In arthropods, annelids, and chordates, both ectodermal and mesodermal tissues are organized into segments. Although these phyla share the trait of segmentation, a widely accepted phylogenetic hypothesis places them into the three separate clades of the bilatarians. Since each of these clades also contains many unsegmented phyla, it is debatable whether segmentation in arthropods, annelids and chordates is homologous. A limitation of previous studies comparing segmentation between these three groups is that most of the existing data is on mesoderm segmentation in vertebrates and ectoderm segmentation in Drosophila. Homologous mechanisms of segmentation, such as in the mesoderm of vertebrates and arthropods, could be missed in these studies. To further investigate the evolution of segmentation, I have examined mesoderm segmentation in the arthropod Parhyale hawaiensis, an amphipod crustacean. In Parhyale, segments are added progressively from anterior to posterior, similar to most other arthropods. Additionally, Parhyale is an ideal system for experiemental manipulations. For example, lineage tracing, cell ablation, and gene knock-down reagents are easily targeted to the segmental mesoderm and ectoderm.

My dissertation focuses on two aspects of Parhyale mesoderm segmentation. First, whether the mesoderm and the ectoderm require signals from each other in order to segment. Second, whether the Parhyale homologs of the snail family of transcriptional repressors play a role in mesoderm segmentation. My first research project tests a hypothesis that mesoderm segmentation is autonomous and induces segmentation in the overlying ectoderm. Using ablations, I show that the ectoderm segments normally without the mesoderm. When I ablate the ectoderm, the mesoteloblasts, the mesodermal stem cells, continue to undergo normal rounds of division. However, they fail to migrate properly. Additionally, the progeny of the mesoteloblasts, the mesoblasts, neither proliferate nor express known markers of mesoderm segmentation. This suggests that the ectoderm provides a permissive signal, allowing mesoblasts to divide, and, possibly, an instructive signal, patterning the mesoblast lineage.

My second research project addresses whether Parhyale homologs of the snail family of transcriptional repressors play a role in mesoderm segmentation and/or specification, similar to their role in other species. There are four known homologs of snail in Parhyale. Since one homolog was previously characterized, I characterized the three other homologs. In addition, I performed analysis of all four family members to determine their relationship with each other as well as snail homologs in other species. The snail family has two branches in bilatarians, snail and scratch. I found that there are three snail homologs and one scratch homolog in Parhyale, which I named Ph-snail1, Ph-snail2 and Ph-snail3, and Ph-scratch, respectively. Since snail plays a major role in early mesoderm development in other animals, I expected one or more Parhyale snail homologs to be expressed in early mesoderm. I found that Ph-snail1 is the only Parhyale snail gene expressed in the mesoderm, and is expressed later in development than expected. Ph-scratch is not expressed during early development and germband elongation. Ph-snail2 and Ph-snail3 are expressed in the ectoderm, suggestive of a role in nervous system development.

In order to investigate the role of Ph-snail1 (Ph-sna1) in the mesoderm, I knocked down Ph-sna1 transcripton using targeted siRNAs. The expression pattern of Ph-sna1 suggests three potential roles in the mesoteloblasts: specification, migration, and/or cell-cycle progression. By injecting siRNAs against Ph-sna1 into the mesoteloblast precursor cell, I found that mesoteloblasts form but undergo fewer divisions than controls. This is consistent with the hypothesis that Ph-sna1 is involved in mesoteloblast cell cycle progression. In addition, when siRNAs for Ph-sna1 are injected at the one cell stage, the embryo fails to develop after the initiation of germ band formation due to knock-down of Ph-sna1 in the anterior head ectoderm.

My thesis work has refined our understanding of the role of the ectoderm and snail homologs in Parhyale mesoderm segmentation. Comparison of segmentation in Parhyale to segementation in the arthropod Drosophila show that, although the role of snail homologs differ in mesoderm segmentation, the interactions between the segmental ectoderm and mesoderm are similar. These interactions differ between arthropods, vertebrates and annelids, though, suggesting that either their common ancestor was not segmented, or that segmentation in these groups has diverged to the point where the germ layer containing the primary information for segmentation has been reversed.

