DNA methylation differences during development distinguish sympatric morphs of Arctic charr (Salvelinus alpinus).

Changes in DNA methylation in specific coding or non-coding regions can influence development and potentially divergence in traits within species and groups. While the impact of epigenetic variation on developmental pathways associated with evolutionary divergence is the focus of intense investigation, few studies have looked at recently diverged systems. Phenotypic diversity between closely related populations of Arctic charr (Salvelinus alpinus), which diverged within the last 10,000 years, offers an interesting ecological model to address such effects. Using bisulphite sequencing, we studied general DNA methylation patterns during development in the four sympatric morphs of Arctic charr from Lake Thingvallavatn. The data revealed strong differences between developmental timepoints and between morphs (mainly along the benthic-limnetic axis), both at single CpG sites and in 1000 bp-regions. Genes located close to differentially methylated CpG sites were involved in nucleosome assembly, regulation of osteoclast differentiation, and cell-matrix adhesion. Differentially methylated regions were enriched in tRNA and rRNA sequences, and half of them were located close to transcription start sites. The expression of 14 genes showing methylation differences over time or between morphs was further investigated by qPCR and nine of these were found to be differentially expressed between morphs. Four genes (ARHGEF37-like, H3-like, MPP3 and MEGF9) showed a correlation between methylation and expression. Lastly, histone gene clusters displayed interesting methylation differences between timepoints and morphs, as well as intragenic methylation variation. The results presented here provide a motivation for further studies on the contribution of epigenetic traits, such as DNA methylation, to phenotypic diversity and developmental mechanisms.

Differential gene expression during early development in recently evolved and sympatric Arctic charr morphs.

Phenotypic differences between closely related taxa or populations can arise through genetic variation or be environmentally induced, leading to altered transcription of genes during development. Comparative developmental studies of closely related species or variable populations within species can help to elucidate the molecular mechanisms related to evolutionary divergence and speciation. Studies of Arctic charr (Salvelinus alpinus) and related salmonids have revealed considerable phenotypic variation among populations and in Arctic charr many cases of extensive variation within lakes (resource polymorphism) have been recorded. One example is the four Arctic charr morphs in the ∼10,000 year old Lake Thingvallavatn, which differ in numerous morphological and life history traits. We set out to investigate the molecular and developmental roots of this polymorphism by studying gene expression in embryos of three of the morphs reared in a common garden set-up. We performed RNA-sequencing, de-novo transcriptome assembly and compared gene expression among morphs during an important timeframe in early development, i.e., preceding the formation of key trophic structures. Expectedly, developmental time was the predominant explanatory variable. As the data were affected by some form of RNA-degradation even though all samples passed quality control testing, an estimate of 3'-bias was the second most common explanatory variable. Importantly, morph, both as an independent variable and as interaction with developmental time, affected the expression of numerous transcripts. Transcripts with morph effect, separated the three morphs at the expression level, with the two benthic morphs being more similar. However, Gene Ontology analyses did not reveal clear functional enrichment of transcripts between groups. Verification via qPCR confirmed differential expression of several genes between the morphs, including regulatory genes such as AT-Rich Interaction Domain 4A (arid4a) and translin (tsn). The data are consistent with a scenario where genetic divergence has contributed to differential expression of multiple genes and systems during early development of these sympatric Arctic charr morphs.

Differential expression of the aryl hydrocarbon receptor pathway associates with craniofacial polymorphism in sympatric Arctic charr.

