas, and its open reading frame of CgMFCP was of 1128 bp, encoding a polypeptide of 376 amino acids with a signal peptide, a Methyltransf_FA domain and a transmembrane region. CgMFCP was clustered with FAMeTs from insecta and crustacea of arthropod. The mRNA transcripts of CgMFCP were detected in different tissues, with the extremely high expression level in haemocytes, which was 131.36-fold (p < 0.05) of that in gills. The expression level of CgMFCP protein was verified to be highly expressed in haemocytes. The expression level of CgMFCP mRNA in primarily cultured haemocytes significantly up-regulated at 3 h, 24 h and 48 h post LPS stimulation, which was 3.25-fold (p < 0.01), 2.04-fold (p < 0.05) and 3.59-fold (p < 0.01) compared to that in blank group. After the oysters were stimulated with Vibrio splendidus in vivo, the expression level of CgMFCP mRNA in haemocytes was also significantly up-regulated at 3 h, 12 h, and 24 h, which was 4.22-fold (p < 0.05), 4.39-fold (p < 0.05) and 6.35-fold (p < 0.01) of that in control group, respectively. By flow cytometry analysis, anti-rCgMFCP can label 95% of oyster haemocytes. And by fluorescence microscope analysis, CgMFCP was mainly distributed in cytomembrane of haemocytes. The recombinant CgMFCP (rCgMFCP) exhibited higher affinity towards MAN and LPS in a dose-dependent manner, while relatively lower affinity to PGN and poly (I:C). rCgMFCP also displayed binding activities towards Gram-negative bacteria (Vibrio anguillarum and V. splendidus), Gram-positive bacteria (Staphylococcu aureu) and fungi (Pichia pastoris). These results collectively indicated that CgMFCP specifically expressed in haemocytes and functioned as a pattern recognition receptor by binding to various microbes in oyster C. gigas, which provided insight into the function of Methyltransf_FA domain containing proteins.

Juvenile hormone acts through FoxO to promote Cdc2 and Orc5 transcription for polyploidy-dependent vitellogenesis.

Vitellogenin (Vg) is a prerequisite for egg production and embryonic development after ovipositioning in oviparous animals. In many insects, juvenile hormone (JH) promotes fat body cell polyploidization for the massive Vg synthesis required for the maturation of multiple oocytes, but the underlying mechanisms remain poorly understood. Using the migratory locust Locusta migratoria as a model system, we report here that JH induces the dephosphorylation of Forkhead box O transcription factor (FoxO) through a signaling cascade including leucine carboxyl methyltransferase 1 (LCMT1) and protein phosphatase 2A (PP2A). JH promotes PP2A activity via LCMT1-mediated methylation, consequently triggering FoxO dephosphorylation. Dephosphorylated FoxO binds to the upstream region of two endocycle-related genes, cell-division-cycle 2 (Cdc2) and origin-recognition-complex subunit 5 (Orc5), and activates their transcription. Depletion of FoxO, Cdc2 or Orc5 results in blocked polyploidization of fat body cells, accompanied by markedly reduced Vg expression, impaired oocyte maturation and arrested ovarian development. The results suggest that JH acts via LCMT1-PP2A-FoxO to regulate Cdc2 and Orc5 expression, and to enhance ploidy of fat body cells in preparation for the large-scale Vg synthesis required for synchronous maturation of multiple eggs.

A mitochondrial phosphatase PTPMT1 is essential for the early development of silkworm, Bombyx mori.

