Maintaining the integrity of genetic material and preventing changes over time is essential for normal cellular function. This genomic stability is directly affected by the DNA replication process. Replication must be both accurate and efficient; mutations that affect DNA replication can cause genomic instability and changes in the genetic makeup of the cell. Through a genomic instability screen in mice, researchers discovered a single base pair mutation in a highly conserved gene required for unwinding DNA during DNA replication. The presence of this single base pair substitution, called Chaos3, in both copies of the gene causes female mice to develop mammary tumors. We have found that in the yeast Saccharomyces cerevisiae, the corresponding Chaos3 mutation decreases activation or “firing” of some replication origins—the sites where DNA replication begins. Chaos3 does not affect all early firing origins in the genome; rather, origins near centromeres are specifically affected, thereby delaying replication of those centromeres, causing chromosome loss. We found that when an affected origin is replaced with an unaffected one, firing levels are restored to wild type function and that chromosome loss is rescued. To further understand what components are essential for timely DNA replication, and why only a subset of origin sequences are sensitive to the Chaos3 allele, I am focusing on the origin sequences directly. I am mutating the origin sequence itself and separately deleting different genes whose products have potential interactions with origin sequences. My research aims to advance the understanding of the role these genes play in the activation of origins for timely DNA replication and how the Chaos3 mutation may be interrupting normal function of these processes. This knowledge can help identify key molecular mechanisms that drive cancer development in higher eukaryotes.

Copy number variation (CNV) is associated with genetic disorders in humans. One particular kind of CNV is a gene triplication in which the central copy is inverted. How such structures arise is poorly understood but is of great interest because of their association with cancer and other genetic disorders. We find similar amplified structures in budding yeast Saccharomyces cerevisiae, and therefore, we can use yeast to understand the mechanism that generates them. The Brewer and Dunham labs have proposed Origin Dependent Inverted Repeat Amplification (ODIRA) as a model that explains inverted CNVs. To understand which proteins/enzymes contribute to amplification, we are implementing gene deletions to observe the effect on the production of these ODIRA amplification events. ODIRA events are rare and thus large sample sizes are required to detect them. To streamline their identification, I am developing a system that uses color as a visual indicator of CNV. When a single copy of the bacterial gene, VioA, is expressed in yeast it produces light purple colonies; in multiple copies, the colonies are a darker shade of purple. I am inserting a single copy of the VioA gene into a region of the yeast genome that undergoes inverted triplication events. Simply scanning plates for dark purple colonies will enable me to screen for ODIRA events quickly and measure their frequency. Using this visual indicator will allow me to rapidly screen different yeast mutants and determine the role they play in the ODIRA amplification mechanism.

In genomics, large-scale experiments commonly exploit DNA libraries, transforming them into a biological system to assay their effects. In these experiments, smaller DNA constructs provide increased transformation efficiency, such that larger libraries can be screened. Previously, we observed a 3-fold increase in the DNA transformation rate in the fruit fly Drosophila melanogaster when we reduced the size of the DNA construct to approximately 1/6 its initial size. Further improvement of this transformation rate will be dependent on a number of features, including the ease of production of tens of micrograms of minicircles, the ability to create a library of minicircle constructs, and the presence of a PhiC31 attB site to allow integration into the D. melanogaster genome. There are no existing minicircle production methods that address all these needs; thus, we are developing a DNA minicircle production approach that should address them. We use asymmetric PCR to produce single-stranded DNA (ssDNA) that is then circularized. We use these ssDNA circles as templates for rolling circle amplification to generate large quantities of double-stranded DNA (dsDNA). Following debranching and digestion of the DNA, we use a serine recombinase PhiC31 and its recombination directionality factor gp3 to recombine small dsDNA fragments into dsDNA minicircles. To date, we have produced linear single-stranded DNA using asymmetric PCR, ligated the linear ssDNA into circular ssDNA, and performed in vitro recombination using PhiC31 and gp3 to make minicircles from small dsDNA sequences. We are now working to combine all parts of the procedure into one continuous workflow and to measure the method’s efficiency and yield. This procedure will be vital for large-scale genomic manipulations of D. melanogaster, and should thereby improve the scale of these experiments. The approach should ultimately increase our understanding of genetic variation as we can assay it in D. melanogaster.