BACKGROUND: The developmental basis of craniofacial morphology hinges on interactions of numerous signalling systems. Extensive craniofacial variation in the polymorphic Arctic charr, a member of the salmonid family, from Lake Thingvallavatn (Iceland), offers opportunities to find and study such signalling pathways and their key regulators, thereby shedding light on the developmental pathways, and the genetics of trophic divergence. RESULTS: To identify genes involved in the craniofacial differences between benthic and limnetic Arctic charr, we used transcriptome data from different morphs, spanning early development, together with data on craniofacial expression patterns and skeletogenesis in model vertebrate species. Out of 20 genes identified, 7 showed lower gene expression in benthic than in limnetic charr morphs. We had previously identified a conserved gene network involved in extracellular matrix (ECM) organization and skeletogenesis, showing higher expression in developing craniofacial elements of benthic than in limnetic Arctic charr morphs. The present study adds a second set of genes constituting an expanded gene network with strong, benthic-limnetic differential expression. To identify putative upstream regulators, we performed knowledge-based motif enrichment analyses on the regulatory sequences of the identified genes which yielded potential binding sites for a set of known transcription factors (TFs). Of the 8 TFs that we examined using qPCR, two (Ahr2b and Ap2) were found to be differentially expressed between benthic and limnetic charr. Expression analysis of several known AhR targets indicated higher activity of the AhR pathway during craniofacial development in benthic charr morphotypes. CONCLUSION: These results suggest a key role of the aryl hydrocarbon receptor (AhR) pathway in the observed craniofacial differences between distinct charr morphotypes.

Bones in motion: Ontogeny of craniofacial development in sympatric arctic charr morphs.

BACKGROUND: The impressive diversity in the feeding apparatus often seen among related fish species clearly reflects differences in feeding modes and habitat utilization. Such variation can also be found within species. One example of such intraspecific diversity is the Arctic charr in Lake Thingvallavatn, where four distinct morphs coexist: two limnetic, with evenly protruding jaws, and two benthic, with subterminal lower jaws. We used these recently evolved morphs to study the role of ontogenetic variation in shaping craniofacial diversity. RESULTS: The segmental development of the pharyngeal arches and the order of events in craniofacial development is the same as has been described for teleosts, emphasizing the conserved nature of this process. However, our morphometric analyses reveal differences between morphs. Hatching is accompanied by increase in size and allometric shape changes in Arctic charr. Ontogenetic trajectories of craniofacial shape also differ significantly between morphs. CONCLUSIONS: The results point to hatching as a significant developmental event in Arctic charr and possibly other fishes. Also, the developmental origins of limnetic and benthic specializations in the craniofacial elements of Arctic charr may stem from events around hatching. This calls for investigations of the mechanisms and consequences of hatching, in the context of development and evolution. Developmental Dynamics 244:1168-1178, 2015. © 2015 Wiley Periodicals, Inc.

MicroRNA-200c-141 and ∆Np63 are required for breast epithelial differentiation and branching morphogenesis.

The epithelial compartment of the breast contains two lineages, the luminal- and the myoepithelial cells. D492 is a breast epithelial cell line with stem cell properties that forms branching epithelial structures in 3D culture with both luminal- and myoepithelial differentiation. We have recently shown that D492 undergo epithelial to mesenchymal transition (EMT) when co-cultured with endothelial cells. This 3D co-culture model allows critical analysis of breast epithelial lineage development and EMT. In this study, we compared the microRNA (miR) expression profiles for D492 and its mesenchymal-derivative D492M. Suppression of the miR-200 family in D492M was among the most profound changes observed. Exogenous expression of miR-200c-141 in D492M reversed the EMT phenotype resulting in gain of luminal but not myoepithelial differentiation. In contrast, forced expression of ∆Np63 in D492M restored the myoepithelial phenotype only. Co-expression of miR-200c-141 and ∆Np63 in D492M restored the branching morphogenesis in 3D culture underlining the requirement for both luminal and myoepithelial elements for obtaining full branching morphogenesis in breast epithelium. Introduction of a miR-200c-141 construct in both D492 and D492M resulted in resistance to endothelial induced EMT. In conclusion, our data suggests that expression of miR-200c-141 and ∆Np63 in D492M can reverse EMT resulting in luminal- and myoepithelial differentiation, respectively, demonstrating the importance of these molecules in epithelial integrity in the human breast.

Transcriptional dynamics of a conserved gene expression network associated with craniofacial divergence in Arctic charr.

