Neurogenetic and genomic approaches reveal roles for Dpr/DIP cell adhesion molecules in Drosophila reproductive behavior

Drosophila reproductive behaviors are directed by fruitless neurons (fru P1 isoforms). A reanalysis of genomic studies shows that genes encoding dpr and DIP Immunoglobulin superfamily (IgSF) members are expressed in fru P1 neurons. Each fru P1and dpr/DIP (fru P1 ∩ dpr/DIP) overlapping expression pattern is similar in both sexes, with dimorphism in neuronal morphology and cell number. Behavioral studies of fru P1 ∩ dpr/DIP perturbation genotypes point to the mushroom body functioning together with the lateral protocerebral complex. Functionally, we find that perturbations of sex hierarchy genes and DIP-ε changes sex-specific morphology of fru P1 ∩ DIP-α neurons. A single-cell RNA-seq analysis shows that the DIPs have high expression in a restricted set of fru P1 neurons, whereas the dprs are expressed in larger set of neurons at intermediate levels, with a myriad of combinations

C. elegans EIF-3.K Promotes Programmed Cell Death through CED-3 Caspase

Programmed cell death (apoptosis) is essential for the development and homeostasis of metazoans. The central step in the execution of programmed cell death is the activation of caspases. In C. elegans, the core cell death regulators EGL-1(a BH3 domain-containing protein), CED-9 (Bcl-2), and CED-4 (Apaf-1) act in an inhibitory cascade to activate the CED-3 caspase. Here we have identified an additional component eif-3.K (eukaryotic translation initiation factor 3 subunit k) that acts upstream of ced-3 to promote programmed cell death. The loss of eif-3.K reduced cell deaths in both somatic and germ cells, whereas the overexpression of eif-3.K resulted in a slight but significant increase in cell death. Using a cell-specific promoter, we show that eif-3.K promotes cell death in a cell-autonomous manner. In addition, the loss of eif-3.K significantly suppressed cell death-induced through the overexpression of ced-4, but not ced-3, indicating a distinct requirement for eif-3.K in apoptosis. Reciprocally, a loss of ced-3 suppressed cell death induced by the overexpression of eif-3.K. These results indicate that eif-3.K requires ced-3 to promote programmed cell death and that eif-3.K acts upstream of ced-3 to promote this process. The EIF-3.K protein is ubiquitously expressed in embryos and larvae and localizes to the cytoplasm. A structure-function analysis revealed that the 61 amino acid long WH domain of EIF-3.K, potentially involved in protein-DNA/RNA interactions, is both necessary and sufficient for the cell death-promoting activity of EIF-3.K. Because human eIF3k was able to partially substitute for C. elegans eif-3.K in the promotion of cell death, this WH domain-dependent EIF-3.K-mediated cell death process has potentially been conserved throughout evolution

Genetic analysis of a potential engulfment gene ina-1 and a proapoptotic gene cM9 in Caenorhabditis elegans