Gene arrangements are observed in human diseases such as cancer and developmental disorders. In developmental disease, gene triplication with an inverted central copy is observed, while a hallmark of cancer are palindromes, or inverted repeats of DNA. Therefore, testing one mechanism of gene arrangement in an easily studied organism like the  budding yeast Saccharomyces cerevisiae may shed some light on the human genome and disease generation. Yeast cells grown for >200 generations in sulfate limiting media are enriched for triplication of the high affinity sulfate transporter SUL1 with the center copy inverted, as amplification confers a selective advantage. To explain how such a triplication occurs, the Brewer and Dunham Labs proposed a model called Origin Dependent Inverted-Repeat Amplification (ODIRA). The ODIRA mechanism requires a DNA origin of replication and short, inverted repeats flanking the SUL1 gene. During DNA replication, an error of the replication fork, or fork regression, causes annealing of leading and lagging strands to create a hairpin intermediate. The intermediate then replicates and recombines into the genome, forming interstitial triplications, with the middle copy inverted. While the proposed mechanism explains the observed triplication in yeast, the specific proteins involved are not yet known. The genes I've chosen to test are the DNA helicases MPH1 which prevents cross-over between ectopic sequences, and RRM3 which relieves replication fork pauses. Both are predicted to regulate necessary steps of the ODIRA mechanism, making them good candidates for genes that may be involved in these triplications. By deleting each gene, I can then measure the frequency of ODIRA events in those strains and compare them to wild-type strains. Increased ODIRA events in the knockout strains may implicate their role in the ODIRA mechanism and prompt further study of these genes and how they might affect copy number variation in humans.

Prostate cancer has a significant heritable component. It is estimated that 10-15% of patients with advanced prostate cancer carry an inherited predisposing genetic mutation, and these patients generally present with a younger age of onset and a strong family history of cancer. The standard in the field of oncology is to conduct short-read DNA sequencing on such patients to find predisposing mutations. While short-read sequencing does well to identify simple mutations that cause disease in many families, our lab concluded that short-read sequencing misses critical mutations in many prostate cancer susceptibility genes. We hypothesized that prostate cancer in many families is due to complex inherited mutations such as genomic deletions, inversions, and mobile element insertions that are not detectable by conventional genomic technologies such as short-read sequencing. To test this hypothesis, our lab specifically recruited prostate cancer patients who, despite having family histories of cancer, did not have any mutations detected via conventional genetic sequencing methods. This project utilizes Nanopore long-read DNA sequencing, which reads DNA in longer fragments and can reliably detect complex mutations. My role is to conduct long-read sequencing on DNA samples from these patients, then analyze the DNA sequence for mutations. I have sequenced 32 patients so far and identified 4 complex mutations through long-read sequencing which were missed by other approaches. These complex mutations include insertions of repeat sequences and duplications which disrupt gene function in BRCA1 and BRCA2. This suggests that, consistent with our hypothesis, some patients who do not have mutations found via conventional sequencing methods do indeed carry causative mutations in well-established prostate cancer risk genes. By finding these mutations, patients can receive more targeted and effective cancer treatment, and undiagnosed family members stand a better chance of catching cancers at earlier stages.