Juvenile hormone (JH) plays important roles in the control of many biological processes in insects, such as development, reproduction, and polyphenism. JH is primarily produced in the corpora allata (CA) by specific JH biosynthetic enzymes under strict temporal regulation. In a previous study, we identified a novel putative JH biosynthetic gene, protein tyrosine phosphatase, mitochondrial 1 (PTPMT1), from silkworm, Bombyx mori, whose expression is nearly exclusive in the CA and is correlated with JH synthetic activities during late larval development. In this study, to reveal the function of PTPMT1 in vivo, we generated PTPMT1 knockout silkworms using TALEN. In the knockout mutants, no signs indicating defects in JH activity were observed. Instead, PTPMT1 knockout silkworms showed embryonic lethality, developmental arrest, and 3rd-instar lethality not only in mutants lacking total enzymatic activity but also in mutants lacking mitochondrial translocation signals. Moreover, in PTPMT1 knockout embryos, the expression of two genes encoded by the mitochondrial genome, CYTB and ND3, was decreased, indicating a mitochondrial disorder. These results suggested that PTPMT1 plays conserved vital role(s) reported in vertebrates in insect mitochondria.

miR-34 modulates wing polyphenism in planthopper.

Polyphenism is a successful strategy adopted by organisms to adapt to environmental changes. Brown planthoppers (BPH, Nilaparvata lugens) develop two wing phenotypes, including long-winged (LW) and short-winged (SW) morphs. Though insulin receptor (InR) and juvenile hormone (JH) have been known to regulate wing polyphenism in BPH, the interaction between these regulators remains largely elusive. Here, we discovered that a conserved microRNA, miR-34, modulates a positive autoregulatory feedback loop of JH and insulin/IGF signaling (IIS) pathway to control wing polyphenism in BPH. Nlu-miR-34 is abundant in SW BPHs and suppresses NlInR1 by targeting at two binding sites in the 3'UTR of NlInR1. Overexpressing miR-34 in LW BPHs by injecting agomir-34 induces the development towards SW BPHs, whereas knocking down miR-34 in SW BPHs by injecting antagomir-34 induces more LW BPHs when another NlInR1 suppressor, NlInR2, is also suppressed simultaneously. A cis-response element of Broad Complex (Br-C) is found in the promoter region of Nlu-miR-34, suggesting that 20-hydroxyecdysone (20E) might be involved in wing polyphenism regulation. Topic application of 20E downregulates miR-34 expression but does not change wing morphs. On the other hand, JH application upregulates miR-34 expression and induces more SW BPHs. Moreover, knocking down genes in IIS pathway changes JH titers and miR-34 abundance. In all, we showed that miRNA mediates the cross talk between JH, 20E and IIS pathway by forming a positive feedback loop, uncovering a comprehensive regulation mechanism which integrates almost all known regulators controlling wing polyphenism in insects.

NUMB regulates the endocytosis and activity of the anaplastic lymphoma kinase in an isoform-specific manner.

NUMB is an evolutionarily conserved protein that plays an important role in cell adhesion, migration, polarity, and cell fate determination. It has also been shown to play a role in the pathogenesis of certain cancers, although it remains controversial whether NUMB functions as an oncoprotein or tumor suppressor. Here, we show that NUMB binds to anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase aberrantly activated in several forms of cancer, and this interaction regulates the endocytosis and activity of ALK. Intriguingly, the function of the NUMB-ALK interaction is isoform-dependent. While both p66-NUMB and p72-NUMB isoforms are capable of mediating the endocytosis of ALK, the former directs ALK to the lysosomal degradation pathway, thus decreasing the overall ALK level and the downstream MAP kinase signal. In contrast, the p72-NUMB isoform promotes ALK recycling back to the plasma membrane, thereby maintaining the kinase in its active state. Our work sheds light on the controversial role of different isoforms of NUMB in tumorigenesis and provides mechanistic insight into ALK regulation.

Isolation functional characterization of allatotropin receptor from the cotton bollworm, Helicoverpa armigera.