細胞計畫性的死亡是多細胞生物發生過程中的正常程序用來消滅不需要的細胞；而細胞吞噬則是必須的程序用以清除因細胞自我死亡所產生的細胞屍體。今年來的研究證明這些訊息傳遞路徑在物種間是有保守性的。Integrins 是一個很大的家族由異分子組成的細胞表面受器，參與細胞黏合、細胞遷移與細胞訊息傳遞過程。ina-1是線蟲integrin 的α次分子，主要影響神經及DTC細胞的遷移。我們發現當ina-1 缺失時，會導致線蟲細胞屍體數目增多，且我們證明此一增多的情況不是因為integrin 缺失導致細胞有anoikis 的性狀，並且這些細胞屍體都是由正常的細胞死亡產生的。這些結果顯示ina-1突變所產生的細胞屍體增加可能為影響了細胞吞噬的過程。我們進一步的去尋找ina-1與其他已知參與在細胞吞噬作用的其他成員之間的關係，發現ina-1可能可以定義一個新的細胞吞噬訊息傳遞路徑。 M9是人類肌肉相關蛋白。我們發現當cM9（線蟲的M9同源基因）突變時，在胚胎發育過程會產生比較少的細胞屍體。並且cM9突變種在腹節神經細胞裡有多餘的細胞逃過細胞死亡的命運而存活下來。因此，我們認為cM9應該為促進細胞死亡的基因。除此之外，我們在觸覺神經中過量表現ced-3(caspase)的情況下過量表現cM9，發現比起單獨ced-3所引發的觸覺神經細胞死亡，在ced-3和cM9一起過量表現有更多的神經細胞死亡。此一結果暗示cM9引發的細胞死亡可能是caspase independent。During the development of multicellular organisms, programmed cell death (apoptosis) is a normally occurring process used to eliminate unnecessary cells. Engulfment is an essential process for clearance of cell corpse generated by apoptosis. Recent studies demonstrate that the molecular control of these processes is evolutionally conserved in metazoans. Integrins are a large family of heterodimeric cell surface receptors involved in cell adhesion, cell migration and signal transduction. INA-1 is C. elegans integrin α subunit that plays roles in neural development and DTC migration. We found that ina-1 mutations increased number of cell corpses in embryos and that this ina-1-mediated enhancement in cell-corpse number is through programmed cell death. This implies that ina-1 might be important in the engulfment process to remove cell corpses. Interestingly, ina-1 acts synergistically with previously identified engulfment genes, suggesting that ina-1 may act in parallel to these genes during programmed cell death and may define a new engulfment pathway in C. elegans. M9 is human muscle specific protein. We found that animals bear a mutation in cM9 (which is C. elegans homolog of M9) decreased numbers of cell corpses throughout embryogenesis. We further showed that cM9 mutant animals had extra surviving cells in the ventral cord in the engulfment-defective ced-2 mutant background. Together these data suggested that cM9 function as a positive mediator during apoptosis in C. elegans. We also showed that cM9 acts synergistically with ced-3 (which is a caspase) to promote cell-killing, suggesting that cM9 may mediate apoptosis through a ced-3 independent pathway.Table of contents Table of contents…………………………………………………………………1 Abstract…………………………………………………………………………… 5 中文摘要…………………………………………………………………………….6 Introduction………………………………………………………………………..7 Experimental Procedures………………………………………………………16 Strains and Genetics………………………………………………………….16 Molecular Constructs……………………...................................................16 Transgenic Animals……………………………………………………..........17 Heat Shock Experiments……………………………………………………..18 Bacteria-mediated RNAi………………………………………………….......19 Results……………………………………………………………………………...20 ina-1 is likely a novel engulfment gene…………………………………..20 The ina-1 mutation and ina-1(RNAi) significantly increased numbers of cell corpses throughout embryogenesis………………………………………….20 The extra cell-corpses observed in ina-1 mutants are generated by programmed cell death………………………………………………………..20 ina-1 might define a new engulfment pathway……………………………...23 cM9 is a novel pro-apoptotic gene in C. elegans………………………..24 cM9 mutation decreased cell-corpse numbers throughout embryogenesis…………………………………………………………...24 cM9 mutations promote extra cell survived in ventral cord neuron cells…25 Overexpression of cM9 in the whole embryos does not increase embryonic lethality…………………………………………………………………………..26 cM9 acts cell autonomously to kill touch receptor neurons and the killing effect is enhanced by ced-3 overexpression………………………………...26 Intermediate filament may have a minor role in cell-killing…………………27 Discussion………………………………………………………………………....28 ina-1 may function in the engulfment process………………………………28 Caspase-independent apoptosis – more than one way to kill a cell………28 References…………………………………………………………………………31 Figures and tables………………………………………………………………..37 Figure 1. The genetic pathway of apoptosis in C. elegans………………37 Figure 2. The ina-1 mutation and ina-1(RNAi) significantly increased numbers of cell corpses throughout embryogenesis……………………….38 Figure 3. Cell-corpse numbers in ina-1 mutants in the presence or absence of dcDAPK mutation…………………………………………………39 Figure 4. Cell-corpse numbers in ina-1 mutants in the presence or absence of p53 (cep-1) mutation……………………………………………..40 Figure 5. The extra cell corpses in ina-1(RNAi) are generated by central programmed cell death………………………………………………………..41 Figure 6. Cell-corpse numbers in ina-1 mutants in the presence or absence of ced-4(n2273) mutation…………………………………………...42 Figure 7. Cell-corpse numbers in ina-1 mutants in the presence or absence of dcM9(gk126) mutation…………………………………………...43 Figure 8. Cell-corpse numbers in ina-1 mutants in the presence or absence of psr-1(tm469) mutation……………………………………………44 Figure 9. Two parallel and partially redundant signaling pathways control the engulfment of apoptotic cells in C. elegans……………………………..45 Figure 10. The cm9 mutation decreased numbers of cell corpses throughout embryogenesis……………………………………………………46 Figure 11. Engulfment or apoptosis mutations lead to the survival of cells programmed to die in the ventral cord……………………………………….47 Figure 12. cM9 acts cell autonomously to kill touch receptor neurons and the killing effect is enhanced by ced-3 overexpression…………………….48 Table 1. ina-1(RNAi) enhances numbers of persistent cell corpses in ced-1, ced-2, and ced-5 mutant backgrounds………………………………49 Table 2. The dcM9(gk126) mutation results in the survival of cells that normally die in the ventral cord……………………………………………….50 Table 3. Overexpression of cm9 under the control of heat-shock promoters does not enhance embryonic lethality in wild type or in ced-3 overexpression backgrounds…………………………………………………51 Table 4. Intermediate filament a-2 and c-1 may have a minor role in cell-killing in a ced-2(n1994) background……………………………………52 Appendix………...…………………………………………………………………53 Appendix 1. The evolutionary tree of integrin α subunits and β subunits……………………………………………………………………..…..53 Appendix 2. The 11 intermediate filaments in C. elegans……………….54 Appendix 3. ina-1 does not involve in the ced-2, 5, 10, 12 engulfment pathway…………………………………………………………………………55 Appendix 4. ina-1 does not involve in the ced-2, 5, 10, 12 engulfment pathway…………………………………………………………………………56 Appendix 5. ina-1 does not involve in the ced-2, 5, 10, 12 engulfment pathway…………………………………………………………………………57 Appendix 6. ina-1 does not involve in the ced-1, 6, 7 engulfment pathway…………………………………………………………………………58 Appendix 7. ina-1 does not involve in the ced-1, 6, 7 engulfment pathway…………………………………………………………………………59 Appendix 8. ced-10 Mutations Reduced Numbers of Persistent Cell Corpses in Strong ced-2, ced-5, and ced-12 Mutants……………………..60 Appendix 9. ced-10 mutations reduced numbers of persistent cell corpses in strong ced-5 mutants throughout embryogenesis……………………….61 Appendix 10. A. Persistance of Pn.aap cell corpse in ced-5 and ced-10ced-5 mutants. B. The Pn.aap surviving rate in ced-10 double mutants is higher than ced-2 ced-5 mutants alone…………………………6