Because the germline eventually gives rise to gametes, which have the potential to become the

next generation, it is arguably one of the most important cell types and understanding how the

germline is specified and maintained throughout an organism's lifetime is a fundamental

question in biology. This dissertation investigates the underlying mechanisms of germline

specification, maintenance, and regeneration in the amphipod crustacean Parhyale hawaiensis.

First I identified additional germline markers, Ph-piwi1, Ph-piwi2, Ph-tudor, and Ph-gustavus in

Parhyale. Then I characterized the Parhyale adult germline stem cell (GSC) niche by using a

combination of molecular markers (for germline and stem cell niche components) and using cell

proliferation assays. I generated an antibody to Parhyale Vasa, which was instrumental in

confidently identifying germline cells. In the Parhyale adult ovaries, this GSC niche likely

resides within the germline ridge. Understanding where the putative GSC niche is important for

evolutionary comparisons and can give clues as to how germline regeneration occurs. Finally, I

investigate the potential role of bone morphogenetic protein (BMP) signaling in Parhyale

germline maintenance and regeneration. This study provides insight into germline specification,

the GSC niche, and adult regeneration in the amphipod crustacean, Parhyale hawaiensis.

Pause for a moment, close your eyes, and picture a few of the most beautiful living organisms that come to your mind... What did you see? Perhaps the creatures were bright and shiny. Perhaps they were colorful and charismatic. Perhaps one of the beautiful creatures you pictured was a butterfly. Indeed, the diversity of colorful patterns in butterflies have captivated humans for centuries. Moreover, their wings have influenced studies in a variety of scientific fields, including evolutionary biology, ecology, and biophysics.Lepidopteran wings are covered with thousands of flat overlapping scales, each one of which derives from a single cell. The scales on an adult are cuticular projections that serve as the unit of color for the wing. Each scale can generate color through pigmentation, which results from molecules that selectively absorb certain wavelengths, or due to light interacting with physical nanoarchitecture on the scales, known as structural color. Thus far, researchers have made progress in understanding genetic pathways responsible for pigment production and the early transcription factors and signaling molecules that demarcate wing pattern positions. It remains less clear, however, what precise genes and pathways give rise to an individual wing scale cell, how such a novel cell type evolved, or what factors modulate cuticular micro- and nanostructures that generate specific optical properties. To better understand processes underlying scale and nanostructure development in Lepidoptera, my dissertation focuses on a unique optical strategy: wing transparency. The wings of butterflies and moths are typically covered with thousands of flat, overlapping scales that endow the wings with colorful patterns. Yet, numerous species of Lepidoptera have evolved highly transparent wings, which often possess scales of altered morphology and reduced size, and the presence of membrane surface nanostructures that dramatically reduce reflection. This trait has been interpreted as an adaptation in the context of camouflage, in which numerous lineages independently evolved transparent wings as a form of crypsis to reduce predation. In order to unravel the biological processes of wing transparency, I engaged in an interdisciplinary collaboration (including the labs of Nipam Patel, Marianne Elias, Doris Gomez and Serge Berthier) at the interface of physics, developmental biology and evolutionary ecology. Working in parallel with our collaborators, we aimed to investigate the structure, development and evolution of wing transparency in butterflies and moths by implementing experimental and phylogenetic comparative methods. We revealed a diversity of structural features that underlie transparent wings, notably modifications of scale morphology, size, and density, and the presence of finely-tuned nanostructures on the surface of the wing membrane that generate anti-reflective properties. We were able to characterize developmental processes of wing micro- and nanostructure formation of glasswing butterflies that were raised in the field and in the lab, and additionally utilized museum specimens and data to identify correlations between light transmission (a quantitative measure of transparency) and structural features. Together, our results provide insight into the development, ecology and evolutionary history of terrestrial transparency within Lepidoptera, highlighting multiple lineages that have independently evolved clearwing phenotypes, as well as potential trade-offs related to thermoregulation, water repellency and predation pressure. One of my main experimental systems became the so-called ‘glasswing butterfly’ Greta oto, which has thin, vertically oriented scales and nanopillars