MEKK3 is required for endothelium function but is not essential for tumor growth and angiogenesis

Mitogen-activated protein kinase kinase kinase 3 (MEKK3) plays an essential role in embryonic angiogenesis, but its role in tumor growth and angiogenesis is unknown. In this study, we further investigated the role of MEKK3 in embryonic angiogenesis, tumor angiogenesis, and angiogenic factor production. We found that endothelial cells from Mekk3-deficient embryos showed defects in cell proliferation, apoptosis, and interactions with myocardium in the heart. We also found that MEKK3 is required for angiopoietin-1 (Ang1)-induced p38 and ERK5 activation. To study the role of MEKK3 in tumor growth and angiogenesis, we established both wild-type and Mekk3-deficient tumor-like embryonic stem cell lines and transplanted them subcutaneously into nude mice to assess their ability to grow and induce tumor angiogenesis. Mekk3-deficient tumors developed and grew similarly as control Mekk3 wild-type tumors and were also capable of inducing tumor angiogenesis. In addition, we found no differences in the production of VEGF in Mekk3-deficient tumors or embryos. Taken together, our results suggest that MEKK3 plays a critical role in Ang1/Tie2 signaling to control endothelial cell proliferation and survival and is required for endothelial cells to interact with the myocardium during early embryonic development. However, MEKK3 is not essential for tumor growth and angiogenesis. cardiovascular; mitogen-activated protein kinase; embryonic development     

Sox2 is required for maintenance and regeneration, but not initial development, of hair cells in the zebrafish inner ear

Sox2 has been variously implicated in maintenance of pluripotent stem cells or, alternatively, early stages of cell differentiation, depending on context. In the developing inner ear, Sox2 initially marks all cells in the nascent sensory epithelium and, in mouse, is required for sensory epithelium formation. Sox2 is eventually downregulated in hair cells but is maintained in support cells, the functional significance of which is unknown. Here we describe regulation and function of sox2 in the zebrafish inner ear. Expression of sox2 begins after the onset of sensory epithelium development and is regulated by Atoh1a/b, Fgf and Notch. Knockdown of sox2 does not prevent hair cell production, but the rate of accumulation is reduced due to sporadic death of differentiated hair cells. We next tested the capacity for hair cell regeneration following laser ablation of mature brn3c:gfp-labeled hair cells. In control embryos, regeneration of lost hair cells begins by 12 h post-ablation and involves transdifferentiation of support cells rather than asymmetric cell division. In contrast, regeneration does not occur in sox2-depleted embryos. These data show that zebrafish sox2 is required for hair cell survival, as well as for transdifferentiation of support cells into hair cells during regeneration.     

Myocellular triacylglycerol breakdown in females but not in males during exercise

The resting content and use of myocellular triacylglycerol (MCTG) during 90 min of submaximal exercise [60% of peak oxygen uptake (VO(2 peak))] were studied in 21 eumenorrheic female and 21 male subjects at different training levels [untrained (UT), moderately trained (MT), and endurance trained (END)]. Males and females were matched according to their VO(2 peak) expressed relative to lean body mass, physical activity level, and training history. All subjects ingested the same controlled diet for 8 days, and all females were tested in the midfollicular phase of the menstrual cycle. Resting MCTG, measured with the muscle biopsy technique, averaged 48.4 +/- 4.2, 48.5 +/- 8.4, and 52.2 +/- 5.8 mmol/kg dry wt in UT, MT, and END females, respectively, and 34.1 +/- 4.9, 31.6 +/- 3.3, and 38.4 +/- 3.0 mmol/kg dry wt in UT, MT, and END males, respectively (P < 0.001, females vs. males in all groups). Exercise decreased MCTG content in the female subjects by an average of 25%, regardless of training status, whereas in the male groups MCTG content was unaffected by exercise. The arterial plasma insulin concentration was higher (P < 0.05) and the arterial plasma epinephrine concentration was lower (P < 0.05) in the females than in the males at rest and during exercise. MCTG use was correlated to the resting concentration of MCTG (P < 0.001). We conclude that resting content and use of MCTG during exercise are related to gender and furthermore are independent of training status.     

