Browsing by Subject "Developmental biology"

As vertebrate embryos grow and develop into adults, their organs must acquire mass and mature tissue architecture to maintain proper homeostasis. While juvenile growth encompasses a significant portion of life, relatively little is known about how individual cells proliferate, with respect to one another, to orchestrate this final maturation. For its simplicity and ease of genetic manipulations, the teleost zebrafish (Danio rerio) was used to understand how the proliferative outputs of individual cells generate an organ from embryogenesis into adulthood.

To define the proliferative outputs of individual cells, a multicolor clonal labeling approach was taken that visualized a large number of cardiomyocyte clones within the zebrafish heart. This Brainbow technique utilizes Cre-loxP mediated recombination to assign cells upwards of ~90 unique genetic tags. These tags are comprised of the differential expression of 3 fluorescent proteins, which combine to give rise to spectrally distinct colors that represent these genetic tags. Tagging of individual cardiomyocytes was induced early in development, when the wall of the cardiac ventricle is a single myocyte thick. Single cell cardiomyocyte clones within this layer expanded laterally in a developmentally plastic manner into patches of variable shapes and sizes as animals grew into juveniles. As maturation continued into adulthood, a new lineage of cortical muscle appeared at the base of the ventricle and enveloped the ventricle in a wave of proliferation that fortified the wall to make it several myocytes thick. This outer cortical layer was formed from a small number (~8) of dominant cortical myocyte clones that originated from trabecular myocytes. These trabecular myocytes were found to gain access to the ventricular surface through rare breaches within the single cell thick ventricular wall, before proliferating over the surface of the ventricle.

These results demonstrated an unappreciated dynamic juvenile remodeling event that generated the adult ventricular wall. During adult zebrafish heart regeneration, the primary source of regenerating cardiomyocytes stems from this outer wall of muscle. Regenerating cardiomyocytes within this outer layer of muscle are specifically marked by the cardiac transcription factor gene gata4, which they continue to express as they proliferate into the wound area.

Using heart regeneration to guide investigation of juvenile cortical layer formation, we found that both processes shared similar molecular and tissue specific responses including expression and requirement of gata4. Additional markers suggested that juvenile hearts were under stress and that this stress could play a role to initiate cortical morphogenesis. Indeed, experimental injury or a physiologic increase in stress to juvenile hearts caused the ectopic appearance of cortical muscle, demonstrating that injury could trigger premature morphogenesis.

These studies detail the cardiomyocyte proliferative events that shape the heart and identify molecular parallels that exist between regeneration and cortical layer formation. They show that adult zebrafish heart regeneration utilizes an injury/stress responsive program that was first used to remodel the heart during juvenile growth.

Sex determination is a critical biological process for all sexually reproducing animals. Despite its significance, evolution has provided a vast array of mechanisms by which sexual phenotype is determined and elaborated even within amniote vertebrates. The most prevalent systems of sex determination in this clade are genetic and temperature dependent sex determination. These two systems are sometimes consistent within large groups of species, such as the mammals who nearly ubiquitously utilize XY genetic sex determination, or they can be much more mixed as in reptiles that use genetic or temperature dependent systems and even both simultaneously. The turtles are a particularly diverse group in the way they determine sex with multiple different genetic and temperature based systems having been described. We investigated the nature of the temperature based sex determination system in Trachemys scripta elegans to ascertain whether it behaved as a purely temperature based system or if some other global source of sex determining information might be apparent within thermal regions insufficient to fully induce male or female development. These experiments found that sex determination in this species is much more complex and early acting than previously thought and that each gonad within an individual has the same sexual fate established enough that it can persist even without further communication between. We established a best practice for the assembly and annotation of de novo whole transcriptomes from T. scripta RNA-seq and utilized the technique to quantify the gene regulatory events that occur across the thermal sensitive period.

Evidence is entirely lacking on the resolution of TSD when eggs are incubated at the pivitol temperature in which equal numbers or males and females are produces. We have produced a timecourse data set that allowed for the elucidation of the gene expression events that occur at both the MPT and FPT over the course of the thermal sensitive period. Our data suggests that early establishment of a male or female fate is possible when temperature is sufficiently strong enough as at MPT and FPT. We see a strong pattern of mutually antagonistic gene expression patterns emerging early and expanding over time through the end of the period of gonad plasticity. In addition, we have identified a strong pattern of differential expression in the early embryo at stages prior to the formation of the gonad. Even without the known systemic signaling attributed to sex hormones emanating from the gonad, the early embryo has a clear male and female gene expression pattern. We discuss how this early potential masculinization or feminization of the embryo may indicate that the influence of temperature may extend beyond the determination of gonadal sex or even metabolic adjustments and how this challenges the well-defined paradigm in which gonadal sex determines peripheral sexual characteristics.

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.

