How cell movements are coordinated during morphogenesis is not well understood. We focus on epiboly, which describes the thinning and spreading of a multilayered cell sheet. The first phase of epiboly involves the doming of the yolk cell up into the overlying blastoderm. We previously showed that over-expression of a dominant– negative eomesodermin a construct inhibits doming. Here I report my analysis of embryos lacking both maternal and zygotic Eomesodermin A (MZeomesa). eomesafh105 mutant embryos (1) exhibit a doming delay, (2) have defective yolk cell microtubules, (3) have tightly packed deep cells with more bleb – like protrusions and (4) express early endoderm markers abnormally. Despite these phenotypes, the majority of MZeomesa embryos are able to complete epiboly and form endodermal derivatives. In both Xenopus and mice, Eomesodermin has also been implicated in the regulation of gastrulation movements and cell fate specification, suggesting a conserved role for Eomesodermin throughout vertebrate development.

How cell movements are coordinated during morphogenesis is not well understood. We focus on epiboly, which describes the thinning and spreading of a multilayered cell sheet. The first phase of epiboly involves the doming of the yolk cell up into the overlying blastoderm. We previously showed that over-expression of a dominant– negative eomesodermin a construct inhibits doming. Here I report my analysis of embryos lacking both maternal and zygotic Eomesodermin A (MZeomesa). eomesafh105 mutant embryos (1) exhibit a doming delay, (2) have defective yolk cell microtubules, (3) have tightly packed deep cells with more bleb – like protrusions and (4) express early endoderm markers abnormally. Despite these phenotypes, the majority of MZeomesa embryos are able to complete epiboly and form endodermal derivatives. In both Xenopus and mice, Eomesodermin has also been implicated in the regulation of gastrulation movements and cell fate specification, suggesting a conserved role for Eomesodermin throughout vertebrate development.

The definitive endoderm gives rise to several specialized organs, including the thymus. Improper development of the definite endoderm or its derivatives can lead to human disease; in the case of the thymus, immunodeficiency or autoimmune disorders. Human pluripotent stem cells (hPSCs) have emerged as a system to model human development, as study of their differentiation allows for elucidation of the molecular basis of cell fate decisions, under both healthy and impaired conditions. Here, we first developed a CRISPR-effector system to control endogenous gene expression in hPSCs, a novel approach to manipulating hPSC state. Next, the human-specific, loss-of-function phenotypes of candidate transcription factors driving hPSC-to-definitive endoderm differentiation were analyzed through combined CRISPR-perturbation and single-cell RNA-sequencing. This analysis revealed the importance of TGFβ mediators in human definitive endoderm differentiation as well as identified an unappreciated role for FOXA2 in human foregut development. Finally, as the differentiation of definitive endoderm to thymic epithelial progenitors (TEPs) is of particular interest, a single-cell transcriptomic atlas of murine thymus development was generated in anticipation of identifying factors driving later stages of TEP differentiation. Taken together, this dissertation establishes a CRISPR-effector system to interrogate gene and regulatory element function in hPSC differentiation strategies, details the role of specific transcription factors in human endoderm differentiation, and sets the groundwork for future investigations to characterize hPSC-derived TEPs and the factors driving their differentiation.

