Browsing by Subject "Sea urchin"

Specification and differentiation of a cell is accomplished by changing its gene expression profiles. These processes require temporally and spatially regulated transcription factors (TFs), to induce the genes that are necessary to a specific cell type. In each cell a set of TFs interact with each other or activate their targets; as development progresses, transcription factors receive regulatory inputs from other TFs and a complex gene regulatory network (GRN) is generated. Adding complexity, each TF can be regulated not only at the transcriptional level, but also by translational, and post-translational mechanisms. Thus, understanding a developmental process requires understanding the interactions between TFs, signaling molecules and target genes which establish the GRN.

In this thesis, two genes, FoxN2/3, a TF and FGFR1, a component of the FGF signaling pathway are investigated. FoxN2/3 and FGFR1 have different mechanisms that function in sea urchin development; FoxN2/3 regulates gene expression and FGFR1 changes phosphorylation of target proteins. However, their ultimate goals are the same: changing the state of an earlier GRN into the next GRN state.

First, we characterize FoxN2/3 in the primary mesenchyme cell (PMC) GRN. Expression of foxN2/3 begins in the descendants of micromeres at the early blastula stage; and then is lost from PMCs at the mesenchyme blastula stage. foxN2/3 expression then shifts to the secondary mesenchyme cells (SMCs) and later to the endoderm. Here we show that, Pmar1, Ets1 and Tbr are necessary for activation of foxN2/3 in the descendants of micromeres. The later endomesoderm expression is independent of the earlier expression of FoxN2/3 in micromeres and independent of signals from PMCs. FoxN2/3 is necessary for several steps in the formation of larval skeleton. A number of proteins are necessary for skeletogenesis, and early expression of at least several of these is dependent on FoxN2/3. Furthermore, knockdown (KD) of FoxN2/3 inhibits normal PMC ingression. PMCs lacking FoxN2/3 protein are unable to join the skeletogenic syncytium and they fail to repress the transfating of SMCs into the skeletogenic lineage. Thus, FoxN2/3 must be present for the PMC GRN to control normal ingression, expression of skeletal matrix genes, prevention of transfating, and control fusion of the PMC syncytium.

Second, we show that the FGF-FGFR1 signaling is required for the oral-aboral axis formation in the sea urchin embryos. Without FGFR1, nodal is induced in all of the cells at the early blastula stage and this ectopic expression of nodal requires active p38 MAP kinase. The loss of oral restriction of nodal expression results in the abnormal organization of PMCs and the larval skeleton; it also induces ectopic expression of oral-specific genes and represses aboral-specific genes. The abnormal oral-aboral axis formation also affected fgf and vegf expression patterns; normally these factors are expressed in two restricted areas of the ectoderm between the oral and the aboral side, but when FGFR1 is knocked down, Nodal expands, and in response the expression of the FGF and VEGF ligands expands, and this in turn affects the abnormal organization of larval skeleton.

Embryonic development occurs with precisely timed morphogenetic cell movements directed by complex gene regulation. In this orchestrated series of events, some epithelial cells undergo extensive changes to become free moving mesenchymal cells. The transformation resulting in an epithelial cell becoming mesenchymal is called an epithelial to mesenchymal transition (EMT), a dramatic cell biological change that occurs throughout development, tissue repair, and disease. Extensive in vitro research has identified many EMT regulators. However, most in vitro studies often reduce the complicated phenotypic change to a binary choice between successful and failed EMT. Research utilizing models has generally been limited to a single aspect of EMT without considering the total transformation. Fully understanding EMT requires experiments that perturb the system via multiple channels and observe several individual components from the series of cellular changes, which together make a successful EMT.