Pathologies of sexual development are common in humans and reflect the precarious processes of sex determination and sexual differentiation. The gonad forms as a bipotential organ, and recent results from the Capel lab revealed that it is initially balanced between testis and ovarian fates by opposing and antagonistic signaling networks. In XY embryos, this balance is disrupted by the transient expression of the Y-linked gene, Sry, which activates genes that promote the testis pathway and oppose the ovarian pathway. While the roles of a few genes have been defined by mutation, current evidence suggests that the interactions of many genes and signaling pathways are involved in the establishment of sexual fate. For example, most cases of disorders of sexual development (DSDs) are unexplained by mutations in known sex determination genes. In addition, recent microarray studies in the mouse revealed that nearly half the transcriptome is expressed in the gonad at the time of sex determination (Embryonic day 11.5, or E11.5), and as many as 1,500 genes are expressed in a sexually dimorphic pattern at this early stage. Thus the sexual fate decision in the developing gonad likely depends on a complex network of interacting factors that converge on a critical threshold.

To begin to elucidate the transcription network topology underlying sex determination, we exploited two inbred mouse strains with well-characterized differences in sex reversal. The common inbred strain C57BL/6J (B6) is uniquely sensitive to XY male-to-female sex reversal in response to a number of genetic perturbations, while other strains, including 129S1/SvImJ (129S1) and DBA/2J (D2) are resistant to sex reversal. We hypothesized that these strain differences in gonad phenotype likely result from underlying expression differences in the gonad at the critical timepoint of E11.5. Using microarrays, we identified significant, reproducible differences in the transcriptome of the E11.5 XY gonad between B6 and 129S1 indicating that the reported sensitivity of B6 to sex reversal is consistent with a higher expression of a female-like transcriptome in B6 XY gonads. Surprisingly, a well-characterized master regulator of the testis pathway, Sox9, was found to be upregulated in the sensitive B6 background, which may serve as a compensatory mechanism to counteract the female-leaning transcriptome and activate the testis pathway in wild type B6 XY gonads.

We extended our expression analysis to a large set of F2 XY gonads from B6 and 129S1 intercrosses. From each pair of gonads, we analyzed the expression of 56 sex-associated genes by nanoliter-scale quantitative RT-PCR (qRT-PCR). The expression levels of most genes were highly variable across the F2 population, yet strong correlations among genes emerged. We employed a First-Order Conditional Independence (FOCI) algorithm to estimate the F2 coexpression network. From this unbiased analysis of XY expression data, we uncovered two subnetworks consisting of primarily male and female genes. Furthermore, we predicted roles for genes of unknown function based on their connectivity and position within the network.

To identify the genes responsible for these strain expression differences, we genotyped each F2 embryo at 128 single nucleotide polymorphisms (SNPs) located evenly throughout the 19 autosomes and X chromosome. We then employed linkage analysis to detect autosomal regions that control the expression of one or more of the 56 genes in the F2 population. These regions are termed expression quantitative trait loci, or eQTLs. We identified eQTLs for many sex-related genes, including Sry and Sox9, the key regulators of male sex determination. In addition, we identified multiple prominent trans-band eQTLs that controlled the expression of many genes. My work represents the first eQTL analysis of a developing vertebrate organ, the mouse gonad. This systems-level approach revealed the complex transcription architecture underlying sex determination, and provides a mechanistic explanation for sensitivity to sex reversal seen in some individuals.

The vasculature has a crucial function in normal physiology, enabling the transport of oxygen and nutrients to cells throughout the body. In turn, endothelial cells, which form the inner-most lining of blood vessels, are key regulators of vascular function. In addition to forming a barrier which separates the circulation from underlying tissues, endothelial cells respond to diverse extracellular cues and produce a variety of biologically-active mediators in order to maintain vascular homeostasis. Disruption of normal vascular function is a prominent feature of a variety of pathological conditions. Thus, elucidating the signaling pathways regulating endothelial function is critical for understanding the role of endothelial cells in both normal physiology and pathology, as well as for potential development of therapeutic interventions.

In this dissertation, we use a combination of pharmacological inhibition and knockdown studies, along with generation of endothelial conditional knockout mice, to demonstrate an important role of the Abelson (Abl) family of non-receptor tyrosine kinases (Abl and Arg) in vascular function. Specifically, loss of endothelial expression of the Abl kinases leads to late-stage embryonic and perinatal lethality in conditional knockout mice, indicating a crucial requirement for Abl/Arg kinases in normal vascular development and function. Endothelial Abl/Arg-null embryos display focal regions of vascular loss and tissue damage, as well as increased endothelial cell apoptosis. An important pro-survival function for the Abl kinases is further supported by our finding that either microRNA-mediated Abl/Arg depletion or pharmacological inhibition of the Abl kinases increases endothelial cell susceptibility to stress-induced apoptosis in vitro. The Abl kinases are activated in response to treatment with the pro-angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). We show that both VEGF- and bFGF-mediated endothelial cell survival is impaired following Abl kinase inhibition.

These studies have uncovered a previously unappreciated role for the Abl kinases in the regulation of the angiopoietin/Tie2 signaling pathway, which functions to support endothelial cell survival and vascular stability. Loss of Abl/Arg expression leads to reduced mRNA and protein levels of the Tie2 receptor, resulting in impaired activation of intracellular signaling pathways by the Tie2 ligand angiopoietin-1 (Angpt1), as well as decreased Angpt1-mediated endothelial cell survival following serum-deprivation stress. Notably, we found that the Abl kinases are activated following Angpt1 stimulation, suggesting a unique dual role for Abl and Arg in Angpt/Tie2 signaling, potentially modulating Tie2 downstream signaling responses, as well as regulating Tie2 receptor expression.

