Doctor of Philosophy

dissertationZinc finger nucleases (ZFNs) are chimeric, modular proteins used for targeted mutagenesis in many model organisms and human cell lines. ZFNs bind to the designed target through a DNA binding domain and make a double-strand break (DSB) at the site. This DSB can then be repaired by the cell's repair machinery. Previous studies have revealed ZFN-induced toxicity in many cases which most likely occurs due to nonspecific cleavage by the ZFNs at secondary targets. The nonspecific DSBs can lead to unwanted secondary mutations. However, if cleavage at secondary sites is extensive, the DNA repair system is overwhelmed, leading to death of the organism or cells. The work in this dissertation describes the attempts made to develop an unbiased and comprehensive method to define these secondary targets for a pair of ZFNs in vitro in the Drosophila melanogaster genome. The strategy involves capturing all ends made by a ZFN in the Drosophila genome and then subjecting them to deep sequencing in order to define these sequences and the frequencies at which the target is cut. Towards this end, a protocol was developed to capture all ZFN-induced ends in the Drosophila genome in vitro using the rosy (ry) ZFNs. The stepwise development of the protocol is detailed here. The method was then applied to a pair of ZFNs targeting yellow (y) which are known to be toxic in Drosophila. The ends generated at the designed ZFN target were successfully captured. However, capture of other ZFN-induced ends failed. The probable causes of the failure of this strategy were also investigated and are discussed. Additional work involved the nematode Caenorhabditis elegans. ZFNs have been used successfully in nematode somatic tissue. However, attempts to generate targeted offspring using ZFNs have been futile due to widespread silencing of the transgenes in the germline. This dissertation also describes attempts to overcome this germline silencing and express ZFNs in the nematode germline

Genetic Requirement of talin1 for Proliferation of Cranial Neural Crest Cells during Palate Development

Background: Craniofacial malformations are among the most common congenital anomalies. Cranial neural crest cells (CNCCs) form craniofacial structures involving multiple cellular processes, perturbations of which contribute to craniofacial malformations. Adhesion of cells to the extracellular matrix mediates bidirectional interactions of the cells with their extracellular environment that plays an important role in craniofacial morphogenesis. Talin (tln) is crucial in cell-matrix adhesion between cells, but its role in craniofacial morphogenesis is poorly understood. Methods: Talin gene expression was determined by whole mount in situ hybridization. Craniofacial cartilage and muscles were analyzed by Alcian blue in Tg(mylz2:mCherry) and by transmission electron microscopy. Pulse-chase photoconversion, 5-ethynyl-2’-deoxyuridine proliferation, migration, and apoptosis assays were performed for functional analysis. Results: Expression of tln1 was observed in the craniofacial cartilage structures, including the palate. The Meckel’s cartilage was hypoplastic, the palate was shortened, and the craniofacial muscles were malformed in tln1 mutants. Pulse-chase and EdU assays during palate morphogenesis revealed defects in CNCC proliferation in mutants. No defects were observed in CNCC migration and apoptosis. Conclusions: The work shows that tln1 is critical for craniofacial morphogenesis in zebrafish. Loss of tln1 leads to a shortened palate and Meckel’s cartilage along with disorganized skeletal muscles. Investigations into the cellular processes show that tln1 is required for CNCC proliferation during palate morphogenesis. The work will lead to a better understanding of the involvement of cytoskeletal proteins in craniofacial morphogenesis

Genetic Requirement of talin1 for Proliferation of Cranial Neural Crest Cells during Palate Development

Background:. Craniofacial malformations are among the most common congenital anomalies. Cranial neural crest cells (CNCCs) form craniofacial structures involving multiple cellular processes, perturbations of which contribute to craniofacial malformations. Adhesion of cells to the extracellular matrix mediates bidirectional interactions of the cells with their extracellular environment that plays an important role in craniofacial morphogenesis. Talin (tln) is crucial in cell-matrix adhesion between cells, but its role in craniofacial morphogenesis is poorly understood. Methods:. Talin gene expression was determined by whole mount in situ hybridization. Craniofacial cartilage and muscles were analyzed by Alcian blue in Tg(mylz2:mCherry) and by transmission electron microscopy. Pulse-chase photoconversion, 5-ethynyl-2’-deoxyuridine proliferation, migration, and apoptosis assays were performed for functional analysis. Results:. Expression of tln1 was observed in the craniofacial cartilage structures, including the palate. The Meckel’s cartilage was hypoplastic, the palate was shortened, and the craniofacial muscles were malformed in tln1 mutants. Pulse-chase and EdU assays during palate morphogenesis revealed defects in CNCC proliferation in mutants. No defects were observed in CNCC migration and apoptosis. Conclusions:. The work shows that tln1 is critical for craniofacial morphogenesis in zebrafish. Loss of tln1 leads to a shortened palate and Meckel’s cartilage along with disorganized skeletal muscles. Investigations into the cellular processes show that tln1 is required for CNCC proliferation during palate morphogenesis. The work will lead to a better understanding of the involvement of cytoskeletal proteins in craniofacial morphogenesis

