Identification and Characterization of MicroRNA Modulators in Caenorhabditis Elegans: A Dissertation

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that posttranscriptionally regulate gene expression primarily through binding to the 3’ untranslated region (3’UTR) of target mRNAs, and are known to play important roles in various developmental and physiological processes. The work presented in this thesis was centered on understanding how Caenorhabditis elegans miRNAs are modulated by genetic, environmental, or physiological factors and how these small RNAs function to maintain the robustness of developmental processes under stressful conditions. To identify modulators of the miRNA pathway, I developed sensitized genetic backgrounds that consist of a panel of miRNA gene mutants and miRNA biogenesis factor mutants with partially penetrant phenotypes. First, I found that upon infection of Caenorhabditis elegans with Pseudomonas aeruginosa, an opportunistic pathogen of diverse plants and animals, let-7 family miRNAs are engaged in reciprocal regulatory interactions with the p38 MAPK innate immune pathway to maintain robust developmental timing despite the stress of pathogen infection. These let-7 family miRNAs, along with other developmental timing regulators, are also integrated into innate immune regulatory networks to modulate immune responses. Next, I demonstrated that loss-of-function mutations of Staufen (stau-1), a double-stranded RNA-binding protein, increase miRNA activity for several miRNA families, and this negative modulation of Staufen on miRNA activity acts downstream of miRNA biogenesis, possibly by competing with miRNAs for binding to target mRNA 3’UTRs. In summary, these studies provide a better understanding on how miRNAs are modulated by various environmental and cellular components, and further support the role of the miRNA pathway in conferring robustness to developmental processes under these perturbations

Cis-Regulation of Gremlin1 Expression during Mouse Limb Bud Development and its Diversification during Vertebrate Evolution

Embryonic development and organogenesis rely on tightly controlled gene expression, which is achieved by cis-regulatory modules (CRMs) interacting with distinct transcription factors (TFs) that control spatio-temporal and tissue-specific gene expression. During organogenesis, gene regulatory networks (GRNs) with selfregulatory feedback properties coordinately control growth and patterning and provide systemic robustness against genetic and/or environmental perturbations. During limb bud development, various interlinked GRNs control outgrowth and patterning along all three limb axes. A paradigm network is the epithelial-mesenchymal (e-m) SHH/GREM1/AER-FGF feedback signaling system which controls limb bud outgrowth and digit patterning. The BMP antagonist GREMLIN1 (GREM1) is central to this e-m interactions as its antagonism of BMP activity is essential to maintain both AER-Fgf and Shh expression. In turn, SHH signaling upregulates Grem1 expression, which results in establishment of a self-regulatory signaling network. One previous study provided evidence that several CRMs could regulate Grem1 expression during limb bud development. However, the cis-regulatory logics underlying the spatio-temporal regulation of the Grem1 expression dynamics remained obscure. From an evolutionary point of view, diversification of CRMs can result in diversification of gene regulation which can drive the establishment of morphological novelties and adaptions. This was evidenced by the observed differences in Grem1 expression in different species that correlates with the evolutionary plasticity of tetrapod digit patterning. Hence, a better understanding of spatio-temporal regulation of the Grem1 expression dynamics and underlying cis-regulatory logic is of interest from both adevelopmental and an evolutionary perspective. Recently, multiple candidate CRMs have been identified that might be functionally relevant for Grem1 expression during mouse limb bud development. For my PhD project, I genetically analyzed which of these CRMs are involved in the regulation of the spatial-temporal Grem1 expression dynamics in limb buds. Therefore, we generated various single and compound CRM mutant alleles using CRISPR/Cas9. Our CRMs allelic series revealed a complex Grem1 cis-regulation among a minimum of six CRMs, where a subset of CRMs regulates Grem1 transcript levels in an additive manner. Surprisingly, phenotypic robustness depends not on threshold transcript levels but the spatial integrity of the Grem1 expression domain. In particular, interactions among five CRMs control the characteristic asymmetrical and posteriorly biased Grem1 expression in mouse limb buds. Our results provide an example of how multiple seemingly redundant limb-specific CRMs provide phenotypical robustness by cooperative/synergistic regulation of the spatial Grem1 expression dynamics. Three CRMs are conserved along the phylogeny of extant vertebrates with paired appendages. Of those, the activities of two CRMs recapitulate the major spatiotemporal aspects of Grem1 expression in mouse limb buds. In order to study their functions in species-specific regulation of Grem1 expression and their functional diversification in tetrapods, I tested the orthologous of both CRMs from representative species using LacZ reporter assays in transgenic mice, in comparison to the endogenous Grem1 expression in limb buds of the species of origin. Surprisingly, the activities of CRM orthologues display high evolutionary plasticity, which correlates better with the Grem1 expression pattern in limb buds of the species of origin than its mouse orthologue. This differential responsiveness to the GRNs in mouse suggests that TF binding site alterations in CRMs could underlie the spatial diversification of Grem1 in limb buds during tetrapod evolution. While the fish fin and tetrapod limb share some homologies of proximal bones, the autopod is a neomorphic feature of tetrapods. The Grem1 requirement for digit patterning and conserved expression in fin buds prompted us to assess the enhancer activity of fish CRM orthologues in transgenic mice. Surprisingly, all tested fish CRMs are active in the mouse autopod primordia providing strong evidence that Grem1 CRMs are active in fin buds and that they predate the fin-to-limb transition. Our results corroborate increasing evidence that CRMs governing autopodial gene expression have been co-opted during the emergence of tetrapod autopod. Furthermore, as part of a collaboration with Dr. S. Jhanwar, I contributed to the study of shared and species-specific epigenomic and genomic variations during mouse and chicken limb bud development. In this analysis, Dr. S. Jhanwar identified putative enhancers that show higher chicken-specific sequence turnover rates in comparison to their mouse orthologues, which defines them as so-called chicken accelerated regions (CARs). Here, I analyzed the CAR activities in comparison to their mouse orthologues by transgenic LacZ reporter assays, which was complemented by analysis of the endogenous gene expression in limb buds of both species. This analysis indicates that diversified activity of CARs and their mouse orthologues could be linked to the differential gene expression patterns in limb buds of both species