Further, we show an important contribution of the Abl family kinases to the regulation of endothelial permeability responses both in vitro and in vivo. The Abl kinases are activated in response to a diverse group of permeability-inducing factors, including VEGF and the inflammatory mediators thrombin and histamine. We show that inhibition of Abl kinase activity, using either the ATP-competitive inhibitor imatinib or the allosteric inhibitor GNF-2, protects against disruption of endothelial barrier function by the permeability-inducing factors in vitro. VEGF-induced vascular permeability similarly is decreased in conditional knockout mice lacking endothelial Abl expression, as well as following treatment with Abl kinase inhibitors in vivo. Mechanistically, we show that loss of Abl kinase activity is accompanied by activation of the barrier-stabilizing GTPases (guanosine triphosphatases) Rac1 and Rap1, as well as inhibition of agonist-induced Ca2+ mobilization and generation of acto-myosin contractility.

Taken together, these results demonstrate involvement of the Abl family kinases in the regulation of endothelial cell responses to a broad range of pro-angiogenic and permeability-inducing factors, as well as a critical requirement for the endothelial Abl kinases in normal vascular development and function in vivo. These findings have implications for the clinical use of Abl kinase inhibitors.

Bone development and homeostasis are governed by a number of developmental signals, transcription factors and cellular metabolism. This process is also dependent on the orchestration of multiple cell types including osteoblasts, chondrocytes, skeletal stem cells and osteoclasts. Osteoblasts are the principal bone forming cells responsible for producing and secreting the type I collagen rich extracellular bone matrix. Protein synthesis is an energetically and biosynthetically demanding process. This requires copious amounts of ATP and amino acids amongst other metabolites. However, the precise mechanisms and systems that osteoblasts utilize to meet these synthetic demands are poorly understood. Previous studies have shown amino acid consumption is increased in osteoblasts during differentiation. This process is regulated by transcription factors ATF4 and FOXO. Additionally, osteogenic signals like WNT and PTH can stimulate amino acid uptake. For example, WNT signaling can rapidly stimulate glutamine uptake and metabolism required for osteoblast differentiation. Unfortunately, transporters mediating glutamine uptake in osteoblasts are unknown. Moreover, the mechanism by which WNT stimulates increased glutamine consumption is also unknown. We identified two amino acid transporters, Slc7a7 and Slc1a5, as the primary glutamine transporters in response to WNT. Slc7a7 is responsible for the rapid WNT-induced glutamine uptake via the -catenin dependent pathway. Conversely, Slc1a5 sustains basal glutamine uptake, which is regulated by ATF4 downstream of the mTORC1 pathway. In summary, these data demonstrate the biphasic role of WNT signaling in regulating glutamine consumption, by two amino acid transporters Slc7a7 and Slc1a5, during osteoblast differentiation. While we have shown the importance of glutamine in bone cells, the role of other amino acids is not clear. Proline has long been considered as a critical amino acid due to its enrichment in collagens. Furthermore, PTH stimulates proline consumption in osteoblasts. The transport of proline is characterized by its dependency on sodium and sensitivity to MEAIB. However, the precise transport system responsible for proline import is not known. Here we identified the amino acid transporter Slc38a2, which encodes SNAT2, as the primary proline transporter in osteoblasts. Deletion of Slc38a2 results in defects in both intramembranous and endochondral ossifications. The phenotype is associated with defective osteoblast differentiation highlighted by reduction of proline enriched proteins (e.g. RUNX2, OSX and COL1A1). Slc38a2 provides proline to support osteoblast differentiation through two mechanisms. First, majority of proline is directly incorporated into proteins and does not contribute to amino acid biosynthesis. Second, proline oxidation regulates bioenergetics required for osteoblast differentiation. These findings highlight the multifaceted functions of proline, which is provided by Slc38a2, in osteoblast differentiation and bone formation. Collectively, my work demonstrates the critical role of amino acid transporters in osteoblast differentiation and provides novel insights in their potential applications in treatments of bone diseases like osteoporosis and bone fracture.

Mutations in myosin VIIa (MyoVIIa), an unconventional myosin, have been shown to cause Usher Syndrome Type 1B in humans. Usher Syndrome Type 1B is characterized by congenital sensorineural deafness, vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. Mouse model studies show that sensorineural deafness and vestibular dysfunction in MyoVIIa mutants is caused by disruption in the structure of microvilli-like projections (stereocilia) of hair cells in the cochlea and vestibular organ. MyoVIIa has also been shown to affect adaptation of mechanoelectrical transduction channels in stereocilia.

In Drosophila melanogaster mutations in MyoVIIa encoded by crinkled (ck) cause defects in hair and bristle morphogenesis and deafness. Here we study the formation of bristles and hairs in Drosophila melanogaster to investigate the molecular basis of ck/MyoVIIa function and its regulation. We use live time-lapse confocal microscopy and genetic manipulations to investigate the requirement of ck/MyoVIIa function in various steps of morphogenesis of hairs and bristles. Here we show that null or near null mutations in ck/MyoVIIa lead to the formation of 8-10 short and thin hairs (split hairs) per epithelial cell that are likely the result of the failure of association of hair-actin bundles that in wild-type cells come together to form a single hair.