In this study, we have taken a novel approach to understand how the sea urchin embryo coordinates an EMT. We use systems level methods to describe the dynamics of EMT by directly observing phenotypic changes created by shifting transcriptional network states over the course of primary mesenchyme cell (PMC) ingression, a classic example of developmental EMT. We systematically knocked down each transcription factor in the sea urchin's PMC gene regulatory network (GRN). In the first assay, one fluorescently labeled knockdown PMC precursor was transplanted onto an unperturbed host embryo and we observed the resulting phenotype in vivo from before ingression until two hours post ingression using time-lapse fluorescent microscopy. Movies were projected for computational analyses of several phenotypic changes relevant to EMT: apical constriction, apical basal polarity, motility, and de-adhesion.

A separate assay scored each transcription factor for its requirement in basement membrane invasion during EMT. Again, each transcription factor was knocked down one by one and embryos were immuno-stained for laminin, a major component of basement membrane, and scored on the presence or absence of a laminin hole at the presumptive entry site of ingression.

The measured results of both assays were subjected to rigorous unsupervised data analyses: principal component analysis, emergent self-organizing map data mining, and hierarchical clustering. This analytical approach objectively compared the various phenotypes that resulted from each knockdown. In most cases, perturbation of any one transcription factor resulted in a unique phenotype that shared characteristics with its upstream regulators and downstream targets. For example, Erg is a known regulator of both Hex and FoxN2/3 and all three shared a motility phenotype; additionally, Hex and Erg both regulated apical constriction but Hex additionally affected invasion and FoxN2/3 was the lone regulator of cell polarity. Measured phenotypic changes in conjunction with known GRN relationships were used to construct five unique subcircuits of the GRN that described how dynamic regulatory network states control five individual components of EMT: apical constriction, apical basal polarity, motility, de-adhesion, and invasion. The five subcircuits were built on top of the GRN and integrated existing fate specification control with the morphogenetic EMT control.

Early in the EMT study, we discovered one PMC gene, Erg, was alternatively spliced. We identified 22 splice variants of Erg that are expressed during ingression. Our Erg knockdown targeted the 5'UTR, present in all spliceoforms; therefore, the knockdown uniformly perturbed all native Erg transcripts (∑Erg). Specific function was demonstrated for the two most abundant spliceoforms, Erg-0 and Erg-4, by knockdown of ∑Erg and mRNA rescue with a single spliceoform; the mRNA expression constructs contained no 5'UTR and were not affected by the knockdown. Different molecular phenotypes were observed, and both spliceoforms targeted Tbr, Tel, and FoxO, only Erg-0 targeted FoxN2/3 and only Erg-4 targeted Hex. Neither targeted Tgif, which was regulated by ∑Erg knockdown sans rescue. Our results suggest the embryo employs a minimum of three unique roles in the GRN for alternative splicing of Erg.

Overall, these experiments increase the completeness and descriptive power of the GRN with two additional levels of complexity. We uncovered five sub-circuits of EMT control, which integrated into the GRN provide a novel view of how a complex morphogenetic movement is controlled by the embryo. We also described a new functional role for alternative splicing in the GRN where the transcriptional targets for two splice variants of Erg are unique subsets of the total set of ∑Erg targets.

A general goal of biology is to understand how two or more sets of traits in an organism are related - for example, disease state and genetics, physiology and behavior, or phenotypic variation and gene function. Many of the early advancements in statistical analysis dealt with relating measured traits when one could be represented as a single number. However, many traits are inherently multi-dimensional, and technologies are advancing for rapidly measuring many types of such highly complex traits. Making efficient use of these new, larger datasets requires new statistical models for to biological inference. In this thesis, I develop a method for relating two very different types of traits in sea urchin larvae: morphological shape, and developmental gene expression. In particular, I develop an approach for regression modeling using shape as a response variable. I use this method to address the question of whether variation in the expression of regulatory genes during development predicts later morphological variation in the larvae. I propose a hierarchical random effects factor regression model with shape as a response variable for relating morphology and gene expression when the individuals in each dataset are related, but not identical. I fit an approximation to the general model by breaking it into three discrete steps. I find that gene expression can explain ~25% of mean symmetric form variation among cultures of related larvae, and identify several groups of related genes that are correlated with aspects of morphological variation.

