Abstract
Regulation of Vertebrate Ladybird Genes
Development of the vertebrate central nervous system is a complex process that relies on the accurate spatiotemporal distribution of signaling centers during embryogenesis. These signals provide cells with positional information, which is integrated via transcription factors and gene regulatory elements to generate a specific downstream gene expression profile that confers specific cellular functions. It is of interest to determine how cells acquire their unique spatiotemporal gene expression patterns. The wide variety of expression profiles established along the dorsoventral axis of the neural tube provides a great system to address this question. Recent advances in zebrafish transgenic technology, along with the phenomenon of a fish-specific genome duplication event, have been exploited here to provide an efficient way of identifying and characterizing gene regulatory elements. An identified neuronal-specific enhancer near the ladybird locus has been incorporated into a transgenic zebrafish strain driving fluorescent reporter protein expression in a subset of dorsal interneurons.
Genetic Variants Among G-Protein-Coupled Receptors and Implications for Cardiovascular Disease
Single-nucleotide polymorphisms, particularly nonsynonymous variants, provide opportunities for deciphering pathophysiological protein influence. An ideal group for realizing these opportunities is the G protein–coupled receptor (GPCR) superfamily. Principles from rhodopsin are applied to receptors for thromboxane (hTP) and prostacyclin (hIP), which play opposing cardiovascular roles. We report the identification and characterization of naturally occurring genetic variants in these receptors. Initially, we analyzed zinc interactions with rhodopsin and Retinitis pigmentosa mutations. These studies identified two conserved zinc-binding motifs mediating receptor destabilization. Site-directed mutagenesis confirmed disruption of native histidine interactions by zinc- binding. Chelation reduced the destabilizing influence of these sites in both wild-type and a common Retinitis pigmentosa mutation. Based on rhodopsin zinc-coordination, we identified similar hTP sites, but not hIP. Saturation binding demonstrates disruption of hTP, but not hIP, with a clear dose–response effect. We sequenced variants from ∼1000 cardiovascular patients. From 33 variants, we focused on 6 within the ligand-binding pocket, the ligand recognition domain, and the G-protein coupling region. Transmembrane variants (V 80 E and A 160 T) altered binding, with V 80 E decreasing affinity, and A 160 T increasing affinity. Other variants (C 68 S, E 94 V, V 176 E, and V 217 I) produced nonsignificant dysfunctions. Altered activation was observed, highlighting agonist and antagonist differences. Finally, mutational hot spots observed within these receptors, where mutations preferentially occur, were investigated. We searched for nucleotide patterns in regions flanking the variants. From 12 million human single-nucleotide polymorphisms, significant flanking motifs included CpG-island transition contributions, similar R- and K-type motifs (A/G and G/T, respectively), and an absence of immediately preceding nucleotide influence on S-type variants (C/G). Extending this to other species, we find that some of these patterns are conserved across species (human, chimpanzee, opossum, mouse, honey bee, bovine, canine, zebrafish, chicken, and rice). This analysis offers the potential to prospectively define sites prone to specific nucleotide alterations. Our studies identified and characterized naturally occurring genetic variants in counterbalancing prostanoid receptors associated with cardiovascular disease. We developed a semiautomated system for predicting and testing the structural influence of genetic variants in GPCRs. Collectively, our findings provide useful experimental tools to streamline the interpretation of novel GPCR variants that may aid in treatments for cardiovascular disease and a plethora of other human disorders.
Revealing New Roles for Sterol-Sensing Domain Proteins Dispatched 1 and Npc1 in Zebrafish Development and Disease
The vertebrate head skeleton is primarily derived from cranial neural crest cells (CNCC). The loss of Hedgehog (Hh) signaling in the vertebrate embryo causes severe craniofacial defects, in part due to defects in CNCC-derived tissues. Craniofacial defects are characteristic of the Hh-associated spectral disorder holoprosencephaly in humans. The Hh signaling pathway is conserved in zebrafish and inactivating the pathway in mutant embryos leads to severe skeletal loss, yet the direct requirements for Hh signaling in zebrafish craniofacial development are largely unknown. Dispatched 1 (Disp1) is a sterol sensing domain (SSD) protein that positively regulates the Hh-signaling pathway. SSD family members are involved in the intracellular movements of cholesterol and cholesterol-linked molecules and Disp1 primary action is to release cholesterol-linked Hh ligands from their site of synthesis. In this dissertation, I show that chameleon mutant zebrafish, lacking a functional Disp1, exhibit severe cranial cartilage and muscle defects and that their postmigratory CNCC are defective in both patterning and differentiation. By inhibiting Hh signaling at different developmental stages, I found that Hh signaling is required during gastrulation and the late pharyngula stage to promote proper CNCC development. By designing a Gli reporter transgenic fish to determine Hh-responding cells, I determined that the Hh requirement for CNCC development is likely influencing the surrounding epithelium cells that interact with CNCC mesenchyme. Npc1 is an SSD protein that is closely related in protein structure to Disp1 and Patched1, the Hh-signaling receptor, and is involved in sterol trafficking from late endosomes to other cellular compartments. Inactivating mutations in Npc1 lead to increased levels of cellular sterols but fewer amounts of sterol derivatives due to reductions in sterol metabolism. The identification of sterol derivatives and sterol-like compounds that modulate the Hh-signaling pathway has provided new potentials for therapeutics. By identifying and cloning the zebrafish npc1 gene, I was able to test its function in sterol homeostasis in the zebrafish embryo. Reducing its function with morpholinos led to the mislocalization of cellular cholesterol, but did not impact levels of Hh signaling in morphants. npc1 morphants exhibited a delay in epiboly, likely a consequence in a reduction of sterol metabolism to hormones in the embryo.