The myosin super family of motor proteins is divided into 17 classes by virtue of differences in the sequence of their motor domain, which presumably affect their physiological functions. In addition, substantial variety in the overall structure of their tail plays an important role in the differential regulation of myosin function. In this study we show that ck/MyoVIIa, that has two MyTH4 FERM domains in its tail separated by an SH3 domain, requires both MyTH4 FERM repeats for efficient association of hair-actin bundles to form hairs. We also show that the "multiple hair" phenotype of over-expression of ck/MyoVIIa requires both MyTH4 FERM domain function but not the tail-SH3 domain. We further demonstrate that the tail-SH3 domain of ck/MyoVIIa plays a role in keeping actin bundles, which run parallel to the length of the growing bristle, separate from each other. Our data also suggests that the tail-SH3 domain plays a role in the association of the actin filament bundles with the membrane and regulates F-actin levels in bristles.

We further demonstrate that over-expression of Quail (villin) can rescue the hair elongation defects seen in ck/MyoVIIa null or near null mutants but does not rescue the split hair defects. We show that over-expression of Alpha-actinin-GFP, another actin bundling protein, phenocopies the multiple hair phenotype of ck/MyoVIIa over-expression. Over-expression of Alpha-actinin-GFP in a ck/MyoVIIa null or near null background shows that Alpha-actinin-GFP cannot rescue the split or short hair phenotype of ck/MyoVIIa loss-of-function. However, cells over-expressing Alpha-actinin-GFP in a ck/MyoVIIa null or near null background have more than the normal 8-10 split hairs, suggesting that Alpha-actinin-GFP over-expression causes the formation of more than the normal complement of hair-actin bundles per cell, resulting in a multiple hair phenotype. We show that Twinfilin, an actin monomer sequestering protein implicated in negatively regulating F-actin bundle elongation in stereocilia in a MyoVIIa-dependent manner, is required for F-actin bundle stability.

In addition, we use yeast two-hybrid strategies to identify Slam as a protein that directly binds to ck/MyoVIIa. We show that Slam, a novel membrane-associated protein, likely functions to regulate ck/MyoVIIa function during hair and bristle morphogenesis. We show that over-expression of Slam and loss-of-function mutations in Slam phenocopy ck/MyoVIIa loss-of-function split and short hair phenotype. We also show that disruption of Slam and RhoGEF2 association causes split hair defects similar to ck/MyoVIIa loss-of-function phenotype suggesting that Slam probably regulates ck/MyoVIIa function via RhoGEF2.

Together our results show that ck/MyoVIIa plays an important role in regulating the actin cytoskeleton that underlies actin-based cellular protrusions like hairs and bristles.

Endopolyploid cells (hereafter - polyploid cells), which contain whole genome duplications in an otherwise diploid organism, play vital roles in development and physiology of diverse organs such as our heart and liver. Polyploidy is also observed with high frequency in many tumors, and division of such cells frequently creates aneuploidy (chromosomal imbalances), a hallmark of cancer. Despite its frequent occurrence and association with aneuploidy, little is known about the specific role that polyploidy plays in diverse contexts. Using a new model tissue, the Drosophila rectal papilla, we sought to uncover connections between polyploidy and aneuploidy during organ development. Our lab previously discovered that the papillar cells of the Drosophila hindgut undergo developmentally programmed polyploid cell divisions, and that these polyploid cell divisions are highly error-prone. Time-lapse studies of polyploid mitosis revealed that the papillar cells undergo a high percentage of tripolar anaphase, which causes extreme aneuploidy. Despite this massive chromosome imbalance, we found the tripolar daughter cells are viable and support normal organ development and function, suggesting acquiring extra genome sets enables a cell to tolerate the genomic alterations incurred by aneuploidy. We further extended these findings by seeking mechanisms by which the papillar cells tolerated this resultant aneuploidy.

Basement membranes (BMs) are thin, dense sheets of extracellular matrix that surround most animal tissues and provide structural support. While the role of BMs in the structural support of tissues is well established, how these matrices can structurally support tissues while accommodating dynamic tissue function is not well understood. Using C. elegans, a powerful model organism that allows for live imaging, genetic analysis, and rapid screening, I was able to utilize endogenous knock-in fluorescent proteins, conditional RNAi, optogenetics, and quantitative live imaging to investigate how BM components contribute to the BM’s ability to withstand mechanical load in various circumstances. In Chapter 1, I discuss the known roles of BM, introduce BM proteins of interest, explore gaps in our understanding of BM’s function in withstanding mechanical force, and expand upon the utility of C. elegans as a model system to investigate these questions. In Chapter 2, I show that BM-to-BM linkages can function to resist the mechanical forces involved in egg-laying. In Chapter 3, I explore how BM stretches to accommodate dynamic tissue movement. In Chapter 4, I discuss future directions and the implications of these findings and in Chapter 5 I summarize my conclusions.