Many factors, including gene networks, developmental processes, and the environment mediate the link between the activity of genes and complex phenotypes in higher organisms. While genetic variants are the raw material for evolution, these other factors are critical for determining which variants are actually exposed to natural selection. In this dissertation, I describe three projects in which I investigate how developmental mechanisms and the environment interact to shape phenotypic variation. In each project, I use gene expression as a window into the activity of genes, and as a tool to measure variation in and among developmental mechanisms. Two projects are experimental, focusing on early development in sea urchins, and how environmental stress caused by climate change impacts the expression of genetic variation in phenotypic traits. In these projects, I explicitly incorporate information about the biochemical functions of genes and how they interact in development, and test how such mechanisms shape the impact of genetic and environmental perturbations to development. The third project is methodological, in which I propose a unified statistical framework for inferring previously unknown developmental constraints that may underlie gene expression phenotypes. Together, these projects demonstrate that an understanding of developmental mechanisms can enhance our understanding of the processes that shape variation in populations, and can help predict the biological effects of climate change.

Sea urchins are relegated as background or non-playable characters in Western Science and Western culture on a daily basis. This dissertation shines a spotlight on my non-human relatives and gives readers a chance to learn about the sciences and cultures that sea urchin species play a role in. Along with offering research within the realms of development biology and immunology, there are pieces relevant to Science & Society and general audience blended throughout the chapters. I address two major problems in this work. The first problem is determining how the complex immune system of the Lytechinus variegatus larva develops and reacts to injury. The second, is uncovering what colonial practices have led to the sea urchin research climate today.The first chapter of this dissertation offers a broad Introduction to sea urchins. Moving away from a “cold” or impersonal description, you’ll learn about the diversity of sea urchins that move about the world’s oceans every day. Topics include what urchin’s eat, their etymological origin, their influence on historical and contemporary cultures, what some taste like, and other ranging topics. Parallel to these topics, this chapter contains basic biology on the adult and larval sea urchin, and the development of the embryo’s mesenchyme cell populations, which will be the foci of multiple chapters. Moving into wet lab research, chapter 2 provides the methods used in molecular, cellular, and embryological experiments. The major novel research method is an injury assay where we are able crush or “squish” L. variegatus larvae, causing a reproducible and quick immune cell response. Chapter 3 involves a review and recommendations on how Western Science developmental biology researchers think about and approach studying the larva’s mesenchyme. Using these new practices, we were able to uncover tissue specific candidate markers in silico, by cloning, and through expression data. Chapter 4 is the characterization of the immune-related cytokine family, the macrophage migration inhibitory factors (MIFs) in sea urchins. Utilizing newly published genomes, we were able to provide a fuller description of MIF gene duplications, and confidently clone select MIFs and perturb candidate regulators. The following chapter addresses the conundrum of wound healing and the immune system’s role in the process. By crushing L. variegatus larvae, we have been able to describe and characterize roles of pigment and blastocoelar cells in epithelial and skeletal wound repair. Our conclusions for the respective chapters are, the MIF family has gone through a major gene duplication, and are now able to perform tissue specific and potentially redundant roles in immune cell function; and pigment cells and blastocoelar cell networks are activated quickly in injuries and can remodel tissues in the larvae. The Science & Society, and penultimate chapter of this dissertation takes a deeper look into why Western Scientists study sea urchin, doing so through the lens of colonialism. In outlining personal kinship, obligations, and an interdisciplinary lens, I am able to name the colonial intentions of marine stations and their use of invertebrate species as research materials. For a contemporary example, I name general instances of colonial research methods and propose select ways to promote anti- and decolonial practices in sea urchin science that move beyond metaphors. This project, and the questions it answers and generates moves away from myths of Western Science such as neutrality, and away from a spiritless resource for academic consumption. Along with learning about the immune system, cytokines, and injury response from my non-human, sea urchin relatives, we can also begin to address broad and systemic problems that are faced in sea urchin science and culture. My goal, and what should be thought about early in reading this work, is to learn from, appreciate, reciprocate, respect, and co-create knowledge with sea urchins in a way that doesn’t harm the Land.