MicroRNA-Mediated Regulation and the Fragile X Family of Proteins
The main purpose of this work is to understand how two members of the fragile X family of RNA-binding proteins, fragile X mental retardation protein (FMRP) and FXR1P, are regulated by post-translational modifications and microRNAs (miRNAs), respectively. Both proteins play key roles in normal development and function. The absence of FMRP leads to the cognitive defects seen in Fragile X syndrome, the leading cause of hereditary mental retardation, while loss of FXR1P expression in mice is fatal after birth, likely due to cardiac and muscle abnormalities. Small, genomically encoded miRNAs are involved in almost every biological process, specifically in the regulation of mRNA translation. Although their biogenesis is relatively well defined, it is still unclear how they are recruited to their mRNA targets. FMRP and its autosomal paralogs, FXR1P and FXR2P, in addition to the single Drosophila ortholog, dFmrp, associate physically and functionally with the miRNA pathway. Constitutively phosphorylated FMRP (P-FMRP) is found associated with stalled untranslating polyribosomes and translation of at least one mRNA is downregulated when FMRP is phosphorylated. We hypothesized that translational regulation by P-FMRP is accomplished through association with the miRNA pathway. Accordingly, we developed a phospho-specific antibody to P-FMRP and showed that P-FMRP associates with increased amounts of precursor miRNAs (pre-miRNA) compared to total FMRP. Furthermore, P-FMRP does not associate with Dicer or Dicer containing complexes in coimmunoprecipitation experiments or in an in vitro capture assay using a P-FMRP peptide sequence bound to agarose beads. These data show that Dicer containing complexes bind FMRP at amino acids 496–503 and that phosphorylation disrupts this association with a consequent increase in association with pre-miRNAs. In sum, we propose that, in addition to regulating translation, phosphorylation of FMRP regulates its association with the miRNA pathway by modulating association with Dicer. We present a new model for the effect of phosphorylation on FMRP function, where phosphorylation of FMRP inhibits Dicer binding, leading to the accumulation of pre-miRNAs and possibly a paucity of activating miRNAs. FMRP's autosomal paralog, FXR1P, plays an important role in normal muscle development and has been implicated in fascioscapulohumeral muscular dystrophy, and its absence or misregulation has been shown to cause cardiac abnormalities in mice and zebrafish. To examine miRNA-mediated regulation of FMRP and FXR1P, we studied their expression in a conditional Dicer knockdown cell line, DT40. We found that FXR1P, but not FMRP, increases upon Dicer knockdown and consequent absence of miRNAs, suggesting that FXR1P is regulated by miRNAs, while FMRP is not. Expression of a luciferase reporter bearing the FXR1 3′UTR was significantly increased in the absence of miRNAs, confirming miRNA-mediated regulation of FXR1P. We identified one of the regulatory regions by removing an 8-nucleotide miRNA seed sequence common to miRNAs 25, 32, 92, 363, and 367 in the 3′UTR of FXR1. Accordingly, overexpression of an miRNA, miR-367, containing this common seed sequence decreased endogenous FXR1P expression in HEK-293T and HeLa cell lines. We report for the first time that FXR1P expression is regulated through miRNA binding to the miR-25/32/92/363/367 seed sequence-binding site in the FXR1 3′UTR.