Insect allatotropin (AT) plays multi-functions including regulation of juvenile hormone synthesis, growth, development and reproduction. In the present study, the full-length cDNA encoding the AT receptor was cloned from the brain of Helicoverpa armigera (Helar-ATR). The ORF of Helar-ATR exhibited the characteristic seven transmembrane domains of the G protein-coupled receptor (GPCR) and was close to the ATR of Manduca sexta in the phylogenetic tree. The Helar-ATR expressed in vertebrate cell lines can be activated by Helar-AT and each Helar-ATL in a dose-responsive manner, in the following order: Helar-ATLI > Helar-ATLII > Helar-AT > Helar-ATLIII. Helar-ATLI and Helar-ATLII represented the functional ligands to Helar-ATR in vitro, while Helar-AT and Helar-ATLIII behaved as partial agonists. The in vitro functional analysis suggested that the Helar-ATR signal was mainly coupled with elevated levels of Ca2+ and independent of cAMP levels. Helar-ATR mRNA in larvae showed the highest level in the brain, followed by the thorax ganglion, abdomen ganglion, fat body and midgut. Helar-ATR mRNA levels in the complex of the brain-thoracic-abdomen ganglion on the 2nd day of the larval stage and during later pupal stages were observed to be relatively higher than in the wandering and early pupal stages.

Omics approaches to study juvenile hormone synthesis.

The juvenile hormones (JHs) are a family of insect acyclic sesquiterpenoids produced by the corpora allata (CA), a pair of endocrine glands connected to the brain. They are involved in the regulation of development, reproduction, behavior, caste determination, diapause, stress response, and numerous polyphenisms. In the post-genomics era, comprehensive analyses using functional 'omics' technologies such as transcriptomics, proteomics and metabolomics have increased our understanding of the activity of the minute CA. This review attempts to summarize some of the 'omics' studies that have contributed to further understand JH synthesis in insects, with an emphasis on our own research on the mosquito Aedes aegypti.

The bantam microRNA acts through Numb to exert cell growth control and feedback regulation of Notch in tumor-forming stem cells in the Drosophila brain.

Notch (N) signaling is central to the self-renewal of neural stem cells (NSCs) and other tissue stem cells. Its deregulation compromises tissue homeostasis and contributes to tumorigenesis and other diseases. How N regulates stem cell behavior in health and disease is not well understood. Here we show that N regulates bantam (ban) microRNA to impact cell growth, a process key to NSC maintenance and particularly relied upon by tumor-forming cancer stem cells. Notch signaling directly regulates ban expression at the transcriptional level, and ban in turn feedback regulates N activity through negative regulation of the Notch inhibitor Numb. This feedback regulatory mechanism helps maintain the robustness of N signaling activity and NSC fate. Moreover, we show that a Numb-Myc axis mediates the effects of ban on nucleolar and cellular growth independently or downstream of N. Our results highlight intricate transcriptional as well as translational control mechanisms and feedback regulation in the N signaling network, with important implications for NSC biology and cancer biology.

Juvenile hormone differentially regulates two Grp78 genes encoding protein chaperones required for insect fat body cell homeostasis and vitellogenesis.

Juvenile hormone (JH) has a well known role in stimulating insect vitellogenesis (i.e. yolk deposition) and oocyte maturation, but the molecular mechanisms of JH action in insect reproduction are unclear. The 78-kDa glucose-regulated protein (Grp78) is a heat shock protein 70-kDa family member and one of the most abundant chaperones in the endoplasmic reticulum (ER) where it helps fold newly synthesized peptides. Because of its prominent role in protein folding, and also ER stress, we hypothesized that Grp78 might be involved in fat body cell homeostasis and vitellogenesis and a regulatory target of JH. We report here that the migratory locust Locusta migratoria possesses two Grp78 genes that are differentially regulated by JH. We found that Grp78-1 is regulated by JH through Mcm4/7-dependent DNA replication and polyploidization, whereas Grp78-2 expression is directly activated by the JH-receptor complex comprising methoprene-tolerant and Taiman proteins. Interestingly, Grp78-2 expression in the fat body is about 10-fold higher than that of Grp78-1 Knockdown of either Grp78-1 or Grp78-2 significantly reduced levels of vitellogenin (Vg) protein, accompanied by retarded maturation of oocytes. Depletion of both Grp78-1 and Grp78-2 resulted in ER stress and apoptosis in the fat body and in severely defective Vg synthesis and oocyte maturation. These results indicate a crucial role of Grp78 in JH-dependent vitellogenesis and egg production. The presence and differential regulation of two Grp78 genes in L. migratoria likely help accelerate the production of this chaperone in the fat body to facilitate folding of massively synthesized Vg and other proteins.