coating the wing membrane that enable omnidirectional anti-reflective properties. My collaborators and I employed a multitude of techniques, including confocal and electron microscopy, GC-MS, optical spectroscopy and analytical simulations to characterize wing development, comparing transparent and non-transparent wing regions. We found that during early wing development, scale precursor cell density was reduced in transparent regions, and cytoskeletal organization during scale growth differed between thin, bristle-like scale morphologies within transparent regions and flat, round scale morphologies within opaque regions. We also show that nanostructures on the wing membrane surface are composed of two layers: a lower layer of regularly arranged nipple-like nanostructures, and an upper layer of irregularly arranged wax-based nanopillars composed predominantly of long-chain n-alkanes. By chemically removing wax-based nanopillars, along with optical spectroscopy and analytical simulations, we demonstrate their role in generating anti-reflective properties. These findings provide insight into morphogenesis and composition of naturally organized microstructures and nanostructures, and may provide bioinspiration for new anti-reflective materials. Additionally, I undertook a comparative transcriptomic analysis to identify molecular pathways involved in scale cell development in the buckeye butterfly Junonia coenia and the giant silkmoth Antheraea polyphemus. I also investigated differential expression between two regions within the wing of A. polyphemus: a region we refer to as a transparent ‘window’ in which scale cells do not develop, and an adjacent region that undergoes canonical scale development. I then applied fluorescent in situ hybridization and CRISPR/Cas9 induced knockouts to characterize the spatiotemporal expression and function of genes involved in scale cell development. Comparative RNA-seq between J. coenia and A. polyphemus uncovered genes with similar expression levels during early pupal wing development and scale precursor differentiation, including proneural, cell cycle, and Notch signaling factors. At later pupal stages, when scale cell projections are forming and maturing, I identified genes with similar expression levels related to cytoskeletal organization, melanization, cuticle formation, and chitin-synthesis. Using stage-specific transcriptomic analysis followed by in situ hybridization, I uncover a suite of genes that likely play conserved roles in scale cell patterning and morphogenesis in butterflies and moths. I identified two achaete scute homologs (ASH1, ASH2) expressed at the scale cell precursor stage and loss of function of ASH2 resulted in the loss of scale cells. In contrast, loss of function of the Notch receptor led to overproduction and dense clusters of scale cells, likely due to improper lateral inhibition during scale precursor cell differentiation. I also identified that the ‘window’ scaleless region in A. polyphemus is associated with high expression levels of Wnt ligands, including wingless, and the bHLH transcription factor hairy, a negative regulator of sensory bristles, revealing how putative co-option of neurogenesis regulatory factors could contribute to scale cell patterning in Lepidoptera. Finally, I lay out a new and easy-to-follow protocol for portable, rapid, field-deployable amplicon sequencing through the use of new miniaturized lab equipment, which can be beneficial for biodiversity exploration and educational programs. Human-mediated environmental change is depleting biodiversity faster than it can be characterized, while invasive species cause agricultural damage, threaten human health, and disrupt native habitats. Consequently, the application of effective approaches for rapid surveillance and identification of biological samples is increasingly important to inform conservation efforts. Taxonomic assignments have been greatly advanced using sequence-based applications, such as DNA barcoding, a diagnostic technique that utilizes polymerase chain reaction (PCR) and DNA sequence analysis of standardized genetic regions. However, in many biodiversity hotspots, endeavors are often hindered by a lack of genomic infrastructure and funding for biodiversity research and restrictions on the transport of biological samples. A promising development is the advent of low-cost, miniaturized scientific equipment. Such tools can be assembled into functional laboratories to carry out genetic analyses in situ, at local institutions, field stations, or classrooms.

How animals develop the complex structures and morphologies that result in diversification and speciation is a fundamental question in evolutionary biology that can be addressed through studies of embryogenesis. Gastrulation is an early morphogenetic event that establishes the embryonic tissue layers that will give rise to the various organs and characteristics of the adult. Cells and their interactions are the basis of this process. Outside of Drosophila melanogaster, little is known about the behavior of cells during gastrulation in arthropods.