Regulation of Pigment Cell Specification During Embryonic Development in the Zebrafish
Pigment cells of the zebrafish, Danio rerio, offer an exceptionally tractable system for studying the genetic and cellular bases of cell fate decisions. The pigment cells of zebrafish include the black melanophores, the iridescent iridophores, and the yellow xanthophores. Each of these cell types develops from the neural crest, a pluripotent cell population found in vertebrates and some basal chordates. The central aim of this dissertation is to understand the mechanisms used to specify distinct pigment cell types from a homogenous neural crest cell population. Using genetic markers specific to each pigment cell type, I determined that xanthophores become distinct from a partially restricted melanophore/iridophore bi-potent precursor at 24 hpf. This bi-potent precursor resolves into distinct melanophores and iridophores between 28 and 42 hpf. I labeled and tracked these mitfa + precursor cells with a photoconvertible protein, EosFP to confirm that both melanophores and iridophores can develop from the bi-potent cell. mitfa is a basic helix-loop-helix transcription factor that activates multiple downstream pathways necessary for melanophore development and exerts a repressive effect on iridophore development. Foxd3, a forkhead transcription factor, helps resolve the bi-potent pigment precursor by repressing mitfa expression and promoting iridophore development. Using cell culture and in vivo assays, I demonstrate that Foxd3 represses mitfa by directly binding to its proximal promoter region. Taken together, our data reveal a Foxd3/mitfa transcriptional switch that governs whether a bi-potent neural crest precursor will attain either an iridophore or melanophore fate.
Site-1 Protease in Chrondrogenesis
During mammalian skeletal development, endochondral ossification is preceded by the establishment of a cartilage template through a process termed chondrogenesis. Disruptions in chondrogenesis result in chondrodysplasia, a disorder of the appendicular skeleton that is characterized by disproportionately shortened limbs. A severe chondrodysplasia phenotype is observed when site-1 protease (S1P) is disrupted in both the zebrafish and the mouse, indicating that S1P is essential for normal skeletal development. To investigate the role of S1P during chondrogenesis, ATDC5 cells were used as an in vitro model. Inhibition of S1P activity in these cells decreases the expression of collagens type II and X, while type I collagen and aggrecan remain unaffected. The substrates of S1P are membrane-bound proteins that, when cleaved, release transcription factors promoting cholesterol biosynthesis and the unfolded protein response. Real-time polymerase chain reaction analysis determined that the expression of two substrates, ATF6 and CREB3/Luman, were significantly upregulated during chondrogenesis. However, silencing of CREB3/Luman expression revealed no cartilage-related phenotype.
The Characterization and Utilization of Middle-Range Sequence Patterns Within the Human Genome
Mid-range inhomogeneity (MRI) is the significant enrichment of particular nucleotides in genomic sequences extending from 30 to 10,000 nucleotides. MRI can be observed for all nucleotide pairings (e.g., G + C, A + G, and G + T) as well as for individual bases. Various types of MRI regions are 4–20 times enriched in mammalian genomes compared to their occurrences in random models. We first show how different types of mutations change MRI regions. Human, chimpanzee, and Macaca mulatta genomes were aligned to study the projected effects of substitutions and indels on human sequence evolution within both MRI regions and control regions of average nucleotide composition. Over 18.8 million fixed point substitutions, 3.9 million single-nucleotide polymorphisms (SNPs), and indels spanning 6.9 Mb were procured and evaluated in human—1.8 Mb substitutions and 1.9 Mb indels within MRI regions. Ancestral and mutant alleles for substitutions were determined. Substitutions were grouped according to their fixation within human populations: fixed substitutions (from the human–chimp–macaca alignment), major SNPs (>80% mutant allele frequency within humans), medium SNPs (20%–80%), minor SNPs (3%–20%), and rare SNPs (<3%). Data on short (<3 bp) and medium-length (3–50 bp) insertions and deletions within MRI regions and appropriate control regions were analyzed for their effect on the expansion or diminution of such regions as well as on changing nucleotide composition. MRI regions have comparable levels of de novo mutations to the control genomic sequences. Newer mutations rapidly erode MRI regions, bringing their nucleotide composition toward genome-average levels. However, substitutions that favor the maintenance of MRI properties have a higher chance to spread throughout the human population. Indels have a clear tendency to maintain MRI features but have a smaller impact than substitutions. Overall, the observed fixation bias for mutations helps maintain MRI regions during evolution. Next, we discuss the splicing of large introns in mammals (over 50,000 bp). Large introns must be spliced out of the pre-mRNA in a timely fashion, which involves bringing together distant 50 and 30 splice sites. In Drosophila large introns can be spliced efficiently through a process known as recursive splicing. We computationally demonstrate that vertebrates lack the proper enrichment of RP-sites in their large introns and, therefore, require some other method to aid splicing. Over 15,000 nonredundant, large introns from 6 mammals, 1600 from chicken and zebrafish, and 560 large introns from 5 invertebrates were analyzed. Unlike the studied invertebrates, the studied vertebrate genomes contain consistently abundant amounts of direct and complementary strand interspersed repetitive elements (mainly short interspersed nuclear elements [SINEs] and long interspersed elements [LINEs]) that may form stems with each other within large introns. Indeed, predicted stems were abundant and stable in the large introns of mammals. We hypothesize that stable stems with long loops within large introns allow splice sites to find each other more quickly by folding the intronic RNA upon itself. Finally, we extend and complement existing Markov model algorithms by developing and testing a novel binary-abstracted Markov model (BAMM) algorithm. BAMM can emphasize selected portions of genomic sequence signals according to specific abstraction rules. We present abstraction rules that generalize genomic sequence patterns at the single nucleotide level up to the level of tetranucleotides, using both in-frame data and data of mixed reading frames. We develop context-dependent abstraction rules that emphasize genomic sequence repetition. Unlike traditional Markov models, BAMM can analyze nucleotide patterns on the short-range (<20 bp) up to the mid-range (20–50 bp) scale. Abstraction rules can also be both frame sensitive or independent. We build classifiers for both coding sequences and introns as well as for 50 and 30 UTR data. Using support vector machines, we demonstrate that we can combine multiple BAMM classifiers to get even better exon–intron classification accuracy.