The Arabidopsis root apical meristem (RAM) is a complex tissue capable of generating all the cell types that ultimately make up the root. The work presented in this thesis takes advantage of the versatility of high-throughput sequencing to address two independent questions about the root meristem. Although a lot of information is known regarding the cell fate decisions that occur at the RAM, cortex specification and differentiation remain poorly understood. In the first part of this thesis, I used an ethylmethanesulfonate (EMS) mutagenized marker line to perform a forward genetics screen. The goal of this screen was to identify novel genes involved in the specification and differentiation of the cortex tissue. Mapping analysis from the results obtained in this screen revealed a new allele of BRASSINOSTEROID4 with abnormal marker expression in the cortex tissue. Although this allele proved to be non-cortex specific, this project highlights new technology that allows mapping of EMS-generated mutations without the need to map-cross or back-cross. In the second part of this thesis, using fluorescence activated cell sorting (FACS) coupled with high throughput sequencing, my collaborators and I generated single-base resolution whole genome DNA methylomes, mRNA transcriptomes, and smallRNA transcriptomes for six different populations of cell types in the Arabidopsis root meristem. We were able to discover that the columella is hypermethylated in the CHH context within transposable elements. This hypermethylation is accompanied by upregulation of the RNA-dependent DNA methylation pathway (RdDM), including higher levels of 24-nt silencing RNAs (siRNAs). In summary, our studies demonstrate the versatility of high-throughput sequencing as a method for identifying single mutations or to perform complex comparative genomic analyses.

The mouse embryonic gonad provides an outstanding model to study the complex mechanisms involved in cell fate specification and maintenance. At the bipotential stage, both XX and XY gonads are capable of becoming testes or ovaries upon specific molecular cues. The specification of the supporting cell lineage (as either Sertoli cells in the male or granulosa cells in the female) initiates the testis or ovary program, leading to male or female fate. However, there are significant gaps in our understanding of how the somatic cells in the gonad arise, are competent to differentiate, and determine and maintain their fates. In this dissertation, we addressed these questions.

We found that NUMB (an antagonist of Notch signaling) serves as competence factor for somatic cell differentiation during early gonadogenesis. The asymmetric allocation of NUMB to the basolateral domain of actively dividing coelomic epithelial (CE) cells is indispensable to (1) maintain the totipotent stem cell-like reservoir at the CE domain, and (2) give rise to progenitor cells that can further differentiate into supporting and interstitial cell lineages. Deletion of Numb; Numbl resulted in disruption of cell polarity in the CE domain as well as a reduction of multiple differentiated cell lineages within XX and XY gonads, including supporting cells and male steroidogenic cells, which were most severely affected. We detected elevated Notch downstream signaling in the Numb; Numbl mutant gonads. Moreover, treatment of DAPT (which blocks Notch signaling) rescued the Numb; Numbl mutant phenotypes, strongly suggesting that upregulation of Notch is responsible.

Previous experiments indicate that when supporting cells commit to the male (Sertoli) fate, they must repress the alternative female (granulosa) cell fate. In another line of experiments, we investigated the hypothesis that the Polycomb repressive complex (PRC1) plays a critical role in repressing the female pathway during male gonad patterning. We found that loss of Ring1B (a component of PRC1) led to the disruption of XY gonad development specific to the posterior region of male gonads. Sry, the upstream driver of the male pathway, was not appropriately expressed in the posterior domain, which contained cells expressing female markers and, in some cases, small aggregates of undifferentiated cells. Using ChIP-Seq, we identified potential targets of PRC during male gonad development. Moreover, a key gene in the male pathway, SOX9, interacts with Ring1B, based on immunoprecipitation results, leading to the hypothesis that it may be involved in the recruitment of PRC to its target sites to execute the repression of female genes in male gonads.

Our findings provide insight into how somatic cell fate is determined and maintained during mammalian sex determination. Our results may be valuable for patients with disorders of sexual development with unidentified genetic contributions.

The redundancy inherent in the genetic code allows for multiple codons to encode for a single amino acid. These synonymous codons are used at different frequencies in the genome, with some being used more frequently than others. This phenomenon is known as codon bias. Although codon bias was not originally thought to affect gene expression, we now know that it impacts nearly every step of the central dogma. Previous work identified two Drosophila tissues that express genes enriched in rarely used codons: the larval brain and adult testes. Both tissues contain numerous stem cells which divide many times to produce specialized progeny. In my thesis work, I investigated codon bias regulation in these tissues at the cellular level. In the brain, I found that neural stem cells express very little protein from a rare-codon enriched reporter, while their progeny, the neurons, have high protein expression from the rare-codon enriched reporter. Next, through a targeted genetic screen, I identified a regulator of this response in neurons: the RNA binding protein Orb2. Orb2 stabilizes mRNA from the rare-codon-enriched reporter specifically in neurons, but not in neural progenitors. I then used RNA sequencing to identify mGluR as an endogenous rare codon-enriched gene that requires Orb2 for high mRNA expression and function in social activity. My work suggests that the convergence of rare codons and Orb2 regulation in an mRNA allows precise tuning of gene expression in neurons. Further work during my thesis revealed stage-specific regulation of codon usage during spermatogenesis, and pinpointed specific testis tRNA regulation that likely underlies tissue-specific codon regulation. My work has revealed that codon usage unlocks another layer of gene expression regulation, which is leveraged by the cell to direct temporal and spatial expression regulation during development and function of specialized cell types.

