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Direct conversion of fibroblasts into induced cardiomyocytes (iCMs) holds promising potential to generate functional cardiomyocytes for drug development and clinical applications, especially for direct in situ heart regeneration by delivery of reprogramming genes into adult cardiac fibroblasts in injured hearts. For a decade, many cocktails of transcription factors have been developed to generate iCMs from fibroblasts of different tissues in vitro and some were applied in vivo. Here, we aimed to develop genetic cocktails that induce cardiac reprogramming directly in cultured cardiac fibroblasts isolated from adult mice with myocardial infarction (MICFs), which could be more relevant to heart diseases. We found that the widely used genetic cocktail, Gata4, Mef2c, and Tbx5 (GMT) were inefficient in reprogramming cardiomyocytes from MICFs. In a whole well of a 12-well plate, less than 10 mCherry(+) cells (

Direct conversion of fibroblasts into induced cardiomyocytes (iCMs) holds promising potential to generate functional cardiomyocytes for drug development and clinical applications, especially for direct in situ heart regeneration by delivery of reprogramming genes into adult cardiac fibroblasts in injured hearts. For a decade, many cocktails of transcription factors have been developed to generate iCMs from fibroblasts of different tissues in vitro and some were applied in vivo. Here, we aimed to develop genetic cocktails that induce cardiac reprogramming directly in cultured cardiac fibroblasts isolated from adult mice with myocardial infarction (MICFs), which could be more relevant to heart diseases. We found that the widely used genetic cocktail, Gata4, Mef2c, and Tbx5 (GMT) were inefficient in reprogramming cardiomyocytes from MICFs. In a whole well of a 12-well plate, less than 10 mCherry(+) cells (

To investigate the genetic etiology of anophthalmia and microphthalmia, we used exome sequencing in a Caucasian female with unilateral microphthalmia and coloboma, bilateral optic nerve hypoplasia, ventricular and atrial septal defects, and growth delays. We found two sequence variants in SALL4 - c.[575C>A], predicting p.(Ala192Glu), that was paternally inherited, and c.[2053G>C], predicting p.(Asp685His), that was maternally inherited. Haploinsufficiency for SALL4 due to nonsense or frameshift mutations has been associated with acro-renal ocular syndrome that is characterized by eye defects including Duane anomaly and coloboma, in addition to radial ray malformations and renal abnormalities. Our report is the first description of structural eye defects associated with two missense variants in SALL4 inherited in trans; the absence of reported findings in both parents suggests that both sequence variants are hypomorphic mutations and that both are needed for the ocular phenotype. SALL4 is expressed in the developing lens and regulates BMP4, leading us to speculate that altered BMP4 expression was responsible for the eye defects, but we could not demonstrate altered BMP4 expression in vitro after using small interfering RNAs (siRNAs) to reduce SALL4 expression. We conclude that SALL4 hypomorphic variants may influence eye development.

Xenopus has provided a powerful system to study cellular, developmental, and neuro-biology. The availability of their embryos and the advent of modern molecular techniques allowed investigators to revisit the observations of classical embryologists and begin to determine the molecular mechanisms underlying germ layer formation and axis induction. My thesis work took advantage of the frog Xenopus to first address the developmental role of Noggin, a Bone morphogenic protein (Bmp) antagonist, and then to determine the mechanism of Wnt-induced anterior-posterior patterning of the neural plate. The frog Xenopus, an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. In the first part of this work, I address this problem by genome editing with zinc finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in X. tropicalis induced mutations that are consistent with results of non homologous end joining at the target site, resulting in mosaic loss of fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles. Founder animals yielded progeny that carry insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity among three germline mutant alleles. Breeding an identified null allele to homozygosity resulted in tadpoles with deformaties in the cranial skeleton. Anatomical analysis revealed severe reductions in Meckel's cartilage with joint fusions. Gene expression analysis via in situ hybridization for chondrogenesis regulating factors in noggin mutants revealed a reduction in sox9 and col2a expression domains. Analysis of Bmp targets showed an expansion of hand2, edn1, and msx2 in the pharygeal arches (PAs) of mutants. This suggested a mechanism whereby incresed Bmp signaling inhibits chondrogenesis and ventralizes the