Gene regulatory networks (GRNs) provide a systems-level orchestration of an organism’s genome encoded anatomy. As biological networks are revealed, they continue to answer many questions including knowledge of how GRNs control morphogenetic movements and how GRNs evolve. Morphogenesis is a complex orchestration of movements by cells that are specified early in development.

The activation of an upstream GRN is crucial in order to orchestrate downstream morphogenetic events. In the sea urchin, activation of the endomesoderm GRN occurs after the asymmetric 4th cleavage. Embryonic asymmetric cell divisions often are accompanied by differential segregation of fate-determinants into one of two daughter cells. That asymmetric cleavage of the sea urchin micromeres leads to a differential animal-vegetal (A/V) nuclear accumulation of cell fate determinants, β-Catenin and SoxB1. Β-Catenin protein is localized into the nuclei of micromeres and activates the endomesoderm gene regulatory network, while SoxB1 is excluded from micromeres and enters the nucleus of the macromeres, the large progeny of the unequal 4th cleavage. Although nuclear localization of β-Catenin and SoxB1 shows dependence on the asymmetric cleavage, the mechanics behind the asymmetrical division has not been demonstrated. In Chapter 3, we show that micromere formation requires the small RhoGTPase, Cdc42 by directing the apical/basal orientation of the mitotic spindle at the apical cortex. By attenuating or augmenting sea urchin Cdc42 function, micromere divisions became defective and failed to correctly localize asymmetrically distributed determinants. As a consequence, cell fates were altered and multiple A/V axes were produced resulting in a “Siamese-twinning” phenotype that occurred with increasing frequency depending on the quantitative level of perturbation. Our findings show that Cdc42 plays a pivotal role in the asymmetric division of the micromeres, endomesoderm fate-determinant segregation, and A/V axis formation.

This dissertation also characterizes, at high resolution, the repertoire of cellular movements contributing to three different morphogenetic processes of sea urchin development: the elongation of gut, the formation of the primary mouth, and the migration of the small micromeres (the presumptive primordial germ cells) in the sea urchin, Lytechinus variegatus. Descriptive studies of the cellular processes during the different morphogenetic movements allow us to begin investigating their molecular control.

In Chapter 4, we dissected the series of complex events that coordinate gut and mouth morphogenesis. Until now, it was thought that lateral rearrangement of endoderm cells by convergent extension was the main contributor to sea urchin archenteron elongation and that cell divisions were minimal during elongation. We performed cell transplantations to live image and analyze a subset of labeled endoderm cells at high-resolution in the optically clear sea urchin embryo. We found that the endomesoderm cells that initially invaginate into the sea urchin blastocoel remained contiguous throughout extension, so that, if convergent extension were present, it was not a major contributor to elongation. We also found a prevalence of cell divisions throughout archenteron elongation that increased the number of cells within the gut linearly over time; however, we showed that the proliferation did not contribute to growth, and their spindle orientations were randomized during divisions and therefore did not selectively contribute to the final gut length. When cell divisions were inhibited, we saw no difference in the ability of the cells within the gut to migrate in order to elongate. Also in Chapter 4, we describe our observations of the cell biological processes underlying primary mouth formation at the end of gastrulation. Using time-lapse microscopy, photo-convertible Kaede, and an assay of the basement membrane remodeling, we describe a sequential orchestration of events that leads to the fusion of the oral ectoderm and the foregut endoderm. Our work characterizes, at higher resolution than previously recorded, the temporal sequence and repertoire of the cellular movements contributing to the length of the sea urchin larval gut and tissue fusion with the larval primary mouth.