Since the discovery of DNA in the 19th century, biochemists have been elucidating not only the structure, but unique biochemical environment of each loci. Protein-DNA neighborhoods govern chromatin structure and cellular functions (transcription, replication, etc.). To investigate which proteins and oligonucleotides compose these microenvironments, our lab and collaborators developed DNA oligonucleotide-directed proximity-interactome mapping (DNA O-MAP), a locus purification method using oligo-based ISH probes to recruit horseradish peroxidase (HRP) activity to specific DNA intervals (Liu & McGann et. al. 2024). Once these secondary, HRP-conjugated probes are localized to loci of interest, hydrogen peroxide is added with biotin-tyramide. Hydrogen peroxide activates HRP, forming biotin-tyramide phenoxyl radicals that biotinylate proteins and nucleic acids within 10-75nm. This allows for a scalable, versatile method to investigate these microenvironments. Large scale DNA O-MAP, tiling across several genomic sites, can elucidate insights into biological questions. However, the upstream protocol remains a barrier to its throughput, sensitivity, and reproducibility. In order to ensure this for analysis of tagged proteins, we sought to automate the streptavidin affinity purification protocol onto the Opentrons OT-2 robot. This is where streptavidin-coated magnetic beads capture biotinylated species from lysate. Coupled beads are recaptured with a magnetic rack and pipetting-off of flow-through. Subsequently, several washes cleans up these beads before peptides are digested off via Trypsin/LysC, dried-down, resuspended, and loaded onto a Orbitrap Eclipse LC-MS for proteomic analysis. Purification of streptavidin beads is manually intensive, inherently leading to variation between runs. The Opentrons OT-2 is an open-source liquid handler, allowing our lab to easily transfer methods to others interested in DNA O-MAP. Automating this protocol launches us from technology development to biological application. Here, I present an automated protocol for streptavidin affinity purification and evaluation of its effectiveness via comparison of the automated protocol to our lab's current, manual methods.

Female mammals possess two X chromosomes in every cell, but one is silenced by condensing into a barr body, making its genetic information largely inaccessible. While X inactivation is stable in somatic cells, it is reversible in germ cells, raising the intriguing question of what proteins maintain this silenced state. My project aims to identify the protein composition of both active and inactive X chromosomes in mice. To achieve this, I will use in situ hybridization to target proximal labeling with biotin of X chromosome-associated proteins. This is accomplished by targeting a biotinylation enzyme, such as HRP, to the X chromosomal region, where it will selectively biotinylate neighboring proteins. After affinity purification, these proteins can be identified using mass spectrometry-based quantitative proteomics. To direct the enzyme to the correct location, a two-probe system is employed. The primary oligonucleotide probe complements a specific X chromosome region which also contains landing sites for a secondary probe. Hybridization of the secondary probe which is tagged with HRP enables precise labeling of chromosome-associated proteins. This approach enables in situ biotinylation, preserving proteins in their native context for accurate identification. Since the two X chromosomes are homologous, distinguishing between the active and inactive X requires careful probe design. By utilizing Single Nucleotide Polymorphisms (SNPs) that exist in the X chromosomes, the maternal and paternal X chromosomes can be differentially targeted by primary probes, allowing for homolog specific protein labeling and analysis of their distinct regulatory environments. 

Cell shape opens a powerful window into the genetic and mechanical processes that drive cell behavior and, ultimately, tissue morphogenesis during development. By identifying cell shape, we can track specific cells and their responses to different gene expressions - creating a clearer mapping of which cells are affected by various manipulations. In this project we combine computational tools with quantitative microscopy to measure nucleus shape, and use these readouts to identify different cell types in the pectoral fins of zebrafish embryos. High resolution images of pectoral fin nuclei were taken using confocal microscopy - a technique commonly used when capturing tissue and cell data. Following nucleus identification and segmentation during data pre-processing, the FlowShape analysis package was utilized to extract quantitative "shape vectors" that encode the morphology of each nucleus. We plan to leverage the spherical harmonic weights produced within FlowShape to cluster and identify key shape-types that emerge from the collected nuclei. These shape readouts will serve as the basis for future analyses aimed at classifying different nucleus morphologies within the pectoral fin. Ultimately we hope to use nucleus morphology to predict the expression of key marker genes. This approach provides a powerful method for bridging the gap between the rich gene expression information provided by single-cell RNA-seq atlases, and the dynamical and morphological information produced by in vivo microscopy.