FGF signaling and cell state transitions during organogenesis

Organogenesis is a complex choreography of morphogenetic processes, patterns and dynamic shape changes as well as the specification of cell fates. Although several molecular actors and context-specific mechanisms have already been identified, our general understanding of the fundamental principles that govern the formation of organs is far from comprehensive. The application of the concept of ‘rebuild it to understand it’ from synthetic biology represents a promising alternative to the classical approach of ‘break it to understand it’ in order to distill biological understanding from complex developmental processes. According to this ‘rebuilding’ concept, in this study we sought to develop an experimental approach to induce the formation of organs from progenitor cells ‘on demand’ and to investigate the minimum requirements for such a process. The zebrafish lateral line chain cells are a powerful in vivo model for our study because they are a group of naïve multipotent progenitor cells that display mesenchyme-like features. In order to bring these cells to form organs, we used the well-known role of the FGF signaling pathway as a driver of organogenesis in the lateral line and developed an inducible and constitutively active form of the fibroblast growth factor receptor 1a (chemoFGFR). The cell-autonomous induction of this chemoFGFR in chain cells effectively triggered the formation of fully mature organs and thus enabled spatial and temporal control of the organogenesis process. Next, we asked what it takes to form an organ de novo. We used a combination of real-time microscopy, single cell tracking, polarity quantification, and mosaic analysis to study the cell behaviors that result from chemoFGFR induction. The picture that emerges from these analyses is that de novo organs form through a genetically encoded self-assembly process that is based on the pattern of chemoFGFR induction. In this scenario, cells expressing chemoFGFR aggregate into clusters and epithelialize as they sort out of non-expressing cells. We found that this sorting process occurs through cell rearrangement and slithering, which involves an extensive remodeling of the cell-cell contacts. Chain cells that do not express chemoFGFR can envelop these chemoFGFR expressing cell clusters and form a rim at the cluster periphery. This multi-stage process leads to the establishment of the inside-outside pattern of de novo organs, which is used as a blueprint for cell differentiation. In summary, in this study we provide insights into the mechanisms involved in the self-assembly of organs from a naïve population of progenitor cells

Compensatory responses to Notch signaling perturbation in polyploid vertebrates, Xenopus laevis and Xenopus borealis, during embryonic development

Embryonic development is a robust process during which embryos must respond and compensate for changes in order to achieve consistent patterning; however, there are still questions about the limits and mechanisms of this robustness. Using tetraploid Xenopus laevis as a model, we have previously shown that embryos respond to perturbations of the highly-conserved Notch signaling pathway in a compensatory manner. We have now demonstrated that this response involves changes in the proliferative status of neural progenitors and differentiated neurons over time. Subsequent RNA-seq analysis of Notch perturbed X. laevis embryos revealed that homeologs (duplicated genes originating from whole-genome duplication) respond differentially to this perturbation, suggesting that the polyploidy of X. laevis may contribute to the compensatory abilities. To address this question, we have perturbed Notch signaling in X. borealis, a tetraploid species that is closely related to X. laevis, and characterized the response over time. Similarly to X. laevis, a compensatory response is seen in X. borealis over time based on gene expression in the developing nervous system, but embryos appear morphologically deformed throughout development. This suggests that X. borealis embryos may be more severely affected by this perturbation and do not compensate as well as X. laevis. RNA-seq was performed on Notch perturbed X. borealis embryos to quantitatively and globally assess the transcriptional response over time. Given that there was previously no reference genome or transcriptome for X. borealis, a de novo assembly of the X. borealis transcriptome was constructed to allow for further analysis. Using the X. laevis genome as a reference has allowed for comparative analysis of the changes in homeolog expression in X. laevis and X. borealis embryos following Notch perturbation. These data have revealed differences in the response to Notch perturbation between X. borealis and X. laevis, with X. borealis generally having more differentially expressed genes when compared to X. laevis under the same condition, again suggesting that X. borealis is more severely perturbed and does not compensate as well as X. laevis. To validate and compliment these RNA-seq results, it would be ideal to visualize homeolog expression in situ; however, given the high degree of sequence homology between homeologs, detection of specific homeolog transcripts in situ has presented a challenge for traditional methods. Using two new in situ hybridization technologies (Molecular Instruments Hybridization Chain Reaction v3.0 and Advanced Cell Diagnostics BaseScopeTM assay), we have been able to visualize X. laevis homeolog expression in situ with extreme specificity, which will enable spatial analysis of homeolog expression and a tool to validate RNA-seq findings. In the future, it will be interesting compare the transcriptional response to Notch signaling perturbation across ploidy levels, in addition to within multiple tetraploid species as we have already done. To enable this type of comparative experiment, we have sequenced and assembled the transcriptome of Xenopus andrei, an octoploid frog, representing, to the best of our knowledge, the first publicly available assembled transcriptome of an octoploid vertebrate