An Examination of Dynamic Gene Expression Changes in the Mouse Brain During Pregnancy and the Postpartum Period

The developmental transition to motherhood requires gene expression changes that alter the brain to drive the female to perform maternal behaviors. We broadly examined the global transcriptional response in the mouse maternal brain, by examining four brain regions: hypothalamus, hippocampus, neocortex, and cerebellum, in virgin females, two pregnancy time points, and three postpartum time points. We find that overall there are hundreds of differentially expressed genes, but each brain region and time point shows a unique molecular signature, with only 49 genes differentially expressed in all four regions. Interestingly, a set of “early-response genes” is repressed in all brain regions during pregnancy and postpartum stages. Several genes previously implicated in underlying postpartum depression change expression. This study serves as an atlas of gene expression changes in the maternal brain, with the results demonstrating that pregnancy, parturition, and postpartum maternal experience substantially impact diverse brain regions

The loss of <i>eif-3.K</i> results in reduced programmed cell deaths.

<p>(A) The inactivation of <i>eif-3.K</i> by RNAi or by genetic deletion reduced cell corpse numbers throughout embryogenesis. Cell corpses of the indicated genotype or RNAi treatment were scored at the comma, 1.5-fold, 2-fold, 3-fold and 4-fold embryonic stages. The <i>eif-3.K(gk126)</i> embryos were compared to the wild-type embryos, and the <i>eif-3.K(RNAi)</i> embryos were compared to control(RNAi) embryos at each stage. All comparisons were performed using the unpaired t test (*P<0.05, **P<0.001). Data are presented as the mean ± standard deviation. Error bars represent S.D. Greater than 20 embryos per stage were analyzed. (B) The loss of <i>eif-3.K</i> reduces TUNEL staining in <i>nuc-1</i> embryos. The number of cells exhibiting TUNEL staining was determined in embryos of the indicated genotypes at the 1.5-fold stage. The <i>eif-3.K(gk126)</i> embryos were compared to the wild-type embryos, and the <i>nuc-1(e1392)</i>single mutants were compared to the <i>eif-3.K(gk126); nuc-1(e1392)</i> double mutants. Comparisons were performed using the unpaired t test (*P<0.05, **P<0.001). Data are presented as the mean ± standard deviation. Error bars represent S.D. Greater than 20 embryos of each genotype were analyzed. n.s. indicates no significant difference. (C) A loss of <i>eif-3.K</i> reduces cell corpse number in the germline. Cell corpses in the germline of the <i>ced-1</i> single mutants (white columns) and <i>eif-3.K; ced-1</i> double mutants (black columns) were counted at the indicated times after entry into the adulthood. The y axis represents the average number of cell corpses scored in each gonadal arm. The <i>eif-3.K(gk126); ced-1(e1735)</i> double mutants were compared to the <i>ced-1(e1735)</i> single mutants at the same developmental stage using the unpaired t test (*P<0.05, **P<0.001). Data are presented as the mean ± standard deviation for >20 gonadal arms. Error bars represent S.D. (D) A loss of <i>eif-3.K</i> increases the number of extra surviving cells in weak <i>ced-3</i> mutants. Cells that failed to undergo programmed cell deaths in the anterior pharynx were scored in the indicated animals. All comparisons were performed using the unpaired t test (*P<0.05, **P<0.001). Data are presented as the mean ± standard deviation for >20 larvae. Error bars represent S.D. n.s. indicates no significant difference.</p