Most eukaryotes use histones to package the genome. However, many animals package their sperm genomes using specialized DNA-binding proteins called protamines, which package DNA in sperm more tightly to fit inside the sperm head. Based on the transcriptional silencing role of protamines, we hypothesize that protamines can suppress meiotic drivers, which kill other sperm to bias their own transmission. Previously, we discovered that one protamine gene, Mst77F, is required to suppress meiotic drivers on the Y-chromosome in Drosophila melanogaster. Since drive is generally deleterious for transmitting autosomal alleles due to lower male fertility, theories predict that multiple drive suppressors will arise within populations; Mst77F may represent just one such suppressor. We hypothesized that natural variants in distinct genetic loci interact with as well as impact meiotic drive in Drosophila melanogaster. To identify these natural variants, I crossed wild-type flies to Mst77F knockout flies to generate hemizygous Mst77F flies carrying genetic backgrounds from four different populations. I measured the fertility and drive strength by crossing individual hemizygous males from each genetic background to five wild-type females. Using a genetic crossing scheme, I will test for variation in X-linked targets and Y-linked drive in 5 additional populations. This will reveal insights into the mechanism of Y-linked drive and the basis of X-chromosome susceptibility to drive. My study contributes to a better understanding of the pervasive effects of meiotic drive in natural populations and the unexpected function of protamines.

Pseudomonas aeruginosa commonly colonizes cystic fibrosis (CF) lungs, causing persistent infections even under novel CFTR modulator therapies such as elexacaftor-tezacaftor-ivacaftor (ETI). While antibiotic resistance and patient-specific factors partly explain this persistence, bacterial adaptation to post-ETI conditions likely plays a critical role. Previous findings of functional shifts in bacterial variants point to underlying genotypic changes, yet the genomic basis for P. aeruginosa’s persistence remains insufficiently defined. This work aims to identify the genetic adaptations enabling P. aeruginosa to persist in CF lungs despite the improved airway environment afforded by ETI. We developed a method combining temporal allele frequency shifts and cross-patient recurrence to identify selection. My preliminary analysis revealed algG, a gene involved in alginate biosynthesis, as a promising candidate showing multiple signatures of positive selection. First, algG mutations increased in frequency across two-thirds of sampled individuals. Second, the phylogenetic analysis demonstrated the parallel evolution of algG mutations within individual hosts. Third, statistical testing showed significant enrichment for non-synonymous mutations in algG, indicating protein-altering changes are favored. I am extending this work by developing null models to quantify the significance of observed parallel evolution both within and between hosts, and using protein structural prediction to evaluate the functional impact of identified mutations. This research provides novel insights into bacterial adaptation mechanisms during CF treatment and may guide the development of more effective therapies targeting P. aeruginosa persistence. The findings will enhance our understanding of pathogen evolution within human hosts and have implications for improving treatment outcomes for CF patients. 

While flocculation is a desirable trait for brewing yeast because it eases the removal of cells from beer after fermentation, other modes of cell-to-cell adhesion can be detrimental to the brewing process. Mother-daughter separation defects cause cells to form large aggregated clusters which use more oxygen, produce a lower fermentative yield, and require more head space during fermentation. These defects can be caused by mutations to a number of genes, which makes a targeted genetic approach challenging. In this work, we used experimental evolution to eliminate mother-daughter separation defects present in a widely used brewing strain. Cells with this defect are less buoyant and settle faster than non-adhering cells. We used this property to select against cells with this defect by letting the cultures settle and propagating only cells present in the top layer of the media. We propagated top-layer cells for approximately 300 generations (about two months), collected daily optical density measurements, and conducted settling assays. Over time, we found that large, branched cell clusters decreased in frequency in our top-layer samples while the amount of single cells increased, which we confirmed through microscopy and optical density measurements. We characterized the mutations that drive this strain’s separation defect using whole genome sequencing of the evolved and ancestral populations. This project demonstrates how experimental evolution can be used to select against less desirable traits in commercially important yeast strains. Future research could implement similar or reciprocal methods to evolve for decreased or increased flocculation respectively.