Testicular type Sox9 is not involved in sex determination but might be in the development of testicular structures in the medaka, Oryzias latipes

Testicular type Sox9 is the most upstream conserved gene in the sex determining cascade among vertebrate. However, in medaka, only one Sox9 gene was identified as expressed in the ovary; no other Sox9 gene was reported expressed in the testis. We explored the medaka genome and cloned a novel testicular type Sox9 cDNA. Phylogenetic analysis revealed that both our isolated Sox9 and the already reportedly cloned medaka Sox9 belongs zebrafish Sox9a branch. Therefore, we named our gene Sox9a2. Unexpectedly, Sox9a2 mRNA was expressed in somatic cells surrounding germ cells at similar high levels in both sexes during early gonadal sex differentiation. However, at the initial stage of testicular tubules development, the expression of Sox9a2 was maintained only in XY gonads, and was remarkably reduced in XX gonads. These results suggest that Sox9a2 is not involved in early sex determination and differentiation, but is involved in the later development of testicular tubules in medaka.     

Phosphatidylinositol 3-kinase but not tuberin is required for PDGF-induced cell migration

The loss of function of the tumor suppressor gene TSC2 and its protein product tuberin promotes the development of benign lesions by stimulating cell growth, although the role of tuberin in regulating cell migration and metastasis has not been characterized. In addition, the role of phosphatidylinositol 3-kinase (PI 3-kinase), an important signaling event regulating cell migration, in modulating tuberin-deficient cell motility remains unknown. Using a tuberin-deficient rat smooth muscle cell line, ELT3, we demonstrate that platelet-derived growth factor (PDGF) stimulates cell migration by 3.2-fold, whereas vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-alpha, and basic fibroblast growth factor (bFGF) increase migration by 2.1-, 2.1-, and 2.6-fold, respectively. Basal and PDGF-induced migration in tuberin-deficient ELT3, ELT4, and ERC15 cells was not significantly different from that of tuberin-positive transformed rat kidney epithelial 2, airway smooth muscle, and pulmonary arterial vascular smooth muscle cells. Expression of tuberin in tuberin-deficient ELT3 cells also had little effect on cell migration. In parallel experiments, the role of PI 3-kinase activation in ELT3 cell migration was investigated. LY-294002, a PI 3-kinase inhibitor, decreased PDGF-induced migration in a concentration-dependent manner with an IC(50) of approximately 5 microM. LY-294002 also abrogated ELT3 cell migration stimulated by bFGF and TGF-alpha but not by VEGF and phorbol 12-myristate 13-acetate. Furthermore, transient expression of constitutively active PI 3-kinase (p110*) was sufficient to induce ELT3 cell migration. However, the migration induced by p110* was less than that induced by growth factors, suggesting other signaling pathways are also critically important in modulating growth factor-induced cell migration. These data suggest that PI 3-kinase is required for growth factor-induced cell migration and loss of tuberin appears to have little effect on cell migration.     

Autophagy is activated,but is not required for the G0 function of BCL-2 or BCL-xL

Cell cycle arrest in G0 and autophagy have features in common, but the inter-relationship between the two processes is not well defined. The anti-apoptosis molecules BCL-2 and BCL-xL promote G0 arrest through upregulation of p27 protein, which can also induce autophagy. We tested the hypothesis that autophagy was involved in the cell cycle arrest function of BCL-2 and BCL-xL. We found that in IL-3-dependent FL5.12 cells, NIH3T3 cells, and mouse embryo fibroblasts induced to arrest, treatment with 3-methyladenine did not affect the expected decrease in cell size and ribosomal RNA synthesis, or upregulation of p27 levels. Using the m5-7 ATG5-/- MEF cell line with doxycycline-regulated ATG5 expression, we demonstrated that autophagy was activated during serum withdrawal and contact inhibition, but inhibition of autophagy had no measurable effect on G0 arrest in parental or BCL-xL-expressing cells. Thus, our data indicate that, in cell culture models, autophagy occurs but is not required for entrance into quiescence or for the G0 function of BCL-2 or BCL-xL.     

Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein.

While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.     