Rapid functional analysis of computationally complex rare human IRF6 gene variants using a novel zebrafish model

Large-scale sequencing efforts have captured a rapidly growing catalogue of genetic variations. However, the accurate establishment of gene variant pathogenicity remains a central challenge in translating personal genomics information to clinical decisions. Interferon Regulatory Factor 6 (IRF6) gene variants are significant genetic contributors to orofacial clefts. Although approximately three hundred IRF6 gene variants have been documented, their effects on protein functions remain difficult to interpret. Here, we demonstrate the protein functions of human IRF6 missense gene variants could be rapidly assessed in detail by their abilities to rescue the irf6 -/- phenotype in zebrafish through variant mRNA microinjections at the one-cell stage. The results revealed many missense variants previously predicted by traditional statistical and computational tools to be loss-of-function and pathogenic retained partial or full protein function and rescued the zebrafish irf6 -/- periderm rupture phenotype. Through mRNA dosage titration and analysis of the Exome Aggregation Consortium (ExAC) database, IRF6 missense variants were grouped by their abilities to rescue at various dosages into three functional categories: wild type function, reduced function, and complete loss-of-function. This sensitive and specific biological assay was able to address the nuanced functional significances of IRF6 missense gene variants and overcome many limitations faced by current statistical and computational tools in assigning variant protein function and pathogenicity. Furthermore, it unlocked the possibility for characterizing yet undiscovered human IRF6 missense gene variants from orofacial cleft patients, and illustrated a generalizable functional genomics paradigm in personalized medicine

<i>IRF6</i> missense gene variants can rescue periderm rupture and restore normal craniofacial development.

<p><b>(A-T)</b> Craniofacial morphologies of maternal-null <i>irf6</i> ^<i>-/-</i> embryos rescued by human <i>IRF6</i> missense gene variant mRNA microinjections (100 pg/embryo) at 96 hpf stained with alcian blue. (A-D) Uninjected wild type control. (E-H) p.P12L. (I-L) p.P76L. (M-P) p.T100A. (Q-T) p.P222L. Scale bars = 150 μm, n = 3.</p

Functional characterization of human <i>IRF6</i> missense gene variant protein functions with the zebrafish <i>irf6</i> model.

<p><b>(A)</b> Experimental approach for characterizing protein functions of human <i>IRF6</i> missense gene variants. Variant mRNAs were synthesized and microinjected into maternal-null <i>irf6</i> ^<i>-/-</i> embryos at the one-cell stage and assessed for phenotypic rescue at 24 hpf. <b>(B-C)</b> Protein modeling of the protein-binding domain and C-terminus of IRF6 using ExPASy with crystalline structures of IRF1. (B) is mapped with missense variant amino acid residues (green) whose mRNA rescued the periderm rupture phenotype, while (C) is mapped with missense variant amino acid residues (red) whose mRNA failed to rescue. <b>(D-F)</b> Results for functional rescue of periderm rupture with maternal-null <i>irf6</i> ^<i>-/-</i> embryos for representative human <i>IRF6</i> missense gene variants. Results were classified as rescued if any maternal-null <i>irf6</i> ^<i>-/-</i> embryos injected with variant mRNA remained alive and phenotypically wild type at 24 hpf (50 embryos/round, n = 3). Missense gene variants were categorized by location within the IRF6 protein, and by computational results from PolyPhen-2 and SIFT on whether the <i>in silico</i> predictions agreed on the deleterious effects of the missense gene variants on protein function. Further shown are ACMG guideline pathogenicity predictions (pathogenic, likely pathogenic, uncertain, and benign), and the number of families identified for each variant (all gene variant annotations were based on NM_006147.3).</p

Functional characterization of human <i>IRF6</i> missense gene variant protein functions with the zebrafish <i>irf6</i> model.

<p><b>(A)</b> Experimental approach for characterizing protein functions of human <i>IRF6</i> missense gene variants. Variant mRNAs were synthesized and microinjected into maternal-null <i>irf6</i> ^<i>-/-</i> embryos at the one-cell stage and assessed for phenotypic rescue at 24 hpf. <b>(B-C)</b> Protein modeling of the protein-binding domain and C-terminus of IRF6 using ExPASy with crystalline structures of IRF1. (B) is mapped with missense variant amino acid residues (green) whose mRNA rescued the periderm rupture phenotype, while (C) is mapped with missense variant amino acid residues (red) whose mRNA failed to rescue. <b>(D-F)</b> Results for functional rescue of periderm rupture with maternal-null <i>irf6</i> ^<i>-/-</i> embryos for representative human <i>IRF6</i> missense gene variants. Results were classified as rescued if any maternal-null <i>irf6</i> ^<i>-/-</i> embryos injected with variant mRNA remained alive and phenotypically wild type at 24 hpf (50 embryos/round, n = 3). Missense gene variants were categorized by location within the IRF6 protein, and by computational results from PolyPhen-2 and SIFT on whether the <i>in silico</i> predictions agreed on the deleterious effects of the missense gene variants on protein function. Further shown are ACMG guideline pathogenicity predictions (pathogenic, likely pathogenic, uncertain, and benign), and the number of families identified for each variant (all gene variant annotations were based on NM_006147.3).</p