Mechanistic target of rapamycin (mTOR) functions in a protein complex with raptor to control protein synthesis in eukaryotes. A reduction of function mutation in C. elegans raptor is resistant to hypoxic death. This mutation, a missense at amino acid 1033 in the daf-15 gene, is interesting because the mutation site is conserved in all mammals, suggesting that this work could shed light on hypoxic injury mechanisms in humans. The Crowder lab has discovered that a mutation called tm11331 in a gene involved in purine metabolism blocks the hypoxia resistance of the raptor mutation. We hypothesized that the tm11331 mutation restores normal protein synthesis to the raptor mutant and therefore restores hypoxic sensitivity. For my project, I examined this hypothesis by measuring nucleolus size as an indirect measurement of protein synthesis. Four strains were used in this assay: unmutated (wild-type) worms, worms with the raptor mutation, worms with the tm11331 mutation, and worms with both raptor and tm11331 mutations. From previous experiments, we know that raptor mutants have smaller nucleoli than wild-type worms, indicating that protein synthesis rates are lowered in mutated worms. We would therefore expect that protein synthesis rates and nucleolus size would be restored in worms made hypoxia sensitive by the addition of tm11331. For this assay, all strains contained a fluorescent protein that labelled the nucleoli, allowing me to image nucleoli under fluorescence. I processed each image and measured average nucleolus size in worms from each strain. Our data shows that the tm11331 mutation increased nucleolus size in strains both with and without raptor mutation. In fact, the combination of tm11331 and the raptor mutation was not significantly different from wild type. Thus, our data supports the hypothesis that the tm11331 mutation restores hypoxic sensitivity by normalizing protein synthesis.

To function as a tumor suppressor, BRCA1 (breast cancer 1) must dimerize with BARD1 (BRCA1-associated RING domain protein 1). Due to this critical interaction, loss-of-function BARD1 variants are associated with increased breast and ovarian cancer risk. Genetic testing has identified many rare single-nucleotide variants (SNVs) that cause missense substitutions in BARD1. Currently, 85.6% (1,736 of 2,028) of BARD1 missense SNVs are classified as a variant of uncertain significance (VUS) in ClinVar. A VUS classification prevents clinicians from using genetic test results to guide patient care. Consequently, there is a strong need to functionally assess BARD1 SNVs to help resolve VUS. We applied saturation genome editing (SGE) to functionally assess all possible 12,000 SNVs and 2,300 3-base deletions in BARD1. In SGE, we use CRISPR-Cas9, to edit all possible SNVs into a region of BARD1 in haploid cells. BARD1 is essential for cell growth, therefore, cells edited with loss-of-function variants become depleted from the population. We use DNA sequencing to track which SNVs become depleted from the population after 13 days in culture and are likely loss-of-function. All 14,000 variants have completed the full experimental pipeline. We show that 98% stop-gain, 29.6% splice-region, and 14.3% missense variants are loss of function relative to 1.6% of synonymous/intronic variants. The SGE data also agree strongly with current pathogenic/likely pathogenic and benign/likely benign BARD1 variants in ClinVar. Moreover, I have identified previously known and potential new protein-protein interaction interfaces through mapping our SGE data to the surfaces of BARD1’s structured domains. Ultimately, the functional scores for all BARD1 variants provide key functional evidence needed to reclassify BARD1 VUS and provide new insight into the mechanisms of BARD1 function.

Type 1 Diabetes is characterized by a dysfunctional response of the immune system, with our project focusing on CD8+ T-cells. Studying epigenomics provides us with information about differential gene expression, as well as distal enhancers and their targets. Understanding this genetic background enables more efficient means of treatment. In this paper, we look at three different kinds of sequencing: ATACseq, RNAseq, and Hi-C. Our focus up until this point has primarily rested upon the first, as we have used it to analyze chromatin accessibility across the genome in patients with T1D and healthy controls. To do so, we have found peaks within the reads for both demographics, which we then used to examine the individual peaks for each subject and the consensus peaks between each condition to see which are especially prominent. These peaks are then the focus of our differential expression analysis, which will allow us to understand the areas of significance and perform further exploration: variance calling and footprinting. As we continue with this project, we hope that RNAseq and Hi-C will provide us with information on gene expression levels and the physical structure of chromatin, respectively. The former was run and sequenced within our lab, but the latter is pre-existing data we will be drawing from for this analysis. Understanding the regulatory landscape allows for better informed treatments, not just for T1D but for autoimmune diseases as a whole.