Overexpression of <i>eif-3.K</i> or <i>ced-3</i> in cell death-defective mutants.

a<p>Transgenic animals were subjected to heat-shock (+) or left at 20°C (−).</p>b<p>Transgenic embryos were scored for the number of cell corpses 1.5 hrs after heat shock (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036584#s2" target="_blank">Materials and methods</a>). Data are presented as the mean ± standard deviation from two independent stably transmitting lines.</p>c<p>Greater than 20 embryos were analyzed from each line except for 1.5-fold embryos carrying <i>P_hspced-3</i> (n≥8) after heat shock due to high lethality.</p><p>The transgenic embryos after heat shock were compared to the corresponding transgenic embryos without heat shock. All comparisons were performed using the unpaired t test (*P<0.05, **P<0.001).</p

The loss of <i>eif-3.K</i> partially suppresses cell death induced by the overexpression of <i>egl-1</i>and <i>ced-4,</i> but not <i>ced-3</i>.

<p>(A–D) The percentage of animals missing specific touch neurons are shown for the wild-type (white columns) or <i>eif-3.K(gk126)</i> (black columns) embryos carrying the <i>P_mec-7egl-1</i> (A), <i>P_mec-7ced-4</i> (B), <i>P_mec-7ced-3</i> (C), or <i>P_mec-7acCED-3</i> (D) transgenes. The <i>eif-3.K(gk126)</i> transgenic worms were compared to the analogous wild-type transgenic worms. Comparisons were performed using the unpaired t test (*P<0.05, **P<0.001). Data are presented as the mean ± standard deviation. Error bars represent S.D. n.s. indicates no significant difference. More than 100 animals were scored for each strain. (A)The percentage of animals missing specific touch neurons or missing at least one touch neuron are shown for wild-type control <i>P_mec-7gfp</i> transgenic animals (white columns) or <i>P_mec-7eif-3.K</i> transgenic animals (black columns). More than 100 animals were scored for each strain.</p

Structure and function analysis of <i>eif-3.K</i>.

<p>^aTransgenic animals were subjected to heat-shock (+) or left at 20°C (−).</p>b<p>Transgenic embryos were scored for the number of cell corpses 1.5 hrs after heat shock.</p><p>(see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036584#s2" target="_blank">Materials and methods</a>).</p><p>Data are presented as the mean ± standard deviation from two independent stably transmitting lines. Greater than 20 embryos were analyzed from each line.</p><p>For the <i>P_let-858</i> expressing transgene, <i>eif-3.K</i> mutant embryos carrying the transgene were compared to <i>eif-3.K</i> mutant without the transgene.</p><p>For the <i>P_hsp</i> expressing transgene, the transgenic embryos after heat shock were compared to the corresponding transgenic embryos without heat shock. All comparisons were performed using the unpaired t test (*P<0.05, **P<0.001).</p

The loss of <i>eif-3.K</i> enhances cell survival in the ventral cord of sensitized mutants.

<p>^aAverage numbers of fluorescent cells caused by expression from <i>P_lin-11gfp</i> in P2, 9, 10, 11, and 12-derived regions were determined using DIC microscopy equipped with an ultraviolet light source. Greater than 20 larvae of each genotype were analyzed.</p>b<p>The percentages of animals that had at least one fluorescent cell in the P2, 9, 10, 11, and 12-derived regions were determined.</p