Vengamanaidu Modepalli

Differential temporal expression of milk miRNA during the lactation cycle of the marsupial tammar wallaby ( Macropus eugenii )

BackgroundLactation is a key aspect of mammalian evolution for adaptation of various reproductive strategies along different mammalian lineages. Marsupials, such as tammar wallaby, adopted a short gestation and a relatively long lactation cycle, the newborn is immature at birth and significant development occurs postnatally during lactation. Continuous changes of tammar milk composition may contribute to development and immune protection of pouch young. Here, in order to address the putative contribution of newly identified secretory milk miRNA in these processes, high throughput sequencing of miRNAs collected from tammar milk at different time points of lactation was conducted. A comparative analysis was performed to find distribution of miRNA in milk and blood serum of lactating wallaby.ResultsResults showed that high levels of miRNA secreted in milk and allowed the identification of differentially expressed milk miRNAs during the lactation cycle as putative markers of mammary gland activity and functional candidate signals to assist growth and timed development of the young. Comparative analysis of miRNA distribution in milk and blood serum suggests that milk miRNAs are primarily expressed from mammary gland rather than transferred from maternal circulating blood, likely through a new putative exosomal secretory pathway. In contrast, highly expressed milk miRNAs could be detected at significantly higher levels in neonate blood serum in comparison to adult blood, suggesting milk miRNAs may be absorbed through the gut of the young.ConclusionThe function of miRNA in mammary gland development and secretory activity has been proposed, but results from the current study also support a differential role of milk miRNA in regulation of development in the pouch young, revealing a new potential molecular communication between mother and young during mammalian lactation.

Characterization of the piRNA pathway during development of the sea anemone Nematostella vectensis

ABSTRACT PIWI-interacting RNAs (piRNAs) and associated proteins comprise a conserved pathway for silencing transposons in metazoan germlines. piRNA pathway components are also expressed in multipotent somatic stem cells in various organisms. piRNA functions have been extensively explored in bilaterian model systems, however, comprehensive studies in non-bilaterian phyla remain limited. Here we investigate the piRNA pathway during the development of Nematostella vectensis, a well-established model system belonging to Cnidaria, the sister group to Bilateria. To date, no population of somatic stem cells has been identified in this organism, despite its long life-span and regenerative capacities that require a constant cell-renewal. We show that Nematostella piRNA pathway components are broadly expressed in early developmental stages, while piRNAs themselves show differential expression, suggesting specific developmental roles of distinct piRNA families. In adults, piRNA associated proteins are enriched in the germline but also expressed in somatic cells, indicating putative stem cell properties. Furthermore, we provide experimental evidence that Nematostella piRNAs cleave transposable elements as well as protein-coding genes. Our results demonstrate that somatic expression of piRNA associated proteins as well as the roles of piRNAs in transposon repression and gene regulation are likely ancestral features that evolved before the split between Cnidaria and Bilateria.

Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution

Our current knowledge about the mechanisms of miRNA silencing is restricted to few lineages such as vertebrates, arthropods, nematodes and land plants. miRNA-mediated silencing in bilaterian animals is dependent on the proteins of the GW182 family. Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria.

Marsupial tammar wallaby delivers milk bioactives to altricial pouch young to support lung development

Our research is exploiting the marsupial as a model to understand the signals required for lung development. Marsupials have a unique reproductive strategy, the mother gives birth to altricial neonate with an immature lung and the changes in milk composition during lactation in marsupials appears to provide bioactives that can regulate diverse aspects of lung development, including branching morphogenesis, cell proliferation and cell differentiation. These effects are seen with milk collected between 25 and 100 days postpartum. To better understand the temporal effects of milk composition on postnatal lung development we used a cross-fostering technique to restrict the tammar pouch young to milk composition not extending beyond day 25 for 45 days of its early postnatal life. These particular time points were selected as our previous study showed that milk protein collected prior to ~ day 25 had no developmental effect on mouse embryonic lungs in culture. The comparative analysis of the foster group and control young at day 45 postpartum demonstrated that foster pouch young had significantly reduced lung size. The lungs in fostered young were comprised of large intermediate tissue, had a reduced size of airway lumen and a higher percentage of parenchymal tissue. In addition, expression of marker genes for lung development (BMP4, WNT11, AQP-4, HOPX and SPB) were significantly reduced in lungs from fostered young. Further, to identify the potential bioactive expressed by mammary gland that may have developmental effect on pouch young lungs, we performed proteomics analysis on tammar milk through mass-spectrometry and listed the potential bioactives (PDGF, IGFBP5, IGFBPL1 and EGFL6) secreted in milk that may be involved in regulating pouch young lung development. The data suggest that postnatal lung development in the tammar young is most likely regulated by maternal signalling factors supplied through milk.

Dynamics of venom composition across a complex life cycle

Little is known about venom in young developmental stages of animals. The appearance of toxins and stinging cells during early embryonic stages in the sea anemone Nematostella vectensis suggests that venom is already expressed in eggs and larvae of this species. Here, we harness transcriptomic, biochemical and transgenic tools to study venom production dynamics in Nematostella. We find that venom composition and arsenal of toxin-producing cells change dramatically between developmental stages of this species. These findings can be explained by the vastly different interspecific interactions of each life stage, as individuals develop from a miniature non-feeding mobile planula to a larger sessile polyp that predates on other animals and interact differently with predators. Indeed, behavioral assays involving prey, predators and Nematostella are consistent with this hypothesis. Further, the results of this work suggest a much wider and dynamic venom landscape than initially appreciated in animals with a complex life cycle.