This dissertation focuses on cell behavior during gastrulation in the amphipod crustacean Parhyale hawaiensis. Using a variety of techniques, we make novel observations about cell shape change, the autonomy of cell behavior, and investigate possible molecular regulators of the entire process. I found cell shape change in the rosette that confirms previous hypotheses suggesting that gastrulation in Parhyale proceeds through ingression or invagination. I also found that the rosette and epithelial sheet act autonomously, and that the descendants of different lineages within the rosette are able to internalize independently. Using drug inhibition, I identified rho-kinase as important to epithelial integrity during gastrulation, and discovered a phenotype that may be associated with abnormal cell division. Finally, I investigated several candidate genes in an effort to discover a molecular marker for early mesoderm and ectoderm fate. This study provides valuable information about the process of cell internalization, cell autonomy during gastrulation, and suggests potential highly conserved mechanisms for cell behavior during Parhyale gastrulation.

The coloration of butterflies and moths, Lepidoptera, has been an important force in biological inquiry, providing among the first supporting evidence for biogeography, Darwinian evolution, and models of morphogen diffusion. In nature, color patterns have evolved that aid species’ navigation of many ecological interactions via crypsis, warning coloration, mate signaling, and the multiple forms of mimicry, which often lean heavily upon color to achieve their effect.

Butterflies and moths as a whole have evolved the ability to produce all of the colors visible by humans, as well as into the UV range. As is true for most animals, the repertoire of pigments available for use in Lepidoptera is actually rather restricted – by and large limited to long-wavelength colors red, orange, and yellow, as well as, black and brown pigments. To expand into the short wavelength (violet, blue and green), Leps have repeatedly resorted to manufacturing photonically-active nanostructures. These harness physical properties of light to create the impression of color in an observer without having to manufacture a pigment. Despite knowledge of butterfly structural coloration for centuries, intense study has only taken off following the advent of the electron microscope, and despite interest, studies have been largely limited to descriptive studies and physical estimations of their function.

I have undertaken efforts to understand the developmental and cellular underpinnings of structural coloration in butterflies. In the work presented here I have furthered the understanding of the field with a particular focus on how pigments modulate the diverse structural colors of 2 genera – the Morpho genus of the neotropics and the Achillides sub-genus of Papilio found throughout Oceania, East, and South Asia. In addition, I have addressed how scale ultrastructure is constructed in the developing pupa from a cell biological perspective. These studies have come hand-in-hand with the improvement of live-imaging techniques, which I argue, will be indispensable for future studies on scale development. What has emerged, is the suggestion that the Actin cytoskeleton, is essential for ultrastructural formation of scales including the modulation of nanostructure profiles. What’s more, I have shown that melanin is deployed to tune the saturation of structural color reflections and, in at least one case, to tune the hue of a multilayer-based structural color.

The diversity of animals is a topic of childlike fascination, poetic wonder, and scientific discovery; but how did such great diversity of form and function come to be? To uncover the possible mechanisms by which diverse new body plans are generated over evolutionary time, we turn to understanding the patterns and processes behind how individual bodies are constructed — and deviated — during embryonic development. Critically important to the specification of body plan diversity is the patterning of the anterior-posterior axis and establishment of regional identities by the Hox genes.

Hox genes have a revolutionizing history, complete with striking transformation of form upon their mutation. The rapid discovery of orthologous sets of Hox genes in a diversity of species led to the exciting realization that not only are individual Hox genes conserved across Bilateria, so too are their patterns of expression, mutation, arrangement, and properties in aggregate—changes to which have been implicated as important generators of evolutionary diversification. This apparent conservation enabled many useful generalizations to be gleaned from a limited number of representative species, and paradigms such as posterior prevalence became widespread. Increasingly, however, new technologies enable us to expand the scope of our study to a wider array of species and cases breaking the “rules” of these models are accumulating. This warrants the comprehensive study of Hox genes in aggregate in a greater number of representative species to resolve a fuller picture of the Hox complex and its interactions during the specification of regionalized bodies and the diversification of specialized forms.

By resolving Hox expression patterns in transformed individuals following Hox gene knockout in the appendage-diverse amphipod crustacean Parhyale hawaiensis, we have revealed a number of cross-regulatory interactions among Hox genes that influence the patterning of Hox domains. Furthermore, by amassing a fuller picture of the Hox expression underlying the appendage transformations, we make a more informed model of the functional roles and interactions of the different Hox genes in the designation of limb identities. We have shown that posterior prevalence is not sufficient to predict the appendage transformations in Parhyale and that a modular ‘Hox code’ expressed in individual segments functions to establish segmental identity. We believe this study demonstrates that interactions among Hox genes provide a mechanism for the diversification of appendage morphologies and arrangements over evolutionary time.

