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.he project was funded by The Icelandic Center for Research (grant number: 100204011) to Sigurður S. Snorrason, Arnar Pálsson, Zophonías O. Jónsson and Bjarni K. Kristjánsson. The University of Iceland Doctoral Fund to Jóhannes Guðbrandsson and University of Iceland research fund to Arnar Pálsson, Sigurður S. Snorrason and Zophonías O. Jónsson. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer Reviewe

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.This project was supported by The Icelandic Centre for Research (RANNIS/IRF, Grant 100204), The University of Iceland Research Fund and the Eimskip University Fund.Peer Reviewe

Extensive genetic differentiation between recently evolved sympatric Arctic charr morphs

Publisher's version (útgefin grein)The availability of diverse ecological niches can promote adaptation of trophic specializations and related traits, as has been repeatedly observed in evolutionary radiations of freshwater fish. The role of genetics, environment, and history in ecologically driven divergence and adaptation, can be studied on adaptive radiations or populations showing ecological polymorphism. Salmonids, especially the Salvelinus genus, are renowned for both phenotypic diversity and polymorphism. Arctic charr (Salvelinus alpinus) invaded Icelandic streams during the glacial retreat (about 10,000 years ago) and exhibits many instances of sympatric polymorphism. Particularly, well studied are the four morphs in Lake Þingvallavatn in Iceland. The small benthic (SB), large benthic (LB), planktivorous (PL), and piscivorous (PI) charr differ in many regards, including size, form, and life history traits. To investigate relatedness and genomic differentiation between morphs, we identified variable sites from RNA‐sequencing data from three of those morphs and verified 22 variants in population samples. The data reveal genetic differences between the morphs, with the two benthic morphs being more similar and the PL‐charr more genetically different. The markers with high differentiation map to all linkage groups, suggesting ancient and pervasive genetic separation of these three morphs. Furthermore, GO analyses suggest differences in collagen metabolism, odontogenesis, and sensory systems between PL‐charr and the benthic morphs. Genotyping in population samples from all four morphs confirms the genetic separation and indicates that the PI‐charr are less genetically distinct than the other three morphs. The genetic separation of the other three morphs indicates certain degree of reproductive isolation. The extent of gene flow between the morphs and the nature of reproductive barriers between them remain to be elucidated.This project was supported by The Icelandic Center for Research (RANNIS #100204011) to SSS and coworkers, The University of Iceland Doctoral Fund to JG and University of Iceland research fund to AP, SSS and ZOJ.Peer reviewe

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.This project was supported by The Icelandic Centre for Research (RANNIS/IRF, grant 100204) and The University of Iceland Research Fund.Peer Reviewe

A man and woman crossing the road near Shillidays building, Mildura, Victoria, 1961 [picture] /

Title devised by cataloguer based on information from acquisition documentation.; Also available in an electronic version via the Internet at: http://nla.gov.au/nla.pic-vn3995269; Purchased from the photographer, 2007

The developmental transcriptome of contrasting Arctic charr (Salvelinus alpinus) morphs

Species and populations with parallel evolution of specific traits can help illuminate how predictable adaptations and divergence are at the molecular and developmental level. Following the last glacial period, dwarfism and specialized bottom feeding morphology evolved rapidly in several landlocked Arctic charr Salvelinus alpinus populations in Iceland. To study the genetic divergence between small benthic morphs and limnetic morphs, we conducted RNA-sequencing charr embryos at four stages in early development. We studied two stocks with contrasting morphologies: the small benthic (SB) charr from Lake Thingvallavatn and Holar aquaculture (AC) charr. The data reveal significant differences in expression of several biological pathways during charr development. There was also an expression difference between SB- and AC-charr in genes involved in energy metabolism and blood coagulation genes. We confirmed differing expression of five genes in whole embryos with qPCR, including lysozyme and natterin-like which was previously identified as a fish-toxin of a lectin family that may be a putative immunopeptide. We also verified differential expression of 7 genes in the developing head that associated consistently with benthic v.s.limnetic morphology (studied in 4 morphs). Comparison of single nucleotide polymorphism (SNP) frequencies reveals extensive genetic differentiation between the SB and AC-charr (~1300 with more than 50% frequency difference). Curiously, three derived alleles in the otherwise conserved 12s and 16s mitochondrial ribosomal RNA genes are found in benthic charr. The data implicate multiple genes and molecular pathways in divergence of small benthic charr and/or the response of aquaculture charr to domestication. Functional, genetic and population genetic studies on more freshwater and anadromous populations are needed to confirm the specific loci and mutations relating to specific ecological traits in Arctic charr.This project was supported by The Icelandic Center for Research (grant number: 100204011) to SSS, AP, ZOJ and BKK, The University of Iceland Research/Doctoral Fund to JG and KHK and University of Iceland research fund to AP, SSS and ZOJ.Peer ReviewedRitrýnt tímari

Expression of SPRY2 in virgin, pregnant and lactating mouse mammary gland.