The Role of the Calcium Ion-Dependent Protein Kinase, CaMK-II, in Heart and Kidney Development in the Zebrafish, Danio rerio
Ca2+/calmodulin-dependent protein kinase type II (CaMK-II) is a multifunctional serine/threonine kinase that is ubiquitously expressed throughout the lifespan of metazoans. Mammals encode four genes (a, b, g, and d) that generate over 40 splice variants. CaMK-II is important in a myriad of functions, including ion channel regulation, cell-cycle progression, and long-term potentiation. In adults, alterations in activation of CaMK-II induce cardiac arrhythmias and heart failure. Developmental roles for CaMK-II are not as well understood since mouse knockouts are embryonic lethal. Therefore, the identification of other vertebrate CaMK-II genes will add to our understanding of development. Zebrafish encode seven catalytically active CaMK-II genes (a1, b1, b2, g1, g2, d1, and d2) due to a genome-wide duplication event that occurred ∼250 million years ago. Although, only 20%–30% of all duplicated genes were retained, 75% of CaMK-II-duplicated genes are transcriptionally active, pointing to a critical role for this signaling protein. mRNA expression patterns demonstrate that CaMK-II is expressed in diverse tissues, including retina, pectoral fins, somites, heart, and kidney. Suppression of each gene generates unique phenotypes that mirror the mRNA expression patterns. Of the seven genes, camk2b2 and camk2g1 have the highest maternal contribution in zebrafish, are expressed in mesodermally derived organs, and develop defects similar to human syndromes. In fact, suppression of camk2b2 mimics the phenotype observed in zebrafish mutants of tbx5, the gene mutated in patients with Holt-Oram Syndrome. Camk2g1 morphants also exhibit similar defects as suppression of pkd2, the gene mutated in patients with autosomal dominant polycystic kidney disease. These roles implicate CaMK-II as an integral protein in the development and maintenance of mesodermally derived tissues.
Disruption of the Embryonic Axis in Fish Embryos
Environmental contamination with human-made and petroleum-derived chemicals is a pervasive and insidious issue for both wildlife and human health. My dissertation research focused on the effect of two classes of environmental contaminants, polycyclic aromatic hydrocarbons (PAHs) and phthalates, on early teleost development. PAHs are a class of widespread environmental contaminants that are a major component of fossil fuels and coal-tar creosote, a widely used wood preservative in the United States. PAHs are also formed during the incomplete combustion of organic matter, and are therefore present in atmospheric pollution as well as urban run-off. Phthalate esters are a class of anthropogenic chemicals that are used as plasticizers to impart flexibility to polyvinyl chloride (PVC) resins, and are also added to many consumer products, including cosmetics and packaging ink. The production and use of these chemicals has led to widespread environmental contamination. Using a model vertebrate species, the zebrafish (Danio rerio), I have characterized both the morphological and cellular effects of phenanthrene and dibutyl phthalate on axis determination. I have established that these environmental contaminants are capable of disrupting development in both invertebrate and vertebrate species by disrupting the Wnt/b-catenin signaling pathway. Using Western blot analysis and antibodies specific to the phosphorylation state of GSK-3b, I examined whether these chemicals affect inhibitory phosphorylation at serine 9 on GSK-3b. No change in the phosphorylation state of GSK-3b was detected in exposed embryos. I also tested whether phenanthrene and dibutyl phthalate could directly inhibit GSK-3b kinase activity in an in vitro kinase assay. These environmental contaminants had no direct effect on the kinase activity of GSK-3b. I present evidence from the literature that indicates that phenanthrene and dibutyl phthalate likely act independently of the aryl-hydrocarbon receptor in my studies. The ability