BACKGROUND: Understanding the molecular basis of craniofacial variation can provide insights into key developmental mechanisms of adaptive changes and their role in trophic divergence and speciation. Arctic charr (Salvelinus alpinus) is a polymorphic fish species, and, in Lake Thingvallavatn in Iceland, four sympatric morphs have evolved distinct craniofacial structures. We conducted a gene expression study on candidates from a conserved gene coexpression network, focusing on the development of craniofacial elements in embryos of two contrasting Arctic charr morphotypes (benthic and limnetic). RESULTS: Four Arctic charr morphs were studied: one limnetic and two benthic morphs from Lake Thingvallavatn and a limnetic reference aquaculture morph. The presence of morphological differences at developmental stages before the onset of feeding was verified by morphometric analysis. Following up on our previous findings that Mmp2 and Sparc were differentially expressed between morphotypes, we identified a network of genes with conserved coexpression across diverse vertebrate species. A comparative expression study of candidates from this network in developing heads of the four Arctic charr morphs verified the coexpression relationship of these genes and revealed distinct transcriptional dynamics strongly correlated with contrasting craniofacial morphologies (benthic versus limnetic). A literature review and Gene Ontology analysis indicated that a significant proportion of the network genes play a role in extracellular matrix organization and skeletogenesis, and motif enrichment analysis of conserved noncoding regions of network candidates predicted a handful of transcription factors, including Ap1 and Ets2, as potential regulators of the gene network. The expression of Ets2 itself was also found to associate with network gene expression. Genes linked to glucocorticoid signalling were also studied, as both Mmp2 and Sparc are responsive to this pathway. Among those, several transcriptional targets and upstream regulators showed differential expression between the contrasting morphotypes. Interestingly, although selected network genes showed overlapping expression patterns in situ and no morph differences, Timp2 expression patterns differed between morphs. CONCLUSION: Our comparative study of transcriptional dynamics in divergent craniofacial morphologies of Arctic charr revealed a conserved network of coexpressed genes sharing functional roles in structural morphogenesis. We also implicate transcriptional regulators of the network as targets for future functional studies.

Patterns of miRNA expression in Arctic charr development.

Micro-RNAs (miRNAs) are now recognized as a major class of developmental regulators. Sequences of many miRNAs are highly conserved, yet they often exhibit temporal and spatial heterogeneity in expression among species and have been proposed as an important reservoir for adaptive evolution and divergence. With this in mind we studied miRNA expression during embryonic development of offspring from two contrasting morphs of the highly polymorphic salmonid Arctic charr (Salvelinus alpinus), a small benthic morph from Lake Thingvallavatn (SB) and an aquaculture stock (AC). These morphs differ extensively in morphology and adult body size. We established offspring groups of the two morphs and sampled at several time points during development. Four time points (3 embryonic and one just before first feeding) were selected for high-throughput small-RNA sequencing. We identified a total of 326 conserved and 427 novel miRNA candidates in Arctic charr, of which 51 conserved and 6 novel miRNA candidates were differentially expressed among developmental stages. Furthermore, 53 known and 19 novel miRNAs showed significantly different levels of expression in the two contrasting morphs. Hierarchical clustering of the 53 conserved miRNAs revealed that the expression differences are confined to the embryonic stages, where miRNAs such as sal-miR-130, 30, 451, 133, 26 and 199a were highly expressed in AC, whereas sal-miR-146, 183, 206 and 196a were highly expressed in SB embryos. The majority of these miRNAs have previously been found to be involved in key developmental processes in other species such as development of brain and sensory epithelia, skeletogenesis and myogenesis. Four of the novel miRNA candidates were only detected in either AC or SB. miRNA candidates identified in this study will be combined with available mRNA expression data to identify potential targets and involvement in developmental regulation.

Expression and functional role of sprouty-2 in breast morphogenesis.

Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.

Transcriptional regulation of peripheral glial cell differentiation in the embryonic nervous system of Drosophila.

