Genetic variation in fast-evolving East African cichlid fishes: an evolutionary perspective

Cichlid fishes from the East African Rift lakes Victoria, Tanganyika and Malawi represent a preeminent example of replicated and rapid evolutionary radiation. In this single natural system, numerous morphological (eg. jaw and tooth shape, color patterns, visual sensitivity), behavioral (eg. bower-building) and physiological (eg. development, neural patterning) phenotypes have emerged, much akin to a mutagenic screen. This dissertation encompasses three studies that seek to decipher the underpinnings of such rapid evolutionary diversification, investigated via the genetic variation in East African cichlids. We generated a valuable cichlid genomic resource of five low-coverage Lake Malawi cichlid genomes, from which the general properties of the genome were characterized. Nucleotide diversity of Malawi cichlids was low at 0.26%, and a sample genotyping study found that biallelic polymorphisms segregate widely throughout the Malawi species flock, making each species a mosaic of ancestrally polymorphic genomes. A second genotyping study expanded our evolutionary analysis to cover the entire East African cichlid radiation, where we found that more than 40% of single nucleotide polymorphisms (SNPs) were ancestral polymorphisms shared across multiple lakes. Bayesian analysis of genetic structure in the data supported the hypothesis that riverine species had contributed significantly to the genomes of Malawi cichlids and that Lake Malawi cichlids are not monophyletic. Both genotyping studies also identified interesting loci involved in important sensory as well as developmental pathways that were well differentiated between species and lineages. We also investigated cichlid genetic variation in relation to the evolution of microRNA regulation, and found that divergent selection on miRNA target sites may have led to differential gene expression, which contributed to the diversification of cichlid species. Overall, the patterns of cichlid genetic variation seem to be dominated by the phenomena of extensive sharing of ancestral polymorphisms. We thus believe that standing genetic variation in the form of ancestrally inherited polymorphisms, as opposed to variations arising from new mutations, provides much of the genetic diversity on which selection acts, allowing for the rapid and repeated adaptive radiation of East African cichlids.Ph.D.Committee Chair: Streelman, Todd; Committee Co-Chair: Yi, Soojin; Committee Member: Jordan, King; Committee Member: McDonald, John; Committee Member: Thomas, Jame

Comprehensive mapping of 5-hydroxymethylcytosine epigenetic dynamics in axon regeneration

<p>In contrast to central nervous system neurons, dorsal root ganglia (DRG) neurons can switch to a regenerative state after peripheral axotomy. In a screen for chromatin regulators of the regenerative responses in this conditioning lesion paradigm, we identified Tet methylcytosine dioxygenase 3 (Tet3) as upregulated in DRG neurons, along with increased 5-hydroxymethylcytosine (5hmC). We generated genome-wide 5hmC maps in adult DRG, which revealed that peripheral and central axotomy (leading to no regenerative effect) triggered differential 5hmC changes that are associated with distinct signaling pathways. 5hmC was altered in a large set of regeneration-associated genes (RAGs), including well-known RAGs, such as <i>Atf3, Bdnf</i>, and <i>Smad1</i>, that regulate axon growth potential of DRG neurons, thus supporting its role for RAG regulation. Our analyses also predicted HIF-1, STAT, and IRF as potential transcription factors that may collaborate with Tet3 for 5hmC modifications. Intriguingly, central axotomy resulted in widespread 5hmC modifications that had little overlap with those of peripheral axotomy, thus potentially constituting a roadblock for regeneration. Our study revealed 5hmC dynamics as a previously unrecognized epigenetic mechanism underlying the divergent responses after axonal injury.</p

Decoding the IGF1 signaling gene regulatory network behind alveologenesis from a mouse model of bronchopulmonary dysplasia

Lung development is precisely controlled by underlying gene regulatory networks (GRN). Disruption of genes in the network can interrupt normal development and cause diseases such as bronchopulmonary dysplasia (BPD) - a chronic lung disease in preterm infants with morbid and sometimes lethal consequences characterized by lung immaturity and reduced alveolarization. Here, we generated a transgenic mouse exhibiting a moderate severity BPD phenotype by blocking IGF1 signaling in secondary crest myofibroblasts (SCMF) at the onset of alveologenesis. Using approaches mirroring the construction of the model GRN in sea urchin's development, we constructed the IGF1 signaling network underlying alveologenesis using this mouse model that phenocopies BPD. The constructed GRN, consisting of 43 genes, provides a bird's eye view of how the genes downstream of IGF1 are regulatorily connected. The GRN also reveals a mechanistic interpretation of how the effects of IGF1 signaling are transduced within SCMF from its specification genes to its effector genes and then from SCMF to its neighboring alveolar epithelial cells with WNT5A and FGF10 signaling as the bridge. Consistently, blocking WNT5A signaling in mice phenocopies BPD as inferred by the network. A comparative study on human samples suggests that a GRN of similar components and wiring underlies human BPD. Our network view of alveologenesis is transforming our perspective to understand and treat BPD. This new perspective calls for the construction of the full signaling GRN underlying alveologenesis, upon which targeted therapies for this neonatal chronic lung disease can be viably developed

Bacterial Infection Drives the Expression Dynamics of microRNAs and Their isomiRs