Evolution of miRNA Tailing by 3′ Terminal Uridylyl Transferases in Metazoa

Abstract In bilaterian animals the 3′ ends of microRNAs (miRNAs) are frequently modified by tailing and trimming. These modifications affect miRNA-mediated gene regulation by modulating miRNA stability. Here, we analyzed data from three nonbilaterian animals: two cnidarians (Nematostella vectensis and Hydra magnipapillata) and one poriferan (Amphimedon queenslandica). Our analysis revealed that nonbilaterian miRNAs frequently undergo modifications like the bilaterian counterparts: the majority are expressed as different length isoforms and frequent modifications of the 3′ end by mono U or mono A tailing are observed. Moreover, as the factors regulating miRNA modifications are largely uncharacterized in nonbilaterian animal phyla, in present study, we investigated the evolution of 3′ terminal uridylyl transferases (TUTases) that are known to involved in miRNA 3′ nontemplated modifications in Bilateria. Phylogenetic analysis on TUTases showed that TUTase1 and TUTase6 are a result of duplication in bilaterians and that TUTase7 and TUTase4 are the result of a vertebrate-specific duplication. We also find an unexpected number of Drosophila-specific gene duplications and domain losses in most of the investigated gene families. Overall, our findings shed new light on the evolutionary history of TUTases in Metazoa, as they reveal that this core set of enzymes already existed in the last common ancestor of all animals and was probably involved in modifying small RNAs in a similar fashion to its present activity in bilaterians.

The methyltransferase HEN1 is required in Nematostella vectensis for microRNA and piRNA stability as well as larval metamorphosis

Small non-coding RNAs (sRNAs) such as microRNAs (miRNAs), small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs) regulate the levels of endogenous, viral and transposable element RNA in plants (excluding piRNAs) and animals. These pathways are explored mainly in bilaterian animals, such as vertebrates, arthropods and nematodes, where siRNAs and piRNAs, but not miRNAs bind their targets with a perfect match and mediate the cleavage of the target RNA. Methylation of the 3′ ends of piRNAs and siRNAs by the methyltransferase HEN1 protects these sRNAs from degradation. There is a noticeable selection in bilaterian animals against miRNA-mRNA perfect matching, as it leads to the degradation of miRNAs. Cnidarians (sea anemones, corals, hydroids and jellyfish), are separated from bilaterians by more than 600 million years. As opposed to bilaterians, cnidarian miRNAs frequently bind their targets with a nearly perfect match. Knowing that an ortholog of HEN1 is widely expressed in the sea anemone Nematostella vectensis, we tested in this work whether it mediates the stabilization of its sRNAs. We show that the knockdown of HEN1 in Nematostella results in a developmental arrest. Small RNA sequencing revealed that the levels of both miRNAs and piRNAs drop dramatically in the morphant animals. Moreover, knockdown experiments of Nematostella Dicer1 and PIWI2, homologs of major bilaterian biogenesis components of miRNAs and piRNAs, respectively, resulted in developmental arrest similar to HEN1 morphants. Our findings suggest that HEN1 mediated methylation of sRNAs reflects the ancestral state, where miRNAs were also methylated. Thus, we provide the first evidence of a methylation mechanism that stabilizes miRNAs in animals, and highlight the importance of post-transcriptional regulation in non-bilaterian animals.

Gene expression profiling of postnatal lung development in the marsupial gray short-tailed opossum (Monodelphis domestica) highlights conserved developmental pathways and specific characteristics during lung organogenesis

BackgroundAfter a short gestation, marsupials give birth to immature neonates with lungs that are not fully developed and in early life the neonate partially relies on gas exchange through the skin. Therefore, significant lung development occurs after birth in marsupials in contrast to eutherian mammals such as humans and mice where lung development occurs predominantly in the embryo. To explore the mechanisms of marsupial lung development in comparison to eutherians, morphological and gene expression analysis were conducted in the gray short-tailed opossum (Monodelphis domestica).ResultsPostnatal lung development of Monodelphis involves three key stages of development: (i) transition from late canalicular to early saccular stages, (ii) saccular and (iii) alveolar stages, similar to developmental stages overlapping the embryonic and perinatal period in eutherians. Differentially expressed genes were identified and correlated with developmental stages. Functional categories included growth factors, extracellular matrix protein (ECMs), transcriptional factors and signalling pathways related to branching morphogenesis, alveologenesis and vascularisation. Comparison with published data on mice highlighted the conserved importance of extracellular matrix remodelling and signalling pathways such as Wnt, Notch, IGF, TGFβ, retinoic acid and angiopoietin. The comparison also revealed changes in the mammalian gene expression program associated with the initiation of alveologenesis and birth, pointing to subtle differences between the non-functional embryonic lung of the eutherian mouse and the partially functional developing lung of the marsupial Monodelphis neonates. The data also highlighted a subset of contractile proteins specifically expressed in Monodelphis during and after alveologenesis.ConclusionThe results provide insights into marsupial lung development and support the potential of the marsupial model of postnatal development towards better understanding of the evolution of the mammalian bronchioalveolar lung.