Synthesis and biological activity of FGLamide allatostatin analogs with Phe(3) residue modifications.

A FGLamide allatostatin neuropeptide mimic (H17) is a potential insect growth regulator which inhibits the production of juvenile hormone by the corpora allata. To find more evidence to reveal the structure-activity relationships of the Phe(3) residue in the C-terminal conserved pentapeptide and search for novel analogs with high activity, a series of Phe(3) residue-modified analogs were designed and synthesized using H17 as the lead compound. Bioassay using juvenile hormone (JH) production by corpora allata of the cockroach Diploptera punctata indicated that analogs 4, 11, and 13 showed strong ability to inhibit JH production in vitro, with IC50 of 38.5, 22.5, and 26 nM, respectively. As well, the activity of analog 2 (IC50 : 89.5 nM) proved roughly equivalent to that of H17. Based on the primary structure-activity relationships of Phe(3) residue, we suggest that for analogs containing six-membered aromatic rings, removing the methylene group of Phe(3) or an o-halogen or p-halogen-substituted benzene ring could increase the ability to inhibit biosynthesis of JH. This study will be useful for the design of new allatostatin analogs for insect management. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

TOR Pathway-Mediated Juvenile Hormone Synthesis Regulates Nutrient-Dependent Female Reproduction in Nilaparvata lugens (Stål).

The "target of rapamycin" (TOR) nutritional signaling pathway and juvenile hormone (JH) regulation of vitellogenesis has been known for a long time. However, the interplay between these two pathways regulating vitellogenin (Vg) expression remains obscure. Here, we first demonstrated the key role of amino acids (AAs) in activation of Vg synthesis and egg development in Nilaparvata lugens using chemically defined artificial diets. AAs induced the expression of TOR and S6K (S6 kinase), whereas RNAi-mediated silencing of these two TOR pathway genes and rapamycin application strongly inhibited the AAs-induced Vg synthesis. Furthermore, knockdown of Rheb (Ras homologue enriched in brain), TOR, S6K and application of rapamycin resulted in a dramatic reduction in the mRNA levels of jmtN (juvenile hormone acid methyltransferase, JHAMT). Application of JH III on the RNAi (Rheb and TOR) and rapamycin-treated females partially rescued the Vg expression. Conversely, knockdown of either jmtN or met (methoprene-tolerant, JH receptor) and application of JH III had no effects on mRNA levels of Rheb, TOR and S6K and phosphorylation of S6K. In summary, our results demonstrate that the TOR pathway induces JH biosynthesis that in turn regulates AAs-mediated Vg synthesis in N. lugens.

Transcriptome analysis to identify genes for peptides and proteins involved in immunity and reproduction from male accessory glands and ejaculatory duct of Bactrocera dorsalis.

In the male reproductive system of insects, the male accessory glands and ejaculatory duct (MAG/ED) are important organs and their primary function is to enhance the fertility of spermatozoa. Proteins secreted by the MAG/ED are also known to induce post-mating changes and immunity responses in the female insect. To understand the gene expression profile in the MAG/ED of the oriental fruit fly Bactrocera dorsalis (Hendel), that is an important pest in fruits, we performed an Illumina-based deep sequencing of mRNA. This yielded 54,577,630 clean reads corresponding to 4.91Gb total nucleotides that were assembled and clustered to 30,669 unigenes (average 645bp). Among them, 20,419 unigenes were functionally annotated to known proteins/peptides in Gene Orthology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes pathway databases. Typically, many genes were involved in immunity and these included microbial recognition proteins and antimicrobial peptides. Subsequently, the inducible expression of these immunity-related genes was confirmed by qRT-PCR analysis when insects were challenged with immunity-inducible factors, suggesting their function in guaranteeing fertilization success. Besides, we identified some important reproductive genes such as juvenile hormone- and ecdysteroid-related genes in this de novo assembly. In conclusion, this transcriptomic sequencing of B. dorsalis MAG/ED provides insights to facilitate further functional research of reproduction, immunity and molecular evolution of reproductive proteins in this important agricultural pest.