Multicellular organisms can be made up of hundreds of different cell types, each with their own unique morphology and characteristics to carry out their specific functions. For over 100 years, the sea urchin embryo has been used as a model to examine how cell types are specified during embryonic development. In each cell type of the embryo, transcription factors and signaling molecules must interact to form a gene regulatory network (GRN) which controls cell differentiation. When developmental GRNs are revealed, they provide insights into the stepwise mechanisms of how cells in the embryo differentiate from a multipotent progenitor to a fully differentiated specialized cell type. To understand the evolutionary history of specialized cells, GRNs that control specification of cell types in one species are compared to GRNs of similar cell types in other species. These comparisons provide insights into how ancestral cell types were changed during animal evolution to give rise to specialized cells in extant species. In this thesis, we use a combination of gene expression and perturbation assays to dissect the molecular mechanisms of cell type specification focusing on the sea urchin nervous and digestive system. We then infer evolutionary conclusions about when these cell types and the mechanisms of their differentiation evolved in metazoans.

In Chapter 3, we build a foundation to study how neural cells are specified and evolved in the nervous system by analyzing spatial and temporal gene expression during sea urchin neurogenesis. We report the expression of 23 genes expressed in areas of active neurogenesis in the sea urchin embryo from blastula stage (just before neural progenitors begin their specification sequence) through pluteus larval stage (when much of the nervous system has been patterned and is functional). Though this chapter is largely descriptive, it is essential to better understand what molecules and transcription factors are required for proper neural development in a basal deuterostome, the sea urchin. The expression patterns can be used as a starting point to 1) identify how subtypes of neurons are specified in the embryo 2) build a spatial gene regulatory network for sea urchin neurogenesis, and 3) perform comparative studies with the sea urchin, protostome and vertebrate organisms.

In Chapter 4, we build off the information found in Chapter 3 to examine the molecular mechanisms of neuronal subtype specification in three distinct neural subtypes in the Lytechinus variegatus larva. We show that these subtypes are specified through Delta/Notch signaling and identify a different transcription factor required for the development of each neural subtype. Our results show achaete-scute and neurogenin are proneural for the serotonergic neurons of the apical organ and cholinergic neurons of the ciliary band, respectively. We also show that orthopedia is not proneural but is necessary for the differentiation of the cholinergic/catecholaminergic postoral neurons. Interestingly, these transcription factors are used similarly during vertebrate neurogenesis. We believe the results in this chapter are a starting point for building a neural gene regulatory network in the sea urchin and for finding conserved deuterostome neurogenic mechanisms (Slota and McClay, 2018).

In Chapter 5, we focus on a neuronal cell type found in Chapter 4 to examine the evolutionary origin of the neural crest cell, a transient embryonic stem cell population unique to vertebrates. The mechanism of neural crest evolution has perplexed biologists since its discovery in the 1860s (Huang, 2004). The emergence of this cell type was critical for vertebrate evolution since it gives rise to tissues in the embryo required for complex predatory behaviors such as connective tissues of the head and neck and peripheral sensory neurons (Gans and Northcutt, 1983). In the last decade, two embryonic cell types in the tunicate Ciona intestinalis, have been proposed to be rudimentary neural crest cell types (Abitua et al., 2012; Stolfi et al., 2015). In this chapter, we show that a population of neurons in Lytechinus variegatus, which is a basal deuterostome, shares features with the neural crest-derived spinal neurons and C. intestinalis bipolar tail neurons. Like the neural crest, this cell type arises from the lateral borders of the neuroectoderm, expresses the transcription factor neurogenin and the acid sensing ion channel gene asicl, undergoes migration, requires MAPK signaling for its specification, and gives rise to afferent neurons in the peripheral nervous system. We believe this is an ancient cell type that is homologous to Ciona bipolar tail neurons and therefore the neural crest. We propose that this cell type existed before the split of chordates and the clade that includes sea urchins and acquired a multipotency gene regulatory program in the vertebrate lineage to give rise to the neural crest.

In Chapter 6, we shift focus to cell type specification in the sea urchin digestive system. We find that molecular inputs from tissues outside the gut provide inductive signals that contribute to cell type specification and anterior/posterior patterning of the developing gut. We show that the Wnt signaling ligand, wnt1, which is expressed in a ring of expression surrounding the developing blastopore, provides an inductive signal to the developing endoderm. In Wnt1 knockdown embryos, gastrulation occurs normally but anterior/posterior pattern of gene expression and regionalized cell type specification is lost in the developing mid and hindgut. Wnt1 knockdown results in a loss of transcription factor expression in the hindgut and anus including cdx, foxd, foxi and phb1. Furthermore, wnt1 knockdown results in loss of expression of the pyloric sphincter markers lox and nkx6.1 and the midgut marker gaba transporter (gat). When wnt1 RNA is ectopically expressed, ectoderm is then fated to become endoderm and the embryo becomes a large tripartite gut with an expansion of hindgut and midgut markers. Using a live imaging digestion assay, we then show the consequences to the organism when this gene expression pattern in the gut is lost, namely that larva cannot properly hold food in their gut. We propose that the inductive capabilities of wnt1 is ancient to metazoans and that another signal, possibly a different Wnt ligand, is the activating signal for regional cell type identity in the digestive system in the vertebrate lineage.