of phenanthrene and dibutyl phthalate to modulate the b-catenin signaling pathway represents a novel mechanism of toxicity for these chemicals and warrants concern, both ecologically and potentially for human health. Disregulation of b-catenin signaling has been linked to carcinogenesis, and activating mutations within the Wnt signaling pathway cause 90% of colorectal cancers. The ability of PAHs and dibutyl phthalate to modulate this pathway represents a unique mechanism for carcinogenesis. In addition to exploring the mechanism for contaminant effects on this signaling pathway, I have also translated my results with zebrafish to the estuarine Pacific herring, Clupea pallasi. Pacific herring are important both ecologically and commercially. Because the Wnt/b-catenin signaling pathway is so remarkably well conserved among animal species, and because PAHs and phthalates are such widespread environmental chemicals, these results could have wide-reaching implications for ecosystem health. This research will allow predictions to be made about early life stage failure in the environment, and could contribute to our understanding of unexplained recruitment losses. Lastly, I examined the ability of two known oxidative stressors, lead acetate and hydrogen peroxide, to induce morphological abnormalities that were homologous to those observed with PAHs. PAHs, both singly and in mixtures, have been shown to increase oxidative damage in aquatic vertebrates and invertebrates. I also test whether processed water can protect against the toxic action of these pro-oxidants, alone, or in combination with a known antioxidant (lipoic acid). Embryos exposed to lead acetate or hydrogen peroxide did not exhibit abnormalities characteristic of dorsal-ventral axis disruption, but did show a suite of abnormalities at the larval stage, including spinal curvatures, cardiac edema and yolk sac edema. My results suggest that under certain conditions the processed water medium is an active agent, with the ability to attenuate toxicological responses that differ from the control medium. (Abstract shortened by UMI.)
Extracellular Matrix Proteins: Implications for Angiogenesis
The extracellular matrix (ECM) is an essential requirement for maintaining permanent shape and rigidity in multicellular organisms. The ECM serves two main functions: scaffolding and signaling. Insoluble collagen and soluble proteoglycans, glycosaminoglycans, and glycoproteins allow for water retention and flexibility. The signaling role of the ECM is essential for a multitude of events, including vascular development and angiogenesis. Via interactions with vascular endothelial cells, proteins of the ECM can induce or repress angiogenesis.
Identification of Multipotent Dental Stem Cells in Zebrafish, Danio rerio
Due to its capacity to replace teeth continuously throughout its life, the zebrafish, Danio rerio, provides an excellent animal model for the identification and characterization of multipotent dental stem cells in adult animals. The aim of this study is to identify the dental stem cell niche through bromodeoxyuridine (BrdU) pulse-chase experiment, using the zebrafish animal model. During the pulse period, BrdU is incorporated into DNA of cells undergoing S-phase for DNA synthesis, and animals are removed from the BrdU solution during chase period in which the cells that divide further dilute out BrdU labels. Thirteen immunohistochemical analyses using anti-BrdU of whole-mount and sectioned pulse-chase specimens revealed that a 2 day pulse of 2–4 dpf embryos, followed by a 14 day chase period is long enough to dilute out detectable BrdU label in the pharyngeal teeth region. However, immunohistochemical analyses of 2 day pulse of 2–4 dpf embryos receiving a 8 day chase revealed discrete populations of BrdU label retaining cells in the pharyngeal teeth region that can be speculated to represent the dental stem cell (DSC) niche. Thus, the results support the existence of multipotent dental stem cells that may enable the continuous replacement of zebrafish dentition.