International audienceThe optimal coordination of the transcriptional response of host cells to infection is essential for establishing appropriate immunological outcomes. In this context, the role of microRNAs (miRNAs) – important epigenetic regulators of gene expression – in regulating mammalian immune systems is increasingly well recognised. However, the expression dynamics of miRNAs, and that of their isoforms, in response to infection remains largely unexplored. Here, we characterized the genome-wide miRNA transcriptional responses of human den-dritic cells, over time, to various mycobacteria differing in their virulence as well as to other bacteria outside the genus Mycobacterium, using small RNA-sequencing. We detected the presence of a core temporal response to infection, shared across bacteria, comprising 49 miRNAs, highlighting a set of miRNAs that may play an essential role in the regulation of basic cellular responses to stress. Despite such broadly shared expression dynamics, we identified specific elements of variation in the miRNA response to infection across bacteria, including a virulence-dependent induction of the miR-132/212 family in response to myco-bacterial infections. We also found that infection has a strong impact on both the relative abundance of the miRNA hairpin arms and the expression dynamics of miRNA isoforms. That we observed broadly consistent changes in relative arm expression and isomiR distribution across bacteria suggests that this additional, internal layer of variability in miRNA responses represents an additional source of subtle miRNA-mediated regulation upon infection. Collectively, this study increases our understanding of the dynamism and role of miRNAs in response to bacterial infection, revealing novel features of their internal variability and identifying candidate miRNAs that may contribute to differences in the pathogenicity of mycobacterial infections

Genetic Adaptation and Neandertal Admixture Shaped the Immune System of Human Populations

International audienceHumans differ in the outcome that follows exposure to life-threatening pathogens, yet the extent of population differences in immune responses and their genetic and evolutionary determinants remain undefined. Here, we characterized, using RNA sequencing, the transcriptional response of primary monocytes from Africans and Europeans to bacterial and viral stimuli—ligands activating Toll-like receptor pathways (TLR1/2, TLR4, and TLR7/8) and influenza virus—and mapped expression quantitative trait loci (eQTLs). We identify numerous cis-eQTLs that contribute to the marked differences in immune responses detected within and between populations and a strong trans-eQTL hotspot at TLR1 that decreases expression of pro-inflammatory genes in Europeans only. We find that immune-responsive regulatory variants are enriched in population-specific signals of natural selection and show that admixture with Neandertals introduced regulatory variants into European genomes, affecting preferentially responses to viral challenges. Together, our study uncovers evolutionarily important determinants of differences in host immune responsiveness between human populations

Changes in relative miRNA arm expression upon infection.

<p>(A) Detection and relative expression of miRNA hairpin arms. Stacked bar plot shows the proportion of detected miRNA hairpins for which either one or both mature miRNA sequences were detected. Density plot shows the distribution of log₂ expression ratios for hairpins where both arms were expressed. (B) An example of one miRNA, miR-361, which showed a strong infection-dependent change in relative arm expression. Axes show the expression of the 3p and 5p arms of all sequenced individuals at each time point. In each panel, colours denote the different time points, and infection conditions are plotted in separate panels. The displacement of points towards the right of the plot in infected samples at later time points shows that the change in the expression ratio of the two arms is due to the increased expression of the 3p arm that, in some conditions, becomes dominant.</p

Shared and specific miRNA responses to bacterial infection.

<p>(A) Plots of the temporal dynamics of two core response miRNAs. As we did not have expression profiles for the 48h time point for STP infection, this bacterium was excluded from the analysis. (B) Multidimensional scaling analysis (MDS) representing the distances between the temporal miRNA responses to different bacterial infections. Distances were based on the sum of edit distances, for each miRNA, between bacteria using STEM-assigned model temporal profiles. (C) The expression of miR-132-3p increased following infection with MTB-Rv and MTB-Bj but not BCG. Transformation of BCG with RD1 from MTB resulted in a significant increase in miR-132-3p expression, not significantly different to that induced by MTB-Rv. Both MTB-Ra and MTB-Hk showed significantly lower miR-132-3p expression than virulent mycobacteria. (D) The induction of miR-212-3p was significantly higher following BCG::RD1 infection, compared to infection with control BCG, and was not significantly different to the response to MTB-Rv. Though the difference between the induction of this miRNA upon infection with virulent or avirulent MTB strains was not significant, the tendencies observed were consistent with miR-132-3p. Significance was calculated using a Mann-Whitney test (* = p < 0.05).</p

Differential expression of miRNAs in DCs upon infection with a panel of bacteria.

<p>(A) Numbers of significantly differentially expressed miRNAs upon infection at each time point for each bacterium. As we did not have expression profiles for the 48h time point for STP infection, this point is missing from the plot. (B) Heatmap illustrating the hierarchical clustering of experimental conditions based on the mean expression levels of the 50 most variable miRNAs. (C) Overlap of differentially expressed miRNAs between bacteria using two different significance cut-offs. Left-bar shows overlap using a single cut-off of FDR-adjusted p<0.01 and |log₂ fold change|>1, while the right bar shows the overlap using a secondary cut-off where a miRNA was called as significant if the absolute log₂ fold change was less than 1, if it passed the first more stringent fold-change cut-off upon infection with at least one of the six bacteria. (D and E) Venn diagrams showing the overlap of significantly differentially expressed miRNAs between bacteria of the MTBC (D) and between MTB-Rv and all other non-mycobacterial infections (E).</p