Modification of cis-regulatory sequences is an important means by which developmental processes can evolve and generate changes in patterning and morphology. Understanding how these cis-regulatory enhancer sequences control gene expression is critical not only for understanding the mechanistic basis of development, but also for understanding how the systems are modified or evolve over time.

In this work I describe the discovery and characterization of many new enhancers that regulate genes for which a similar enhancer driving a similar expression pattern was previously known; these are "shadow" enhancers. The genes examined are critical developmental control genes responsible for the patterning and subdivision of the early Drosophila embryo. At first glance, many of these enhancers seem redundant, but are found to be evolutionarily conserved, suggesting a role in fitness and specific developmental function.

Sufficiently controlled tests of shadow enhancer function required the development and use of techniques such as bacterial artificial chromosome (BAC) recombineering and quantitative confocal imaging. Recombineering allowed the precise modification of large genomic regions and an unprecedented level of control in evaluating enhancer function when combined with the use of reporters and genetic rescue experiments. The ability to accurately quantify gene expression was aided by the development of a reporter gene containing an intron, allowing clear visualization of sites of active transcription. These tools facilitated the evaluation of shadow enhancer function.

Evidence is presented which shows that in some cases one enhancer of the shadow enhancer pair can be removed without disrupting core function of the associated gene under normal developmental conditions. The system breaks down, however, under stress such as elevated temperatures during development, but only when one enhancer is missing. These results suggest that shadow enhancers represent a novel mechanism for the canalization of gene expression in varying environmental conditions and genetic backgrounds.

It is shown that in several cases shadow enhancers help ensure complete patterns of transcriptional activation across domains of gene expression. We present a mechanistic model in which increasing the number of enhancers for a given pattern helps ensure proper transcriptional state even allowing for stochastic interruptions of underlying molecular interactions such as enhancer/promoter looping or activator/repressor binding. It is suggested that shadow enhancers play a key role in ensuring robustness and reliability of gene expression patterns in development.

We propose a model whereby cryptic duplication events lead to the birth of shadow enhancers. Such enhancers can provide immediate value for fitness, for example, by ensuring robustness in response to environmental or genetic variations. The second enhancer might facilitate the evolution of novel patterns of gene expression, or become incorporated into the core developmental machinery to produce precise and rapid patterns of gene expression.

The phylum Arthropoda displays some of the most diverse and versatile body plans and appendages. To explore this diversity, the main focus of this thesis is on members of the arthropod clade, the Pancrustacea (crustaceans and hexapods), and the role that body patterning genes, Hox genes, have played in body plan evolution and limb identity. Specifically focusing on the expression boundaries and the role of Hox genes in two orders of crustaceans, Branchiopoda and Amphipoda. In Branchiopoda, the focus was to expand our understanding of the role of the posterior most Hox gene Abdominal-B (Abd-B) in the brine shrimp, Artemia franciscana, this was done through gene expression data and functional experiments. In our gene expression data, a temporal expression pattern was observed of Abd-B, that suggests that Abd-B does play a role in the limbless nature of the abdomen A. francsicana, while our functional experiments (RNAi) preliminarily suggests that Abd-B represses limb formation. In Amphipoda, the focus was to discern the potential causes of the highly reduced and strikingly different body plan of the skeleton shrimp, Caprella mutica. The first question approached was which mechanisms are used to reduce the abdomen, by exploring apoptosis (TUNEL assay) during embryogenesis. Observations through Scanning Electron Microscopy (SEM) were made to understand the sexually dimorphic appendages on the male abdomen. Once it was observed that C. mutica reduced the abdomen through apoptosis, the next question was to understand the limb identity along the Anteroposterior axis (A/P axis). To do this a transcriptome of 4 embryonic time points of C. mutica were used to discern the Hox gene expression boundaries, visualized through in situ Hybridization Chain Reaction (HCR). These Hox gene expression boundaries were then compared to another member of Amphipoda, Parhyale hawaiensis, which has a typical amphipod body plan. This comparison was to tease out the role the Hox genes had in limb identity along the A/P axis and explore the evolutionary relationship of the Hox gene expression pattern in limb identity.