Neuropsychiatric disorders lead to devastating impacts on a patient’s life, affecting physical movement, cognition, and behavior. A pattern observed in patients with neurodegenerative disease includes neurofibrillary tangles in the brain, which may be caused by the abnormal accumulation of the microtubule-associated protein tau (MAPT). Tau is encoded for in the MAPT gene locus on chromosome 17, where the locus commonly interacts with an enhancer to boost transcription. However, some patients have one copy of chromosome 17 with an inversion that breaks this interaction, which is associated with lower risk of disease. This raises the question of whether the MAPT locus interacts with other enhancers that increases tau production. Thus, the goal of this project is to identify genetic variants that influence the 3D interactions between the MAPT locus and potential enhancers in patients heterozygous for the MAPT inversion associated with a lower risk of neuropsychiatric disorders. To accomplish this objective, neuronal nuclei extracted from patients are analyzed using fluorescent in-situ hybridization (FISH) to identify interactions in the MAPT gene locus. We will map a 2 Mb region of chromosome 17 centered on the inversion using FISH probes. This region is broken up into ten 200 kb spots to be individually visualized using fluorescent oligonucleotides through a fluidics system, to create a composite image of all spots. Interactions involving the MAPT locus may be identified by comparing distances between spots, in which gene segments that interact would have a shorter distance compared to segments that do not interact. This would allow us to find genetic variants associated with the chromosome 17 inversion that potentially influence MAPT gene regulation.

The resistance of castration-resistant prostate cancer (CRPC) to androgen receptor signaling inhibitors (ARSIs) continues to be a significant clinical problem. Translation inhibitors are being researched as a potential treatment for AR-independent CRPC after our laboratory discovered that elevated mRNA translation as one of the major contributing factors. We screened pharmaceutical firms' known mRNA translation inhibitors in three human LuCaP models of advanced prostate cancer: AR-low prostate cancer (LuCaP 176), castration-resistant prostate cancer (LuCaP 35CR), and AR-intact castration-sensitive prostate cancer (LuCaP 35CS). We discovered that only a unique eukaryotic translation initiation factor 4E (eIF4E) 5' cap-binding domain inhibitor was able to efficiently target LuCaP 176, whereas the majority of inhibitors were able to stop the growth of LuCaP 35CS/CR. 5' cap binding inhibition increased the efficacy of enzalutamide in AR-low cells by inducing basal to luminal lineage plasticity and post-transcriptionally downregulating basal keratins. Furthermore, in the AR-low basal LuCaP 176 PDX (patient derived xenograft) model, enzalutamide effectiveness was enhanced by eIF4E cap binding domain suppression. These results are consistent with patient data indicating that people with elevated eIF4E levels have faster resistance to ARSI. Overall, our preliminary data demonstrates that translation inhibitors targeting the eIF4E cap binding domain can reverse lineage plasticity through a translation-based mechanism and sensitize AR-low prostate cancers to ARSIs. To further investigate these mechanisms and assess their implications in patient specimens, we are determining how eIF4E cap binding domain inhibition regulates translation of basal keratins and cellular plasticity along with impacting CRPC growth. Our study shows a promising method for reducing ARSI resistance in deadly metastatic CRPC which is to target eIF4E cap-binding. In order to optimize patient outcomes, this study lays the groundwork for eIF4E cap binding domain inhibition as a possible therapeutic and incorporates a cap-binding assay as a biomarker for individualized treatment.