The sea urchin embryo has been used for decades for building developmental GRNs that control the separation of germ layers and for specification of cell types in the early embryo. At later developmental stages however, particularly after gastrulation is complete, little work has been done to build GRNs for specialized cell types required for the complex behavior of the larva. Identifying cell types in the sea urchin, understanding the mechanisms that lead to their specification and differentiation and then comparing that to cell types in other species allows us to understand how cells were modified and specialized during animal evolution.

The formation of the heart is a critical part of development that, if defective, can lead to congenital malformations incompatible with life. An improved understanding of the cellular and molecular processes that build the heart is essential to elucidate the causes of congenital defects and to design appropriate therapies. Relatively little is known about how the cardiac chambers adopt distinct forms to follow their specialized functions. Here, I have used a multicolor genetic labeling system to trace the progeny of zebrafish atrial cardiomyocytes as they expand to form the mature atrial myocardium. By comparing the observed cellular dynamics to those previously mapped in the ventricle, I identified characteristics of chamber development, including wall thickening, wall composition, and internal muscle formation, that contribute to the structural divergence of the chambers. As coronary vessel formation is one such chamber-specific morphogenetic process, I then explored the effect of a chamber-specific growth factor on cardiac development and homeostasis. Using a transgenic reporter and an inducible overexpression tools, I found ectopic expression of this growth factor stimulates cardiomyocyte proliferation. However, overexpression also blocks regeneration, possibly due to the abolition of an endogenous gradient localized to the site of injury. These findings not only provide new details for how the cardiac chambers form, but also demonstrate how understanding developmental phenomena can provide insights into important concepts of regenerative medicine.

Radial glial cells (RGCs) are essential for the generation and organization of neurons in the cerebral cortex. RGCs have an elongated bipolar morphology with basal and apical endfeet that reside in distinct niches. Yet, how this subcellular compartmentalization of RGCs controls cortical development is largely unknown. Here, we employ in vivo proximity labeling, in the mouse, using unfused BirA to generate the first subcellular proteome of RGCs and uncover new principles governing local control of cortical development. We discover a cohort of proteins that are significantly enriched in RGC basal endfeet, with MYH9 and MYH10 among the most abundant. Myh9 and Myh10 transcripts also localize to endfeet with distinct temporal dynamics. Although they each encode isoforms of non-muscle myosin II heavy chain, Myh9 and Myh10 have drastically different requirements for RGC integrity. Myh9 loss from RGCs decreases branching complexity and causes endfoot protrusion through the basement membrane. In contrast, Myh10 controls endfoot adhesion, as mutants have unattached apical and basal endfeet. Finally, we show that Myh9- and Myh10-mediated regulation of RGC complexity and endfoot position non-cell autonomously controls interneuron number and organization in the marginal zone. The first part of this study demonstrates the utility of in vivo proximity labeling for dissecting local control of complex systems, and reveals new mechanisms for dictating RGC integrity and cortical architecture. In the second portion of this work, we have developed a method for purification of endfeet from the embryonic mouse brain and employed it to discover the first global transcriptome of RGC endfeet. Analysis at E15.5 revealed that the network of localized mRNAs is much more extensive than previously appreciated. There are over 3,000 transcripts localized to RGC endfeet and 870 of them are highly enriched in the endfeet compared to the cell body. These data uncovered hundreds of new genes in endfeet and also reinforced our previous findings that cytoskeletal regulators and ECM components are especially important in endfeet. Exploration of the newly discovered localized transcripts will provide valuable insights into additional RGC functions and allow us to assess potential signaling interactions between endfeet and surrounding cells. We also propose a method for subcellular gene knockdown in which we can modulate mRNA levels of a gene of interest in the cell body and endfeet independently in vivo. Through these studies we have discovered vital roles for subcellular gene regulation in RGCs and developed tools to facilitate future studies.

The means by which cells, tissues, and organisms undergo morphogenesis are variable and highly regulated, and the mechanisms that govern cellular changes in response to signaling cues are poorly understood. This study seeks to address the role of a newly characterized protein in zebrafish in translating signaling cues into physical changes within a cell.

The Cordon–bleu (Cobl) gene is widely conserved in vertebrates, with developmentally regulated axial and epithelial expression in mouse and chick embryos. In vitro, Cobl can bind monomeric actin and nucleate formation of unbranched actin filaments, while in cultured cells it can modulate the actin cytoskeleton. However, an essential role for Cobl in vivo has yet to be determined. We have identified the zebrafish cobl ortholog and have used zebrafish as a model to assess the requirements for Cobl in embryogenesis. We find that cobl shows enriched expression in ciliated epithelial tissues during zebrafish organogenesis. The utilization of antibodies developed against Cobl shows that the protein is concentrated along the apical domain of ciliated cells, in close proximity to the apical actin cap.