<p><i>A) Expression of SPRY2 and pEGFR is inversely correlated with cell proliferation.</i> Low expression of SPRY2 is found within the virgin gland with few positive stromal cells (a). Note, increased stromal expression of SPRY2 in pregnant gland accompanied with expression in myoepithelial cell as evidenced by double staining of SPRY2 and the myoepithelial marker CK14 (b). Dramatic increase in SPRY2 expression is seen during lactation (a and b). SPRY2 and EGFR show similar expression pattern at all stages (c) with pEGFR expression seen at terminal buds in pregnant gland. Dramatic increase in pEGFR expression is seen in the lactating gland. Similar expression is found for SPRY2 and pEGFR in lactating gland. Proliferation is increased from virgin to pregnant gland but is reduced during lactation, with only few PCNA positive cells left. Cells counterstained with TOPRO-3, Bar = 100 µm. <i>B) SPRY2 expression is highest during lactation accompanied by activation of Erk/MAPK pathway.</i> Western blot demonstrated the expression differences of SPRY2 in virgin, pregnant and lactating glands. There is over 38 fold increase in SPRY2 expression during lactation compared to virgin state. Total ERK and pERK is also significantly increased during lactation. Actin was used as a loading control. <i>C) SPRY2 activity is peaking during pregnancy in the mouse mammary gland.</i> Using proximity ligation assay it was shown that phosphorylated SPRY2 was significantly more expressed during pregnancy compared to virgin and lactating gland. Bar = 25 µm.</p

Epithelial integrity is disturbed in SPRY2 KD cells when co-cultured with endothelial cells.

<p><i>A) Endothelial cells stimulate growth of D492 cells.</i> When plated in 3D rBM culture with breast endothelial cells (BRENCs), D492 cells can form complex branching colonies from as little as 100–1000 cells compared to 7×10³ –10⁴ in 3D monoculture. <i>B) D492-derived branching structures form bi-layered epithelium with BRENCs positioned extralobular.</i> The branching colonies are bi-layered and polarized structures as evidenced by the expression of the myoepithelial marker CK14 on the outer side and the luminal epithelial marker CK19 on the inner side (upper figure). In co-cultures endothelial cells stay as single cells positioned outside the branching structures as seen with CD31 staining (lower figure, arrows). Bar  =  100 µm. Sections counterstained with TOPRO-3 nuclear stain. <i>C) Phenotypes of D492 in co-culture with BRENCs.</i> In co-culture with endothelial cells D492 cells form both branching- and spindle-like colonies. The branching colonies show strong expression of E-cadherin, while spindle like colonies have undergone EMT as evidenced by cadherin switch from E- to N-cadherin. Staining for the proliferation marker ki67 shows that both the branching and spindle like colonies are viable and growing. Bar  =  100 µm. Sections counterstained with TOPRO-3 nuclear stain. <i>D) Spry2-KD cells show an increase in the spindle-like morphology.</i> While D492^NS cells form about 40% spindle-like colonies there is a significant increase in the D492^Spry2-KD3 cells up to 65%. The D492^Spry2-KD3A form almost exclusively spindle-like colonies in co-culture with endothelial cells.</p

SPRY2 Knockdown in D492 breast epithelial stem cell line.

<p><i>A) D492 cells show significant knockdown of SPRY2.</i> D492 were transfected with non-silencing (NS) shRNA and different version of knockdown (KD) shRNA against SPRY2. KD3 showed most efficient knockdown (70%) measured by western blot. KD3A is a single cell cloned subline from KD3. <i>B) D492SPRY2^-KD3 retains an epithelial phenotype in monolayer culture.</i> No phenotypic differences were observed in monolayer of D492^NS and <i>D492^SPRY2-KD3</i> (upper row). Transfection efficacy was evaluated by GFP (lower row). <i>C) D492^SPRY2-KD cells have acquired increased migration potential.</i> When plated on porous transwell filter <i>D492^SPRY2-KD3</i> showed increased migration compared to D492^NS. Single cell derived clone KD3A from KD3 had the highest migration potential. <i>D) SPRY2 knockdown has no effect on cell proliferation</i>. Monolayer proliferation of D492^NS, D492^SPRY2-KD3 and D492^SPR2-KD3A was evaluated at different time points, as indicated. There was no remarkable difference in the proliferation rate of the NS and KD cells, although at day 4 D492^SPRY2-KD3A seemed to proliferate slightly less.</p

SPRY2 expression is correlated with critical points in branching morphogenesis of D492 breast stem cell line.

<p><i>A) D492 cells generate branching structures when cultured in rBM.</i> When seeded in rBM D492 cells generate TDLU-like structures. By generating in vitro TDLU-like structures it is possible to follow individual steps in the branching morphogenesis process. Until day 8 or 9 cells grow as single colonies. First sign of initial budding occurs at day 10 and 11 (yellow arrows) followed by duct elongation and bifurcation (blue and red arrows), respectively. <i>B) SPRY2 expression shows a dramatic shift during TDLU formation in 3D culture.</i> Colonies were isolated from 3D cultures at different time points as indicated. Initially at day 8 there is relative high expression of SPRY2 mRNA but its expression is reduced during initial budding but increases again during duct elongation and further bifurcation of complex branching. Western blot confirms that SPRY2 levels increase up to day 16 and remain high while pEGFR is slightly decreasing for day 16 to day 19. Actin was used as a loading control. <i>C) pEGFR and SPRY2 are expressed at the growing tips of TDLU-like structures.</i> D492-derived TDLU-like structures generated in 3D culture were stained with antibodies against SPRY2, EGFR, pEGFR, β4-integrin and F-actin. pEGFR was predominantly expressed at the branching tips while total EGFR had a more general distribution. SPRY2 was also expressed at branching tips but not in clefts. Co-staining of SPRY2 and EGFR show strong expression at the branching tips (arrows). F-actin staining gives a general outlook of a branching colony while β4-integrin outlines their connection to the surrounding rBM matrix. Phosphorylated SPRY2 (right) was analyzed using proximity ligation assay as described above. pSPRY2 was predominantly expressed at the branching tips showing similar pattern as total SPRY2. Cells were counterstained with TOPRO-3 nuclear stain. Bar = 100 µm.</p