Requirements for Wnt Signaling in Morphogenesis of the Neural Crest, Neural Tube, and Craniofacial Skeleton of Zebrafish Embryos
How are morphogenesis, growth, and differentiation orchestrated to generate multiple functioning organs during embryonic development? For my thesis studies I have focused on understanding the role of Wnt signaling in several organs and cell types during zebrafish embryonic development. I studied context-specific requirements for Wnt signals to pattern the neural crest (NC)-derived pigment cells and craniofacial skeleton. Additionally, I studied the roles of Ovo transcription factors, putative Wnt targets, in the formation of the central nervous system. The central nervous system rudiment, the neural tube, forms by the rolling into a tube of the neuroectoderm during neurulation. NC cells form adjacent to the neuroectoderm but migrate throughout the embryo to differentiate into a variety of cell types, including cartilage cells that give rise to the craniofacial skeleton and pigment cells of the skin. Differential cadherin expression differentiates the neural tube from NC during neurulation. Additionally, a sequential cadherin code, partially controlled by Wnt signaling, is also required for NC cell migration. Wnt signaling is well known in embryogenesis and cancer, playing multiple roles in several aspects of development. I have isolated and characterized requirements in neural tube morphogenesis for two zebrafish orthologs of the Ovo family of transcription factors, ovo1 and ovo3. I also established zebrafish ovo1 as a target of Wnt signaling required for the migration of the NC-derived pigment cells and provided the first link between Ovo transcription factors and N-cadherin regulation via transcriptional control of Rab Gtpases known to control trafficking of molecules within the cell. Wnt signaling also functions to pattern the NC-derived craniofacial skeleton. Using temporally controlled gain-of-function studies I have shown that Wnt signaling is required for the formation of the lower jaw skeleton by coordinating morphogenesis and growth. My research shows that Dkk1 overexpression inhibits Wnt signaling, which is sufficient to inhibit cell proliferation resulting in craniofacial abnormalities. Although expressed by the adjacent endoderm, Dkk1 acts as an extrinsic cue to organize growth and morphogenesis of the craniofacial skeleton.
Microrobotic Manipulation and Characterization of Biological Cells
Mechanical manipulation and characterization of biological cells have wide applications in genetics, reproductive biology, and cell mechanics. This research focuses on (1) the development of enabling microrobotic systems and techniques for automated cell microinjection and in situ mechanical characterization, and (2) the demonstration of molecule efficacy testing and cell quality assessment with the new technologies. Targeting high-speed cell injection for molecule screening, a first-of-its-kind automated microrobotic cell injection system, is developed for injecting foreign materials (e.g., DNA, morpholinos, and proteins) into zebrafish embryos (∼1.2 mm) and mouse oocytes/embryos (∼100 mm), which overcomes the problems inherent in manual operation, such as long learning curves, human fatigue, and large variations in success rates due to poor reproducibility. Novel cell holding devices are developed for immobilizing a large number of embryos into a regular pattern, greatly facilitating sample preparation and increasing the sample preparation speed. Leveraging motion control and computer vision techniques, the microrobotic system is capable of performing robust cell injection at a high speed with high survival, success, and phenotypic rates. The mouse embryo injection system is applied to molecule testing of recombinant mitochondrial proteins. The efficacy of an anti-apoptotic Bcl-xL (ΔTM) protein is, for the first time, quantitatively evaluated for enhancing the development competence of mouse embryos. For cell quality assessment, this research develops a vision-based technique for real-time cellular force measurement and in situ mechanical characterization of individual cells during microinjection. A microfabricated elastic device and a subpixel computer vision tracking algorithm together resolve cellular forces at the nanonewton level. Experimental results on young and old mouse oocytes demonstrate that the in situ obtained force–deformation data can be used for mechanically distinguishing healthy mouse oocytes from those with cellular dysfunctions. This work represents the first study that quantified the mechanical difference between young and old mouse oocytes, promising a practical way for oocyte quality assessment during microinjection.
A Role for the Transcriptional Repressor RE1 Silencing Transcription Factor in Patterning the Zebrafish Neural Tube
The spatial and temporal control of gene expression is key to generation of specific cellular fates during development. Studies of the transcriptional repressor RE1 silencing transcription factor/neural restrictive silencing factor (REST/NRSF) have provided important insight into the role that epigenetic modifications play in differential gene expression. Functional studies place REST within multiple developmental pathways and transcriptional networks. However, findings between different groups are often incongruent, and little progress has been made on understanding the embryonic lethality of the Rest knockout mice. What emerges from the controversies surrounding REST function is that the cellular context of REST is paramount. Here, zebrafish embryos are used to study REST function within the broader context of a developing organism. The approach was to assay changes in gene expression following Rest knockdown in various backgrounds. This method revealed a novel interaction between zebrafish Rest and the Hedgehog (Hh) signaling pathway. It was observed that Rest knockdown enhances or represses Hh signaling in a context-dependent manner. In wild-type embryos and embryos with elevated Hh signaling, Rest knockdown augments transcription of Hh target genes. Conversely, in contexts where Hh signaling is diminished, Rest knockdown has the opposite effect and Hh target gene expression is further attenuated. Epistatic analysis revealed that Rest interacts with the Hh pathway at a step downstream of Smo. Further, the findings demonstrate that the bifunctional transcription factor Gli2a is key to Rest modulation of the Hh response. The role of Rest as a regulator of Hh signaling has broad implications for many developmental contexts where REST and Hh signaling act.