Tribolium castaneum Transformer-2 regulates sex determination and development in both males and females

Tribolium castaneum Transformer (TcTra) is essential for female sex determination and maintenance through the regulation of sex-specific splicing of doublesex (dsx) pre-mRNA. In females, TcTra also regulates the sex-specific splicing of its own pre-mRNA to ensure continuous production of functional Tra protein. Transformer protein is absent in males and hence dsx pre-mRNA is spliced in a default mode. The mechanisms by which males inhibit the production of functional Tra protein are not known. Here, we report on functional characterization of transformer-2 (tra-2) gene (an ortholog of Drosophila transformer-2) in T. castaneum. RNA interference-mediated knockdown in the expression of gene coding for tra-2 in female pupae or adults resulted in the production of male-specific isoform of dsx and both female and male isoforms of tra suggesting that Tra-2 is essential for the female-specific splicing of tra and dsx pre-mRNAs. Interestingly, knockdown of tra-2 in males did not affect the splicing of dsx but resulted in the production of both female and male isoforms of tra suggesting that Tra-2 suppresses female-specific splicing of tra pre-mRNA in males. This dual regulation of sex-specific splicing of tra pre-mRNA ensures a tight regulation of sex determination and maintenance. These data suggest a critical role for Tra-2 in suppression of female sex determination cascade in males. In addition, RNAi studies showed that Tra-2 is also required for successful embryonic and larval development in both sexes.     

Inbreeding affects sexual signalling in males but not females of Tenebrio molitor

In many species of animals, individuals advertise their quality with sexual signals to obtain mates. Chemical signals such as volatile pheromones are species specific, and their primary purpose is to influence mate choice by carrying information about the phenotypic and genetic quality of the sender. The deleterious effects of consanguineous mating on individual quality are generally known, whereas the effect of inbreeding on sexual signalling is poorly understood. Here, we tested whether inbreeding reduces the attractiveness of sexual signalling in the mealworm beetle, Tenebrio molitor, by testing the preferences for odours of inbred and outbred (control) individuals of the opposite sex. Females were more attracted to the odours produced by outbred males than the odours produced by inbred males, suggesting that inbreeding reduces the attractiveness of male sexual signalling. However, we did not find any difference between the attractiveness of inbred and outbred female odours, which may indicate that the quality of females is either irrelevant for T. molitor males or quality is not revealed through female odours.     

Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function

Cell division, differentiation and morphogenesis are coordinated during embryonic development, and frequently are in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but that undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and use the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is driven substantially by cell migration. We find that the segmentation clock, which regulates somitogenesis, functions normally in the absence of cell cycle progression, and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1(-/-). The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle, somites appear to hyper-epithelialize, as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for the normal segmental arrangement of epithelial borders and internal mesenchymal cells.     

Securin is not required for cellular viability, but is required for normal growth of mouse embryonic fibroblasts

Sister chromatid separation depends on the release of cohesion by the activity of Esp1, a member of the caspase family [1, 2]. In budding yeast, Esp1p is kept inactive by its association with Pds1p, until the onset of anaphase, when Pds1p is ubiquitinated by the APC/Cdc20 complex [3--5] and subsequently degraded by the 26S proteasome. Pds1 is not an essential gene in budding yeast, but is required for cell cycle arrest prior to anaphase in response to the disruption of spindle structures [6, 7]. Thus, Pds1 mutant yeast cells display precocious sister chromatid separation in the presence of nocodazole [6]. Mammalian orthologs of yeast Esp1 and Pds1, separin and securin, have been identified [8], and, as anticipated, a nondegradable mutant form of securin inhibits sister separation when added to mitotic Xenopus egg extracts [8]. Securin was also independently identified as PTTG (pituitary tumor transforming gene), a gene overexpressed in pituitary tumors [9]. The relationship between its overexpression in tumors and its control of sister chromatid cohesion remains ill defined. To explore securin function in mammals, we took a targeted gene disruption approach in mice. Here, we report that securin is neither essential for cell viability nor required for spindle checkpoint function, and mice lacking securin are viable and apparently normal, but mouse embryonic fibroblasts lacking securin grow abnormally in culture.     

Xenopus TACC3/maskin is not required for microtubule stability but is required for anchoring microtubules at the centrosome

Members of the transforming acidic coiled coil (TACC) protein family are emerging as important mitotic spindle assembly proteins in a variety of organisms. The molecular details of how TACC proteins function are unknown, but TACC proteins have been proposed to recruit microtubule-stabilizing proteins of the tumor overexpressed gene (TOG) family to the centrosome and to facilitate their loading onto newly emerging microtubules. Using Xenopus egg extracts and in vitro assays, we show that the Xenopus TACC protein maskin is required for centrosome function beyond recruiting the Xenopus TOG protein XMAP215. The conserved C-terminal TACC domain of maskin is both necessary and sufficient to restore centrosome function in maskin-depleted extracts, and we provide evidence that the N terminus of maskin inhibits the function of the TACC domain. Time-lapse video microscopy reveals that microtubule dynamics in Xenopus egg extracts are unaffected by maskin depletion. Our results provide direct experimental evidence of a role for maskin in centrosome function and suggest that maskin is required for microtubule anchoring at the centrosome.     

