More than royal food - <Emphasis Type="Italic">Major royal jelly protein</Emphasis> genes in sexuals and workers of the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Background

In the honeybee Apis mellifera, female larvae destined to become a queen are fed with royal jelly, a secretion of the hypopharyngeal glands of young nurse bees that rear the brood. The protein moiety of royal jelly comprises mostly major royal jelly proteins (MRJPs) of which the coding genes (mrjp1-9) have been identified on chromosome 11 in the honeybee’s genome.

Results

We determined the expression of mrjp1-9 among the honeybee worker caste (nurses, foragers) and the sexuals (queens (unmated, mated) and drones) in various body parts (head, thorax, abdomen). Specific mrjp expression was not only found in brood rearing nurse bees, but also in foragers and the sexuals.

Conclusions

The expression of mrjp1 to 7 is characteristic for the heads of worker bees, with an elevated expression of mrjp1-4 and 7 in nurse bees compared to foragers. Mrjp5 and 6 were higher in foragers compared to nurses suggesting functions in addition to those of brood food proteins. Furthermore, the expression of mrjp9 was high in the heads, thoraces and abdomen of almost all female bees, suggesting a function irrespective of body section. This completely different expression profile suggests mrjp9 to code for the most ancestral major royal jelly protein of the honeybee.

     

An integrated approach of bioinformatic prediction and in vitro analysis identified that miR-34a targets <Emphasis Type="Italic">MET</Emphasis> and <Emphasis Type="Italic">AXL</Emphasis> in triple-negative breast cancer

Background

Breast cancer is the most prevalent cancer among women, and AXL and MET are the key genes in the PI3K/AKT/mTOR pathway as critical elements in proliferation and invasion of cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs regulating the expression of genes.

Methods

Bioinformatic approaches were used to find a miRNA that simultaneously targets both AXL and MET 3′-UTRs. The expression of target miRNA was evaluated in triple-negative (MDA-MB-231) and HER2-overexpressing (SK-BR-3) breast cancer cell lines as well as normal breast cells, MCF-10A, using quantitative real-time PCR. Then, the miRNA was overexpressed in normal and cancer cell lines using a lentiviral vector system. Afterwards, effects of overexpressed miRNA on the expression of AXL and MET genes were evaluated using quantitative real-time PCR.

Results

By applying bioinformatic software and programs, miRNAs that target the 3′-UTR of both AXL and MET mRNAs were determined, and according to the scores, miR-34a was selected for further analyses. The expression level of miR-34a in MDA-MB-231 and SK-BR-3 was lower than that of MCF-10A. Furthermore, AXL and MET expression in SK-BR-3 and MDA-MB-231 was lower and higher, respectively, than that of MCF-10A. After miR-34a overexpression, MET and AXL were downregulated in MDA-MB-231. In addition, MET was downregulated in SK-BR-3, while AXL was upregulated in this cell line.

Conclusions

These findings may indicate that miR-34a is an oncogenic miRNA, downregulated in the distinct breast cancer subtypes. It also targets MET and AXL 3′-UTRs in triple-negative breast cancer. Therefore, it can be considered as a therapeutic target in this type of breast cancer.

     

Anti-apoptotic effects of IGF-I on mortality and dysmorphogenesis in <Emphasis Type="Italic">tbx5</Emphasis>-deficient zebrafish embryos

Background

Tbx5 deficiency in zebrafish causes several abnormal phenotypes of the heart and pectoral fins. It has been reported that exogenous human growth hormone can enhance expression of downstream mediators in the growth hormone and insulin-like growth factor I (IGF-I) pathway and partially restore dysmorphogenesis in tbx5 morphants. This study aimed to further evaluate the effects of IGF-I on cell apoptosis and dysmorphogenesis in zebrafish embryos deficient for tbx5.

Results

Among the five studied groups of zebrafish embryos (wild-type embryos [WT], tbx5 morphants [MO], mismatched tbx5 morpholino-treated wild-type embryos [MIS], IGF-I-treated wild-type embryos [WTIGF1], and IGF-I-treated tbx5 morphants [MOIGF1]), the expression levels of the ifg1, igf1-ra, ifg-rb, erk1, and akt2 genes as well as the ERK and AKT proteins were significantly reduced in the MO group, but were partially restored in the MOIGF1 group. These expression levels remained normal in the WT, MIS, and WTIGF1 groups. Exogenous human IGF-I also reduced the incidence of phenotypic anomalies, decreased the expression levels of apoptotic genes and proteins, suppressed cell apoptosis, and improved survival of the MOIGF1 group.

Conclusions

These results suggest that IGF-I has an anti-apoptotic protective effect in zebrafish embryos with tbx5 deficiency.

     

Identification of a novel <Emphasis Type="Italic">Drosophila</Emphasis> gene, <Emphasis Type="Italic">beltless</Emphasis>, using injectable embryonic and adult RNA interference (RNAi)

Background

RNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype.

Results

Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts.

Conclusions

We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system.

     

The <Emphasis Type="Italic">flipflop</Emphasis> orphan genes are required for limb bud eversion in the <Emphasis Type="Italic">Tribolium</Emphasis> embryo

Background

Unlike Drosophila but similar to other arthropod and vertebrate embryos, the flour beetle Tribolium castaneum develops everted limb buds during embryogenesis. However, the molecular processes directing the evagination of epithelia are only poorly understood.

Results

Here we show that the newly discovered genes Tc-flipflop1 and Tc-flipflop2 are involved in regulating the directional budding of appendages. RNAi-knockdown of Tc-flipflop results in a variety of phenotypic traits. Most prominently, embryonic limb buds frequently grow inwards rather than out, leading to the development of inverted appendages inside the larval body. Moreover, affected embryos display dorsal closure defects. The Tc-flipflop genes are evolutionarily non-conserved, and their molecular function is not evident. We further found that Tc-RhoGEF2, a highly-conserved gene known to be involved in actomyosin-dependent cell movement and cell shape changes, shows a Tc-flipflop-like RNAi-phenotype.

Conclusions

The similarity of the inverted appendage phenotype in both the flipflop- and the RhoGEF2 RNAi gene knockdown led us to conclude that the Tc-flipflop orphan genes act in a Rho-dependent pathway that is essential for the early morphogenesis of polarised epithelial movements. Our work describes one of the few examples of an orphan gene playing a crucial role in an important developmental process.