Xenopus has provided a powerful system to study cellular, developmental, and neuro-biology. The availability of their embryos and the advent of modern molecular techniques allowed investigators to revisit the observations of classical embryologists and begin to determine the molecular mechanisms underlying germ layer formation and axis induction. My thesis work took advantage of the frog Xenopus to first address the developmental role of Noggin, a Bone morphogenic protein (Bmp) antagonist, and then to determine the mechanism of Wnt-induced anterior-posterior patterning of the neural plate. The frog Xenopus, an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. In the first part of this work, I address this problem by genome editing with zinc finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in X. tropicalis induced mutations that are consistent with results of non homologous end joining at the target site, resulting in mosaic loss of fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles. Founder animals yielded progeny that carry insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity among three germline mutant alleles. Breeding an identified null allele to homozygosity resulted in tadpoles with deformaties in the cranial skeleton. Anatomical analysis revealed severe reductions in Meckel's cartilage with joint fusions. Gene expression analysis via in situ hybridization for chondrogenesis regulating factors in noggin mutants revealed a reduction in sox9 and col2a expression domains. Analysis of Bmp targets showed an expansion of hand2, edn1, and msx2 in the pharygeal arches (PAs) of mutants. This suggested a mechanism whereby incresed Bmp signaling inhibits chondrogenesis and ventralizes the

Embryonic stem cells (ESCs) hold tremendous promise for cell based therapeutics and as a biological tool. As differentiation of ESCs mimics early development, ESCs also serve as an excellent tool to study the underlying mechanisms of many developmental processes and cancer. The transition from ESC to somatic cell requires proper signaling as well as many intricate layers of gene regulation. Clarifying the interplay between these different processes is important for understanding the many transitions that happen during development as well as those that occur upon cancer metastasis., To elucidate some of the molecular mechanisms of ESC differentiation, we explored different areas of regulation within ESCs including cell signaling as well as epigenetic and transcription gene regulation. To best investigate these areas, we developed a recombinant gene technology called the Floxin system, in which we specifically and efficiently tagged genes within their endogenous loci in ESCs. The gene trap and tagged ESC lines were used to identify novel binding partners and investigate disease alleles. Through the Floxin system, we demonstrate that Ofd1, a protein required for ciliogenesis, restrains neural differentiation in embryoid bodies (EBs). We also show that Ofd1 is essential for proper Hh and Wnt signaling and that disease alleles of Ofd1 cannot rescue proper neural differentiation. Secondly, we investigate Polycomb-like 3 (Pcl3), a protein involved in histone modifications and gene repression. We found that Pcl3 incorporates into Polycomb repressive complex 2 (PRC2) and promotes complex function. Diminishment of Pcl3 decreased PRC2 binding as well as ESC self-renewal, indicating that Pcl3 enhances PRC2 targeting and ESC maintenance. Finally, we identify novel binding partners of Sall4, a transcription factor important for ESC pluripotency and reprogramming. Notably, Ofd1, Pcl3, and Sall4 are all associated with human conditions and cancer. Thus besides understanding how the

Embryonic stem cells (ESCs) hold tremendous promise for cell based therapeutics and as a biological tool. As differentiation of ESCs mimics early development, ESCs also serve as an excellent tool to study the underlying mechanisms of many developmental processes and cancer. The transition from ESC to somatic cell requires proper signaling as well as many intricate layers of gene regulation. Clarifying the interplay between these different processes is important for understanding the many transitions that happen during development as well as those that occur upon cancer metastasis., To elucidate some of the molecular mechanisms of ESC differentiation, we explored different areas of regulation within ESCs including cell signaling as well as epigenetic and transcription gene regulation. To best investigate these areas, we developed a recombinant gene technology called the Floxin system, in which we specifically and efficiently tagged genes within their endogenous loci in ESCs. The gene trap and tagged ESC lines were used to identify novel binding partners and investigate disease alleles. Through the Floxin system, we demonstrate that Ofd1, a protein required for ciliogenesis, restrains neural differentiation in embryoid bodies (EBs). We also show that Ofd1 is essential for proper Hh and Wnt signaling and that disease alleles of Ofd1 cannot rescue proper neural differentiation. Secondly, we investigate Polycomb-like 3 (Pcl3), a protein involved in histone modifications and gene repression. We found that Pcl3 incorporates into Polycomb repressive complex 2 (PRC2) and promotes complex function. Diminishment of Pcl3 decreased PRC2 binding as well as ESC self-renewal, indicating that Pcl3 enhances PRC2 targeting and ESC maintenance. Finally, we identify novel binding partners of Sall4, a transcription factor important for ESC pluripotency and reprogramming. Notably, Ofd1, Pcl3, and Sall4 are all associated with human conditions and cancer. Thus besides understanding how the