A Microfluidic Temperature Gradient Device and Its Application to Uncovering Temporal Systems of Robustness in the Developing Embryo of the Nematode C. elegans

Animal development is a complex process, shepherded by systems of robustness to ensure its success. To date, such systems that have been experimentally identified, largely have been found to ensure specific and correct cell identities in terms of gene and protein expression and spatial position. Little is known about systems that supervise, guide, and compensate for variability in the timing of events within development. The developing embryo of the nematode C. elegans follows a highly-stereotyped sequence of events starting at the two-cell stage where an asymmetric first division results in two cells differing in size, genetic and proteomic identity, and lineage dependent rates of division. These first two cells stereotypically divide at different times, with the larger dividing before the smaller. This sequence will later result in a number of key cell-cell interactions necessary for successful development. The rigid stereotype of this sequence and the critical nature of the dependent later events, suggests that the sequence itself may be under the influence of a system of robustness. At this stage such a system would necessarily include communication between the two cells to ensure their coordination in time. The work presented here establishes a method of challenging this system, by placing the C. elegans embryo in a temperature gradient sufficient to push the temperature dependent rate of division of the two cells away from their stereotyped temporal relationship. To achieve this we built and characterized a novel microfluidic temperature gradient device that can establish a 7.5 OC temperature gradient across the ~ 50 μm long developing embryo within biologically permissive temperatures. This temperature gradient establishes a condition that would be considered aberrant by the embryo at this stage, if and only if the two cells are monitoring each other's behavior. We have found that within a temperature gradient, the two cells of the embryo identify the existence of an aberrant condition and compensate for it by slowing their division rates. We find that the fold change in division timing of the two cells is dependent on their orientation in the temperature gradient, and that embryos that survive this condition to hatching generally slow down more than those that do not. We are able to reverse the sequence of divisions between the two cells and although they do not hatch, a surprising percentage undergo morphogenesis and result in a product that looks “wormlike” suggesting even later checkpoints and compensation. We also found that in a fraction of the embryos loaded into the gradient after the first division, the cleavage between the two cells reverses and the two nuclei of the two cells migrate back toward each other. The behavior of the two-cell embryos in the temperature gradient: 1) surviving a high percentage of time in lower gradients, and even a fraction of the time in higher gradients, 2) the slowing down of each cell relative to its expected behavior at the temperature it is experiencing with greater slowing resulting in a greater likelihood of survival, and 3) the entry of embryos into morphogenesis even after violation of the stereotypical sequence of division at the two-cell stage, constitutes evidence for one and possibly two previously unidentified compensation and coordination mechanisms that act to ensure robustness against variation in the timing of events in the development of the early C. elegans embryo

Getting back on track: exploiting canalization to uncover the mechanisms of developmental robustness

Developing embryos can adapt dynamically to noise and variation to generate organs of incredible precision, a process termed ‘canalization’; however, the underlying robustness mechanisms are poorly understood. Technological developments, both in quantitative imaging and high precision perturbation, are now enabling targeted investigation into developmental robustness in vivo. Here, we will first distil the common design features of studies that have exploited the canalization behaviour of specific systems to interrogate developmental adaptation, to provide a general experimental framework for future investigations in other contexts. We will then highlight, using a selection of recent case studies, how this approach is revealing that tissues and embryos can fix themselves in unexpected ways

Understanding the development and growth of the zebrafish (Danio rerio) infraorbital bones

xiv, 136 leaves : ill. (chiefly col.) ; 29 cm.Includes abstract and appendices.Includes bibliographical references (leaves 117-121).This study investigates the development of the neural crest derived infraorbital bones of the zebrafish (Danio rerio). Located around the underside of the orbit, the infraorbital bones ossify intramembranously in a set sequence and are closely associated with the lateral line sensory system. I conducted detailed analyses of the condensation to ossification phases of development of these bones, analysed infraorbital bone development via a series of skeletogenic condensation laser ablation experiments, and the neuromasts associated with the infraorbital bones were ablated at multiple time-points in order to investigate their highly debated inductive potential for infraorbital ossification. The results of this study highlight the developmental robustness of the infraorbital bones. The recovery of ablated structures demonstrates rescue mechanisms that allowed for the series to develop normally following perturbation. The mechanisms of developmental robustness should be investigated further as the infraorbital bones can be used to increase understanding of intramembranous bone patterning