The Drosophila nervous system is ideally suited to study glial cell development and function, because it harbors only relatively few glial cells, and nervous system development is very well conserved during evolution. In the past, enhancer trap studies provided tools allowing to study glial cells with a single-cell resolution and, moreover, disclosed a surprising molecular heterogeneity among the different glial cells. The peripheral nervous system in the embryo comprises only 12 glial cells in one hemisegment and thus offers a unique opportunity to decipher the mechanisms directing glial development. Here, we focus on transcriptional regulators that have been reported to function during gliogenesis. To uncover additional regulators, we have conducted a genetic screen and report the identification of two additional transcriptional regulators involved in the control of peripheral glial migration: nejire and tango.

Construction and characterization of an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein.

The construction of a molecular clone of maedi-visna virus (MVV) expressing the enhanced green fluorescent protein (EGFP) is described. The egfp gene was inserted into the gene for dUTPase since it has been shown that dUTPase is dispensable for MVV replication both in vitro and in vivo. MVV-egfp is infectious and EGFP expression is stable over at least six passages. This fluorescent virus will be a useful tool for monitoring MVV infections.

The eye imaginal disc as a model to study the coordination of neuronal and glial development.

A complex nervous system comprises two distinct cell types, neurons and glial cells, whose development, differentiation and function is mutually interdependent. Many studies contributed to uncovering the basic mechanisms determining neuronal and glial fate and we are progressing enormously towards an understanding of how neurons interconnect to form intricate neuronal networks. However, the mechanisms used to couple neuronal and glial development remain largely obscure. Here we advocate the usefulness of the developing Drosophila compound eye as a new model to study the complex relationship between glial and neuronal cells.

Switch in FGF signalling initiates glial differentiation in the Drosophila eye.

The formation of a complex nervous system requires the intricate interaction of neurons and glial cells. Glial cells generally migrate over long distances before they initiate their differentiation, which leads to wrapping and insulation of axonal processes. The molecular pathways coordinating the switch from glial migration to glial differentiation are largely unknown. Here we demonstrate that, within the Drosophila eye imaginal disc, fibroblast growth factor (FGF) signalling coordinates glial proliferation, migration and subsequent axonal wrapping. Glial differentiation in the Drosophila eye disc requires a succession from glia-glia interaction to glia-neuron interaction. The neuronal component of the fly eye develops in the peripheral nervous system within the eye-antennal imaginal disc, whereas glial cells originate from a pool of central-nervous-system-derived progenitors and migrate onto the eye imaginal disc. Initially, glial-derived Pyramus, an FGF8-like ligand, modulates glial cell number and motility. A switch to neuronally expressed Thisbe, a second FGF8-like ligand, then induces glial differentiation. This switch is accompanied by an alteration in the intracellular signalling pathway through which the FGF receptor channels information into the cell. Our findings reveal how a switch from glia-glia interactions to glia-neuron interactions can trigger formation of glial membrane around axonal trajectories. These results disclose an evolutionarily conserved control mechanism of axonal wrapping, indicating that Drosophila might serve as a model to understand glial disorders in humans.

Simultaneous mutations in CA and Vif of Maedi-Visna virus cause attenuated replication in macrophages and reduced infectivity in vivo.

Maedi-visna virus (MVV) is a lentivirus of sheep sharing several key features with the primate lentiviruses. The virus causes slowly progressive diseases, mainly in the lungs and the central nervous system of sheep. Here, we investigate the molecular basis for the differential growth phenotypes of two MVV isolates. One of the isolates, KV1772, replicates well in a number of cell lines and is highly pathogenic in sheep. The second isolate, KS1, no longer grows on macrophages or causes disease. The two virus isolates differ by 129 nucleotide substitutions and two deletions of 3 and 15 nucleotides in the env gene. To determine the molecular nature of the lesions responsible for the restrictive growth phenotype, chimeric viruses were constructed and used to map the phenotype. An L120R mutation in the CA domain, together with a P205S mutation in Vif (but neither alone), could fully convert KV1772 to the restrictive growth phenotype. These results suggest a functional interaction between CA and Vif in MVV replication, a property that may relate to the innate antiretroviral defense mechanisms in sheep.