Zebrafish periderm rupture assay can detect <i>IRF6</i> missense gene variants with reduced protein function by mRNA dosage titration.

<p><b>(A)</b> Identification and characterization of <i>IRF6</i> missense gene variants in the ExAC and gnomAD databases. p.V274I alleles were identified in all populations. <b>(B)</b> mRNA dosage titration experiement results for a subset of missense gene variants correlating amount of variant mRNA microinjected to the percent of maternal-null <i>irf6</i> ^<i>-/-</i> embryos rescued from rupture and undergoing normal embryonic development at 24 hpf. The missense variants were classified into three categories based on levels of protein function. No variant was identified in ExAC/gnomAD that could not rescue the periderm rupture phenotype. Error bar = 2xSEM, 50 embryos/round, n = 3.</p

Generation and characterization of the zebrafish <i>irf6</i> CRISPR mutant model.

<p><b>(A)</b> Zebrafish <i>irf6</i> gene structure composed of eight exons, a helix-turn-helix DNA-binding domain (yellow), and a SMIR/IAD protein-binding domain (green). The CRISPR gRNA target site was located in exon 6 at the start of the protein-binding domain. Sanger sequencing of the target site revealed a -8bp deletion (Δ8bp) that created a frameshift and premature stop codon, truncating the protein to 29 kD as predicted by in silico translation. Another +5bp insertion was also identified. <b>(B)</b> Breeding pedigree revealed the <i>irf6</i> mutant phenotype in F3 and the importance of maternal transcripts. <b>(C)</b> Top: western blot at the sphere stage (4 hpf) revealed a lack of Irf6 full-length (55 kD) or truncated (29 kD) protein in all maternal <i>irf6</i> ^{<i>Δ8bp/Δ8bp</i>} embryos but not paternal <i>irf6</i> ^{<i>Δ8bp/Δ8bp</i>} embryos or wild type embryos. Bottom: relative gene expression by RT-qPCR revealed a lack of <i>irf6</i> mRNA transcripts in all maternal <i>irf6</i> ^{<i>Δ8bp/Δ8bp</i>} embryos but comparable levels between wild type and paternal <i>irf6</i> ^{<i>Δ8bp/Δ8bp</i>} embryos. Error bar = 2xSEM, n = 3. <b>(D-D’)</b> Wild type embryos at the sphere stage (4 hpf) (D) and at the 30% epiboly stage (5 hpf) (D’). <b>(E-E’)</b> Maternal <i>irf6</i> ^{<i>Δ8bp/Δ8bp</i>} embryos at the sphere stage (4 hpf) (E) and displaying the periderm rupture phenotype at 5 hpf (E’). Scale bar = 250 μm. <b>(F)</b> Cross-sectional schematic through the embryonic midline illustrating the zebrafish embryo epiboly process. Arrows represent cell and yolk directional movements. Wild type embryos experience rapid cellular lamination and yolk doming between 4–5 hpf, while maternal-zygotic <i>irf6</i> ^{<i>Δ8bp/Δ8bp</i>} embryos experience incomplete periderm differentiation and animal pole/yolk separation.</p

Periderm rupture and craniofacial development can be rescued by injection of either zebrafish or human wild type <i>IRF6</i> mRNA.

<p><b>(A-L)</b> Zebrafish embryos at 96 hpf stained with alcian blue for cartilaginous craniofacial elements. Maternal/zygotic-null <i>irf6</i> ^<i>-/-</i> embryos were rescued by microinjection of either zebrafish <i>irf6</i> mRNA (E-H) or human <i>IRF6</i> mRNA (I-L) at the one-cell stage, preventing the periderm rupture phenotype and restoring normal craniofacial development compared to wild type embryos (A-D). Scale bar = 150 μm. <b>(M-N)</b> Dimensional measurements of dissected ethmoid plates at 96 hpf, with length (<i>l</i>) and width (<i>w</i>) denoted by dashed lines on panel (D). The length (M) and width (N) of ethmoid plates from zebrafish and human <i>IRF6</i> mRNA rescued maternal/zygotic-null <i>irf6</i> ^<i>-/-</i> embryos are statistically indistinguishable in dimensions compared those of wild type embryos. Error bar = 2xSEM, n = 12.</p