Aberrant protein levels can lead to pathological states and subsequent disease, traditionally requiring treatment by therapeutics that work by occupying a pocket on a target protein and result in inhibition of the protein's enzymatic function. However, many high-value therapeutic targets do not have enzymatic activity and thus are not amenable to small molecule inhibition. To address this shortcoming and expand the number of druggable proteins, an intense focus on directly altering protein levels within cells has recently emerged. Targeted protein degradation (TPD) or stabilization (TPS) aims to develop therapeutics for previously undruggable targets by leveraging the endogenous protein degradation system within cells, recruiting an effector protein, either an E3 ubiquitin ligase (for TPD) or a deubiquitinase (for TPS), in proximity to a target protein. My research in the Shendure Lab  combines computational de novo protein design and high-throughput screening with the goal of identifying designed proteins capable of mediating TPD and TPS in cells. Specifically, we are collaborating with the UW Institute for Protein Design to design degrader/stabilizer binding proteins to be screened in HEK293 cells. By labeling each designed protein with a unique RNA barcode, we can leverage massively parallel DNA sequencing to characterize 6,000 de novo designed degraders/stabilizers in a single experiment. If successful, this will be the first demonstration of designed proteins that can modulate cellular protein levels, paving the way for a new therapeutic modality.

All organisms develop from a single, symmetrical cell. That symmetry must be broken at several points during embryogenesis to develop into a complex, intricate form of life. The earliest symmetry breaking event in vertebrates is the formation of the dorsal organizer, a signaling center that establishes dorsal-ventral and anterior-posterior axes. β-catenin signaling is highly conserved in the dorsal organizer and utilized during cancer proliferation. However, the mechanisms employed in selective β-catenin stabilization are still not fully understood, due in part to limited vertebrate embryological models. Established model organisms for development, like fish and frogs, pre-pattern their dorsal organizer through maternal determinants, which is lacking in mammalian model organisms who break symmetry with self-organization. Remarkably, the African Turquoise Killifish, Nothobranchius furzeri, lack a pre-pattern. This presents a strong model organism, N. furzeri, to investigate mechanisms of self-organization. In this work, I explore the metabolic shifts and mechanical forces as two potential drivers of selective β-catenin stabilization. To investigate whether fluctuations in intracellular pH (pHi) stabilize β-catenin, I created a Tol-2 mediated transgenic pHi reporter line. Using light sheet microscopy, I observed that pHi fluctuations occur after β-catenin is stabilized in the incipient dorsal organizer. This ruled out pHi as the initializing factor of β-catenin stabilization. Next, I will explore whether mechanical forces drive embryonic symmetry breaking. This model posits that local microtubules-generated forces are transduced by focal adhesions into biochemical signals, enabling selective β-catenin stabilization. To evaluate this model, I will develop a transgenic toolkit to visualize microtubules polymerization and focal adhesion signaling with pharmacological and dominant negative approaches. These experiments will elucidate the mechanism responsible for symmetry breaking in N. furzeri and potentially conserved regulators of Β-catenin signaling, foundational to our understanding of development and cancer research.

Approximately 320 million years ago, teleost fish experienced a whole-genome duplication event, which is theorized to have contributed to developmental and morphological innovations that enhanced the reproductive success of their modern descendants. However, the role of duplicated genes in the genesis of novel cell types remains unknown. Here we show that the African Turquoise Killifish (Nothobranchius furzeri) possesses a novel immune lineage specified prior to gastrulation—a far earlier stage than observed in other teleosts. Surprisingly, through single-cell RNA sequencing, we found that this lineage unexpectedly expresses nanos1b, a duplicated paralog of nanos1, a gene well known for its role in germline development across vertebrates. To verify this novel expression of nanos1b in immune cells before gastrulation, I performed RNA in situ hybridization to visualize the expression of nanos1b, eomes (a mesodermal marker), and lcp1 (a marker of mature immune cells). The results revealed co-expression of nanos1b with both eomes and lcp1, supporting the hypothesis that nanos1b expression links the myeloid lineage to the developing mesoderm. These investigations will help elucidate the pathway through which the killifish embryo fast-tracks the production of immune cells during early development.