Reduction of cobl by antisense morpholinos reveals an essential role in embryonic morphogenesis and organ development. A requirement for Cobl was shown for the proper function of various and ciliated epithelial organs. Cobl appears to direct the elongation of motile cilia in organs such as Kupffer’s vesicle and the pronephros. In Kupffer’s vesicle, the reduction in Cobl coincides with a reduction in the amount of apical F-actin. Additionally, Cobl may play a role during gastrulation cell movements and convergence and extension morphogenesis during early embryonic development. Thus, Cobl may represent a molecular activity that couples developmental patterning signals with local intracellular cytoskeletal dynamics to support elongation of motile cilia and tissue morphogenesis.

Regeneration is a remarkable feat of developmental regrowth and patterning. The blastema is a mass of progenitor cells that enables complete regeneration of amputated salamander limbs or fish fins. Despite years of study, methodologies to identify and track blastemal cell progenies have been deficient, restricting our understanding of appendage regeneration at a cellular and molecular level. To bridge this knowledge gap, gene expression analysis, the generation of transgenic and mutant zebrafish, qualitative and quantitative analyses, morphological measurements, and chemical treatments were used to assess molecular and cellular processes involved in fin regeneration. Two main findings arose from these methods. The first provides evidence that connective tissue progenitors are rapidly organized into a scalable blueprint of lost structures, and that amputation stimulates resident cells to reset proximodistal positional information. The second identifies a fibroblast subpopulation near uninjured fin joints that contributes to the blastemal progenitor population. These findings reveal insights on cellular and molecular mechanisms of appendage regeneration and provide a basis for work exploring how cells in an adult vertebrate bone appendage coordinately rebuild a new structure.

Codon usage bias is a fundamental aspect of the genetic code. For many years synonymous mutations to a coding sequence were considered to be functionally “silent.” We now appreciate that is not the case and that synonymous codon choice can have drastic implications for gene expression and protein production. A major debate in the field remains whether codon usage bias is evolutionarily selected for to drive efficient translation in a tissue-specific manner. Here we perform an organism wide screen in Drosophila using codon modified reporters to reveal tissue-specific responses to codon usage bias. We uncover a strict limit on rare codon usage for protein expression, and this limit coincides with the rareness limit of endogenous genes in Drosophila. We find that rare codon usage near the edge of this limit is sufficient to impart tissue-specific gene expression, notably in the testis and brain. We define a new codon usage metric, the tissue-apparent Codon Adaptation Index (taCAI) to reveal a conserved enrichment in rare codons in endogenous testis genes of both flies and humans. We further demonstrate that rare codons in the evolutionarily young gene, RpL10Aa, are required for female fertility.

The roundworm C. elegans reversibly arrests larval development during starvation, but extended early-life starvation reduces reproductive success. Maternal dietary restriction (DR) buffers progeny from starvation as young larvae, preserving reproductive success. However, the developmental basis of reduced fertility following early-life starvation is unknown, and it is unclear how maternal diet modifies developmental physiology in progeny. We show here that extended starvation in first-stage (L1) larvae followed by unrestricted feeding results in a variety of developmental abnormalities in the reproductive system, including proliferative germ-cell tumors and uterine masses that express neuronal and epidermal cell-fate markers. We found that maternal DR and reduced maternal insulin/IGF signaling (IIS) increase oocyte provisioning of vitellogenin lipoprotein, reducing penetrance of starvation-induced abnormalities in progeny, including tumors. Furthermore, we show that maternal DR and reduced maternal IIS reduce IIS in progeny. daf-16/FoxO and skn-1/Nrf, transcriptional effectors of IIS, are required in progeny for maternal DR and increased vitellogenin provisioning to suppress starvation-induced abnormalities. daf-16/FoxO activity in somatic tissues is sufficient to suppress starvation-induced abnormalities, suggesting cell-nonautonomous regulation of reproductive system development. This work reveals that early-life starvation compromises reproductive development and that vitellogenin-mediated intergenerational insulin/IGF-to-insulin/IGF signaling mediates adaptation to nutrient availability. Using our SIA model, we go on to show that early-life starvation persistently activates PQM-1/SALL2 with pervasive effects on adult gene expression, including prominent effects on membrane biology. Early-life starvation increases fatty-acid synthetase fasn-1/FASN expression in pqm-1/SALL2-dependent fashion, and both genes promote SIA. Lipidomic analysis implicates phosphatidylcholine, and unsaturated phosphatidylcholine supplementation suppresses SIA. The fatty-acid desaturases fat-1 and fat-4 inhibit and promote SIA, respectively, revealing a role of arachidonic acid-containing phosphatidylcholine, the Lands cycle, and eicosanoid signaling. Indeed, fat-4 increases eicosanoid levels in adults subjected to early-life starvation, and N-acetylcysteine treatment suppresses SIA. This work shows that early-life starvation and IIS converge on PQM-1/SALL2 to affect adult lipid metabolism and eicosanoid signaling, affecting stem-cell proliferation and tumor formation.