Systematic Analysis of Asymmetric Nodal Signaling in the Development of Zebrafish Left–Right Patterning
My thesis focuses on the interactions among spaw, lft1, and oep in left–right patterning determination in zebrafish embryonic development. Zebrafish is an ideal model system for studying left–right patterning because, like human beings, zebrafish is vertebrate whose organs display asymmetric patterning along the left–right axis. Additionally, zebrafish possess gene orthologs of the main players discovered in human left–right patterning determination: spaw is the left–right axis-specific nodal in zebrafish, lefty is a Nodal antagonist, while oep is the coreceptor for Nodal signaling. My results indicate ectopic spaw expression induces the expression of Nodal signaling downstream targets in an oep-dependent way, including expression of the other two nodal genes in zebrafish, and this induction can be antagonized by lft1 expression, while oep attenuates this inhibition. While investigating the regulation of these three components in the process of left–right determination, I discovered more intriguing phenomena. All three components (nodal, lefty, and oep) exhibit highly dynamic temporal and spatial patterns of expression during the establishment of the left–right axis. Both spaw and lft1 propagate from posterior to anterior, but the relationship between these two genes in space is dynamic. By quantitatively analyzing the mRNA level in the spaw propagation stripes, I applied a traveling-decay model to calculate the key parameters involved in the process. Further, the dosage of the three components is also essential for proper asymmetric patterning. Insufficient oep levels in Late Zygotic oep mutants, leads to absence of Nodal in the LPM, while ectopic oep can restore it. However, overexpression of oep leads to symmetric/bilateral Nodal in wild-type embryos. Finally, based on all of the experiments and previous discoveries, I formulated a mathematical model based on two-dimensional reaction–diffusion to describe the dynamics of the left–right axis determination system in zebrafish. This model succeeds simulating several phenomena as observed in experiments. Overall, with both experimental and computational approaches, I have revealed that spaw acts as Nodal ligand and that the spatial interaction of the three components is essential for the establishment of asymmetric Nodal signaling. This provides new insights to the research field of left–right patterning.
N-Cadherin-Mediated Cell Adhesion Regulates Proliferation and Differentiation in the Developing Zebrafish Central Nervous System
Cadherins play an important role in morphogenesis and have recently been implicated in the regulation of cell proliferation; however, the mechanisms by which they function are poorly understood. In the vertebrate central nervous system, loss of N-cadherin (N-cad) results in impaired neuroepithelial integrity. Zebrafish N-cad null mutants also exhibit a transient increase in neurons and in cell proliferation in the neural tube. Here, we investigate the cellular and molecular basis for this phenotype, using multiple N-cad alleles with distinct molecular properties. We confirm that cell proliferation is enhanced in N-cad mutants, but contrary to previous findings, we observe that the increase is sustained over multiple stages of development. At the cellular level, loss of N-cad results in a shorter cell cycle. Further, we demonstrate that hyperproliferation is not linked to abnormal beta-catenin localization, suggesting that Wnt signaling is not increased. Our findings indicate that components of Shh signaling, namely, ptc-1 and gli1, are upregulated in the dorsal region of N-cad mutants. In addition, blocking Shh signaling rescues the hyperproliferation phenotype observed in N-cad mutants, suggesting that N-cad regulates cell division by limiting the range of Shh signaling. Despite an increase in the total number of neurons, the neuronal differentiation index is in fact reduced in N-cad mutants, suggesting that the supernumerary neurons are produced as a result of overproliferation rather than enhanced differentiation. Consistent with this observation, the rate of cell cycle exit is unchanged. Intriguingly, we also observe that a small subset of N-cad mutant cells are double positive for mitotic and neuronal differentiation markers, suggesting a role for N-cad in coupling cell cycle exit and differentiation. These findings highlight the role of N-cad in regulating cell proliferation in the developing nervous system and reveal a novel function for N-cad in coupling cell cycle exit and differentiation.