In Chapter 5, the migration of the small micromeres to the coelomic pouches in the sea urchin embryo provides an exceptional model for understanding the genomic regulatory control of morphogenesis. An assay using the robust homing potential of these cells reveals a “coherent feed-forward” transcriptional subcircuit composed of Pax6, Six3, Eya, and Dach1 that is responsible for the directed homing mechanism of these multipotent progenitors. The linkages of that circuit are strikingly similar to a circuit involved in retinal specification in Drosophila suggesting that systems-level tasks can be highly conserved even though the tasks drive unrelated processes in different animals.

The sea urchin gene regulatory network (GRN) describes the cell fate specification of the developing embryo; however, the GRN does not describe specific cell biological events driving the three distinct sequences of cell movements. Our ability to connect the GRN to the morphogenetic events of gastrulation, primary mouth formation, and small micromere migration will provide a framework for characterizing these remarkable sequences of cell movements in the simplest of deuterostome models at an unprecedented scale.

Changes in the expression and function of genes active during metazoan development have played a critical role in the evolution of morphological differences between species and phyla, yet the origins of these changes remain poorly understood. What roles do positive and negative selection play in the evolution of development? How do evolutionary changes accumulate given the degree to which organisms are able to buffer the effects of environmental and genetic perturbations during development? The crucial insight of the Modern Evolutionary Synthesis was that divergence between species arises from variation within populations. Following this principle, I have made use of tools from quantitative and population genetics to investigate three central questions: 1) How much genetic variation is there in the networks of genes that underlie metazoan development? 2) What affect does developmental buffering have on the accumulation of selectable genetic variation? 3) To what extent does selection act to shape patterns of genetic variation among different kinds of genes and at different stages of development? I show that developmental systems can harbor extensive levels of genetic variation, and that the amount of genetic variation in individual genes at different stages of development is related to the extent to which variation in those genes is buffered by genetic interactions. I also show that while selection plays an active role in shaping genetic variation in development, the extent to which variation in a gene is visible to selection depends in predictable ways on a) the biological function of that gene and b) whether the mutations in question influence gene expression or protein function. My results as a whole demonstrate the utility of population level approaches to the study of the evolution of development, and provide key insights into the role that selection plays in generating developmental variation.

Sea urchin larvae possess a beautiful, intricately patterned, calcium-carbonate skeleton. Formation of this complex structure results from two independent processes within the developing embryo: specification of the mesenchymal cells that produce the skeletal rods, and patterning inputs from the ectoderm, which secretes signals directing the growth and shape of the skeleton. To understand patterning of the skeleton therefore, the specification events behind these two processes must be understood separately, and then connected in order to understand how ectoderm signaling directs skeletal growth. While the former processes of mesenchyme specification and mineralization are under study elsewhere, the means by which ectodermal cues directing skeletal growth are activated and localized is not known. Using molecular genetics, including gene knock downs and mis-expression, as well as microsurgical manipulations of early cleavage embryos, I show how a previously undescribed territory within the ectoderm, the border ectoderm (BE) is specified with short range signaling inputs. Then, experiments show that the BE provides signals that initiate, and contribute to the propagation of skeletogenesis. From this dataset, and from biological experiments I outline a model for how the BE patterns and contributes to the directed growth of the skeleton. I also discuss challenges to this model that need to be addressed in future research. In principle, the mechanism proposed herein depends on the integration of information from both the primary and secondary embryonic axes. It requires both short-range signaling by Wnt5 from the endoderm to establish the BE fate, and TGFß signaling from the oral and aboral ectoderm which subdivides the BE into four territories. These findings demonstrate that the short-range signaling cascade that subdivides the embryo into first mesoderm and then endoderm also specifies ectodermal fates. Ultimately, this research paves the way for understanding how the larval skeleton is patterned during embryogenesis and may provide a paradigm for understanding other, more complex, developmental problems.