Surface translocation of pactolus is induced by cell activation and death, but is not required for neutrophil migration and function

Pactolus is a cell surface protein expressed by murine neutrophils. Pactolus is similar to the beta integrins, except it lacks a functional metal ion-dependent adhesion site domain and is expressed without an alpha-chain partner. The majority of the Pactolus protein is held within the cell in dense granules in a highly glycosylated form. This intracellular form of Pactolus can be released to the cell surface following inflammatory activation or ligation of Pactolus on the cell surface. In addition, intracellular Pactolus translocates to the neutrophil surface following induction of apoptosis. Neutrophil activation studies suggest that Pactolus does not serve as an activating or phagocytic receptor for the neutrophil. To further define the function of Pactolus, a Pactolus-null mouse was generated. Pactolus-deficient animals mature appropriately and possess normal numbers of neutrophils, display appropriate migration into sites of inflammation, and combat introduced infections efficiently. These data suggest that Pactolus does not function as a neutrophil phagocytic or adhesion receptor, but may instead serve as a sugar-bearing ligand for lectin recognition by other cells.     

Early weaning in bighorn sheep, Ovis canadensis affects growth of males but not of females

Theories of parental investment and parent-offspring conflictassume that investment involves a cost to the parent and a benefitto the offspring, but for herbivorous mammals, behavioral andnutritional weaning are gradual processes that are difficultto define, and little is known about the consequences of individualvariation during weaning. To study the effects of late maternalcare on offspring fitness, we removed female bighorn sheep (Oviscanadensis) from a marked population in Alberta, Canada, andmonitored the survival, growth, and reproductive success oforphan and nonorphan lambs. Mothers were removed when lambswere 3.5–4.0 months, about 2–4 weeks before thesuspected time of nutritional weaning. Femaleorphans and nonorphanshad the same weight as yearlings, the same probability of producingtheir first lamb at 2 years of age, the same lifetime reproductivesuccess (lambs produced or lambs that survived to early autumn),and the same longevity. Male orphans from most cohorts weresmaller as yearlings compared to nonorphans from the same cohort.They were unable to compensate for this early weight differencein later life: at 4 years, orphan males had smaller horns andwere lighter than nonorphans. Small horn and body size likelylowered the reproductive success of orphaned males comparedto nonorphans from the same cohort. We suggest that in thissexually dimorphic species late maternal care is more importantfor males than for females. Because late maternal care had nomeasurable benefit for daughters, we suggest that parent-offspringconflict over the duration of maternal care may not exist formother-daughter pairs. or mother-son pairs it remains to beshown whether late maternal care involves a cost to the mother,but the assumption of a benefit to the son was met.     

Ena/VASP function in retinal axons is required for terminal arborization but not pathway navigation

The Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins is required for filopodia formation in growth cones and plays a crucial role in guidance cue-induced remodeling of the actin cytoskeleton. In vivo studies with pharmacological inhibitors of actin polymerization have previously provided evidence for the view that filopodia are needed for growth cone navigation in the developing visual pathway. Here we have re-examined this issue using an alternative strategy to generate growth cones without filopodia in vivo by artificially targeting Xena/XVASP (Xenopus homologs of Ena/VASP) proteins to mitochondria in retinal ganglion cells (RGCs). We used the specific binding of the EVH1 domain of the Ena/VASP family of proteins with the ligand motif FP4 to sequester the protein at the mitochondria surface. RGCs with reduced function of Xena/XVASP proteins extended fewer axons out of the eye and possessed dynamic lamellipodial growth cones missing filopodia that advanced slowly in the optic tract. Surprisingly, despite lacking filopodia, the axons navigated along the optic pathway without obvious guidance errors, indicating that the Xena/XVASP family of proteins and filopodial protrusions are non-essential for pathfinding in retinal axons. However, depletion of Xena/XVASP proteins severely impaired the ability of growth cones to form branches within the optic tectum, suggesting that this protein family, and probably filopodia, plays a key role in establishing terminal arborizations.