The Role of Notch Signaling in Hemogenic Endothelial Cell Development
Notch signaling plays important roles in development of embryos by participating in cell-fate decisions and differentiation of many different cell types, including endothelial cells and hematopoietic cells. Hematopoiesis in the embryo occurs in two phases; a transient primitive phase and a definitive phase, which generates hematopoietic stem cells that constitute the whole blood system. Notch has been known to be specifically required for definitive hematopoiesis and proper endothelial cell development. In studying mouse embryonic stem cell (ESC) differentiation, we generated an ESC line that expresses the active form of Notch1 (ICN1) after induction with doxycycline. During embryoid body (EB) differentiation, ICN1 induction increased hematopoietic differentiation with an increase of CD41-VE-cadherin + a4-integrin + CD45-hemogenic endothelial population, which showed hematopoietic and endothelial cell outgrowth in the subsequent culture after sorting. Gene expression analysis showed an upregulation of Foxc2 in this population after ICN1 induction. Genetic studies in the zebrafish showed that Foxc2 and its orthologs are downstream targets of Notch signaling in hemogenic endothelial cell development, and the analysis of Foxc2−/− mouse embryos further confirmed the requirement of Foxc2 in definitive hematopoiesis. In human ESC differentiation, Notch ligand treatment promoted hematopoietic differentiation with an increase of VE-cadherini low a4-integrin+ population. These results indicate that upregulating Notch signaling during ESC differentiation may promote definitive hematopoiesis through hemogenic endothelial cells. In summary, in the zebrafish, mouse, and human, data collected here suggest that Notch signaling plays an important role in hemogenic endothelial cell development. Mouse EBs provided the platform to capture the gene expression profile of hemogenic endothelial cells, which lead to the further analysis of Foxc2 in zebrafish and mouse embryos. Ligand treatment during human EB differentiation showed similar results observed in mouse EB differentiation without genetic modification, suggesting evolutionary conservation of the Notch pathway and its effect on blood development. Further characterization of the emergence of hemogenic endothelial cells during embryo development may help better understand the guided differentiation of hematopoietic stem cells from pluripotent stem cells and open new clinical opportunities.
Gender-Specific Changes in Key Regulators of Neurodevelopment and Autistic Behavioral Pathology in Mice Exposed to Water Chlorination Byproducts
Autism is a heterogeneous group of disorders with no definitive etiology. Out of concern for higher than expected prevalence, the Agency for Toxic Substances and Disease Registry (ATSDR) investigated the municipal water supply in Brick Township, New Jersey. The ATSDR found that two trihalomethanes (THMs), specifically chloroform and bromoform, as well as tetrachloroethylene (perchlorethylene; PCE), were present in concentrations that exceeded allowable maximum contaminant (MCL) values. In a related study, it was found that THMs and PCE act synergistically to increase the level of catalytic protein kinase A (PKA) in neurons of clam embryos. PKA is a key regulator of neurodevelopment, and it is hypothesized that abnormalities in PKA activity could induce both histopathological and biochemical manifestations that are found in autism. Based upon these findings, we hypothesized that THM/PCE exposure induces changes in key regulators of neurodevelopment and behavioral pathology similar to that which is found in autism. In our experiments we found that exposure to THM/PCE induces an increase in the level of catalytically active PKA in zebrafish neurons and increases PKA activity in microglia cell culture. In a mouse model, we found that exposure to THM/PCE via drinking water induces an increase in the activity of PKA in the cerebral cortex of male animals at postnatal day 4 (P4) and P10. Female cortical PKA activity was unaffected by THM/PCE exposure. By P15, male cortical PKA activity is no longer affected by THM/PCE exposure and female cortical PKA activity remains unaffected. Behaviorally, we found that the THM/PCE-exposed males develop autistic like behavioral pathology as they evidence deficits in communication and social behavior and demonstrate both perseverance behavior and anxiety. Again, this finding is gender specific, as female behavior is unaffected by THM/PCE exposure. These findings suggest that these chemicals may be involved in the etiology of autism and that males are more susceptible to this set of insults.
The Function of Signaling Gradients in Zebrafish Somitogenesis
Somitogenesis is the process by which the vertebrate body axis is divided into repeated segments called somites. Somites form in an anterior to posterior wave, with new somites deriving from tissue generated by the growing tail bud. Segment boundary formation is presaged by waves of oscillating gene expression that begin in the tail bud and move anteriorly through presomitic mesoderm until they encounter a determination front. The opposing gradients of FGF/Wnt signaling and retinoic acid (RA) signaling are required for positioning the determination front. However, the molecular interactions at the determination front that transition cells from being immature to being competent to form somites is unknown. To better understand these molecular events, I investigated expression and function of gadd45b and gadd45bl, two highly related genes expressed in the zebrafish anterior presomitic mesoderm. I find that gadd45b and gadd45bl are expressed at the determination front and are sensitive to perturbation of Fgf and RA signaling gradients. Fgf negatively regulates and RA positively regulates gadd45b expression. By depleting Gadd45b and Gadd45bl, I show that they are not necessary for identified a downstream gadd45bl/gadd45bl target, dusp1, which encodes a phosphatase that acts as a negative regulator of Fgf signaling. I showed that dusp1, a gene encoding a phosphatase that negatively regulates Fgf signaling, is a gadd45b/gadd45bl target that requires Gadd45b/Gadd45bl for expression and is sensitive to modulation by addition of exogenous RA. My work sets the stage for future investigations addressing a potential role for Gadd45b and Gadd45bl in creating a permissive environment for initiation of gene transcription as cells respond to the changes in Fgf and RA signaling gradients and prepare to differentiate as muscle cells.