Temporal Coordination of Carbohydrate Metabolism during Mosquito Reproduction.

Hematophagous mosquitoes serve as vectors of multiple devastating human diseases, and many unique physiological features contribute to the incredible evolutionary success of these insects. These functions place high-energy demands on a reproducing female mosquito, and carbohydrate metabolism (CM) must be synchronized with these needs. Functional analysis of metabolic gene profiling showed that major CM pathways, including glycolysis, glycogen and sugar metabolism, and citrate cycle, are dramatically repressed at post eclosion (PE) stage in mosquito fat body followed by a sharply increase at post-blood meal (PBM) stage, which were also verified by Real-time RT-PCR. Consistent to the change of transcript and protein level of CM genes, the level of glycogen, glucose and trehalose and other secondary metabolites are also periodically accumulated and degraded during the reproductive cycle respectively. Levels of triacylglycerols (TAG), which represent another important energy storage form in the mosquito fat body, followed a similar tendency. On the other hand, ATP, which is generated by catabolism of these secondary metabolites, showed an opposite trend. Additionally, we used RNA interference studies for the juvenile hormone and ecdysone receptors, Met and EcR, coupled with transcriptomics and metabolomics analyses to show that these hormone receptors function as major regulatory switches coordinating CM with the differing energy requirements of the female mosquito throughout its reproductive cycle. Our study demonstrates how, by metabolic reprogramming, a multicellular organism adapts to drastic and rapid functional changes.

Juvenile hormone-receptor complex acts on mcm4 and mcm7 to promote polyploidy and vitellogenesis in the migratory locust.

Juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata, coordinates insect growth, metamorphosis, and reproduction. While JH action for the repression of larval metamorphosis has been well studied, the molecular basis of JH in promoting adult reproduction has not been fully elucidated. Methoprene-tolerant (Met), the JH receptor, has been recently shown to mediate JH action during metamorphosis as well as in vitellogenesis, but again, the precise mechanism underlying the latter has been lacking. We have now demonstrated using Met RNAi to phenocopy a JH-deprived condition in migratory locusts, that JH stimulates DNA replication and increases ploidy in preparation for vitellogenesis. Mcm4 and Mcm7, two genes in the DNA replication pathway were expressed in the presence of JH and Met. Depletion of Mcm4 or Mcm7 inhibited de novo DNA synthesis and polyploidization, and resulted in the substantial reduction of vitellogenin mRNA levels as well as severely impaired oocyte maturation and ovarian growth. By using luciferase reporter and electrophoretic mobility shift assays, we have shown that Met directly regulates the transcription of Mcm4 and Mcm7 by binding to upstream consensus sequences with E-box or E-box-like motifs. Our work suggests that the JH-receptor complex acts on Mcm4 and Mcm7 to regulate DNA replication and polyploidy for vitellogenesis and oocyte maturation.

Clueless regulates aPKC activity and promotes self-renewal cell fate in Drosophila lgl mutant larval brains.

Asymmetric cell division of Drosophila neural stem cells or neuroblasts is an important process which gives rise to two different daughter cells, one of which is the stem cell itself and the other, a committed or differentiated daughter cell. During neuroblast asymmetric division, atypical Protein Kinase C (aPKC) activity is tightly regulated; aberrant levels of activity could result in tumorigenesis in third instar larval brain. We identified clueless (clu), a genetic interactor of parkin (park), as a novel regulator of aPKC activity. It preferentially binds to the aPKC/Bazooka/Partition Defective 6 complex and stabilizes aPKC levels. In clu mutants, Miranda (Mira) and Numb are mislocalized in small percentages of dividing neuroblasts. Adult mutants are short-lived with severe locomotion defects. Clu promotes tumorigenesis caused by loss of function of lethal(2) giant larvae (lgl) in the larval brain. Removal of clu in lgl mutants rescues Mira and Numb mislocalization and restores the enlarged brain size. Western blot analyses indicate that the rescue is due to the down-regulation of aPKC levels in the lgl clu double mutant. Interestingly, the phenotype of the park mutant, which causes Parkinson's Disease-like symptoms in adult flies, is reminiscent of that of clu in neuroblast asymmetric division. Our study provides the first clue for the potential missing pathological link between temporally separated neurogenesis and neurodegeneration events; the minor defects during early neurogenesis could be a susceptible factor contributing to neurodegenerative diseases at later stages of life.

RNAi reveals the key role of Nervana 1 in cockroach oogenesis and embryo development.

Na(+), K(+)-ATPases is a heterodimer protein consisting of α- and ß-subunits that control the ion transport through cell membranes. In insects the ß-subunit of the Na(+), K(+)-ATPase, known as Nervana, was characterized as a nervous system-specific glycoprotein antigen from adult Drosophila melanogaster heads. Nervana is expressed ubiquitously in all insect tissues, and in epithelial cells appeared located in a basolateral position as part of the septate junctions. Herein we study two Nervana isoforms from Blattella germanica, a cockroach species with panoistic ovaries. The sequencing and the phylogenetic analysis results suggest that these two isoforms are orthologs of D. melanogaster Nervana 1 and Nervana 2, respectively. Nervana 1 is highly expressed in the ovary of B. germanica, and depleting its expression results in changes in oocyte shape that do not impair oviposition. However, the resulting embryos show different defects and never hatch. These findings highlight the importance of this type of membrane pump in insect oogenesis as well as in embryo development, and its possible regulation by juvenile hormone.

Interaction of Notch signaling modulator Numb with α-Adaptin regulates endocytosis of Notch pathway components and cell fate determination of neural stem cells.

The ability to balance self-renewal and differentiation is a hallmark of stem cells. In Drosophila neural stem cells (NSCs), Numb/Notch (N) signaling plays a key role in this process. However, the molecular and cellular mechanisms underlying Numb function in a stem cell setting remain poorly defined. Here we show that α-Adaptin (α-Ada), a subunit of the endocytic AP-2 complex, interacts with Numb through a new mode of interaction to regulate NSC homeostasis. In α-ada mutants, N pathway component Sanpodo and the N receptor itself exhibited altered trafficking, and N signaling was up-regulated in the intermediate progenitors of type II NSC lineages, leading to their transformation into ectopic NSCs. Surprisingly, although the Ear domain of α-Ada interacts with the C terminus of Numb and is important for α-Ada function in the sensory organ precursor lineage, it was dispensable in the NSCs. Instead, α-Ada could regulate Sanpodo, N trafficking, and NSC homeostasis by interacting with Numb through new domains in both proteins previously not known to mediate their interaction. This interaction could be bypassed when α-Ada was directly fused to the phospho-tyrosine binding domain of Numb. Our results identify a critical role for the AP-2-mediated endocytosis in regulating NSC behavior and reveal a new mechanism by which Numb regulates NSC behavior through N. These findings are likely to have important implications for cancer biology.

Functional genomics of life history variation in a butterfly metapopulation.

In fragmented landscapes, small populations frequently go extinct and new ones are established with poorly understood consequences for genetic diversity and evolution of life history traits. Here, we apply functional genomic tools to an ecological model system, the well-studied metapopulation of the Glanville fritillary butterfly. We investigate how dispersal and colonization select upon existing genetic variation affecting life history traits by comparing common-garden reared 2-day adult females from new populations with those from established older populations. New-population females had higher expression of abdomen genes involved in egg provisioning and thorax genes involved in the maintenance of flight muscle proteins. Physiological studies confirmed that new-population butterflies have accelerated egg maturation, apparently regulated by higher juvenile hormone titer and angiotensin converting enzyme mRNA, as well as enhanced flight metabolism. Gene expression varied between allelic forms of two metabolic genes (Pgi and Sdhd), which themselves were associated with differences in flight metabolic rate, population age and population growth rate. These results identify likely molecular mechanisms underpinning life history variation that is maintained by extinction-colonization dynamics in metapopulations.