The pancreas is located below the liver and adjacent to the small intestine where it connects to the duodenum. It consists of exocrine and endocrine components. The exocrine portion makes enzymes which are deposited in the duodenum to digest fats, proteins, and carbohydrates. Exocrine tissue also makes bicarbonates that neutralize stomach acids. The endocrine portion produces hormones such as insulin and glucagon which are released into the blood stream. These hormones regulate glucose transport into the body's cells and are crucial for energy production. The pancreas is associated with diseases such as cancer, diabetes, Annular pancreas and Nesidioblastosis. Annular pancreas and Nesidioblastosis are congenital malformations associated with excess endocrine tissue of the pancreas and its structures. Understanding the development of the pancreas might lead to insight of these diseases. The pancreas arises from the endoderm. In zebrafish, Nodal signaling activates mix-type and gata genes that then function together to regulate sox32 expression which is necessary and sufficient to induce endoderm formation. Interestingly, sox32 is exclusive to zebrafish and works synergistically with pou5f1 to regulate its own expression and turn on sox17 expression. sox17is evolutionarily conserved from zebrafish to mouse and is necessary for endoderm formation. Signals from within the endoderm and the surrounding mesoderm specify regions in the endoderm to develop into the pancreas and other endodermal organs. Sonic hedgehog (shh) expression in the foregut establishes the anterior boundary of the pancreas primordium while cdx4 expression establishes the posterior boundary, but what regulates these factors is unclear. We determined that two Three Amino Acid Loop Extension (TALE) homeodomain transcription cofactors, Meis3 and Pbx4, regulate shh expression in the anterior endoderm. Disrupting either meis3 or pbx4 reduces shh expression in the anterior endoderm. As a result, anterior ectopic insulin expression occurs outside the normal pancreatic domain. Therefore, we discovered upstream regulatory factors of shhexpression in the anterior endoderm, which is necessary for patterning the endoderm and pancreas primordium. We performed an ENU (N-ethyl-N-nitrosurea) haploid screen to look for endocrine pancreas mutants and to find other factors involved in pancreas development and patterning. From the screen, we characterized two mutants. We identified an aldh1a2 mutant, aldh1a2um22, which blocks the production of Retinoic Acid (RA) from vitamin A. While RA is known to be necessary for differentiation of the pancreas and liver, we also found it to be necessary for intestine differentiation. Two other aldh family genes exist in the zebrafish genome, but our data suggests that aldh1a2is the only Aldh that functions in endoderm differentiation and it is maternally deposited. From the screen, we discovered a second mutant, 835.4, that spontaneously arose within the background. pou5f1 expression is normal in mutant embryos, but sox32 expression is reduced and sox17 expression is lost. Downstream endoderm genes of sox17 are also lost and as a result no endodermal organs develop. Rescue experiments indicate that the mutation is located between sox32 and sox17 in the endoderm pathway. We currently have not been successful at mapping this mutation and therefore are unable to rule out the possibility that it lies in the sox17 gene. However, our data suggest that the mutation occurs in a new gene that is necessary for sox17 expression, potentially working with sox32 and/or pou5f1.

This thesis investigates the applicability of novel approaches designed to study the molecular mechanisms required for the initiation of organogenesis within the early endoderm. The endoderm is the germ layer that gives rise to the gut-tube and associated organs including the thyroid, lung, liver and pancreas. Our laboratory focuses on understanding the molecular mechanisms governing the developmental transition from endoderm to liver and pancreas. Several signaling pathways including Wnt, Retinoic Acid (RA), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor-β (TGFβ) have been implicated in the emergence of the liver bud from the endoderm in the mouse or other vertebrate species. However, neither the exact signals nor the precise roles during budding process have been identified, due to the complexity of specifically altering these essential pathways using traditional genetic approaches during the earliest stages of endoderm organogenesis. These traditional techniques include transgenic, knockout or conditional knockouts strategies. To overcome the difficulties of genetic accessibility, our laboratory has optimized two complementary approaches, electroporation and addition of activators or inhibitors directly to the culture media, to study the earliest stages of organ formation using an ex vivo culture system (whole embryo culture), that allow us for normal embryonic development for up to two days. This ex-vivo technique also provides the opportunity to access and manipulate the endoderm, specifically the liver and pancreas precursor cells, prior to organ specification. Because the endoderm undergoes normal liver and pancreas specification in our ex vivo system by 24 hours after culture begin, we reason that it is possible to manipulate gene expression at the onset of culture. We then determine the effects of this manipulation on liver or pancreas development by molecular and morphological analysis after culture. The first approach we developed is the use of directional electroporation of nucleic acids to manipulate a specific region of the endoderm, particularly on liver and pancreas developmental processes. The second method is global inhibition or activation using inhibitors or growth factors activators, focusing on the TGFβ signaling pathway. These techniques will be performed prior to, or concurrent with, liver and pancreas specification, followed by embryo culture until after the onset of organogenesis. The combination of these techniques constitutes a practical approach to stage-manage the endoderm in a temporally and spatially distinct manner. In addition, it will allow us to alter specific signaling pathways without the labor-intensive generation of genetically modified animals. Indeed, establishment of these methodologies may provide a robust tool for rapid screening of candidate genes and signaling molecules underlying organogenesis in any endodermally derived organ in mouse embryos.

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

Au cours de la préimplantation, l'embryon de souris produit deux lignages cellulaires, le trophectoderme (TE), et la masse cellulaire interne (MCI) qui elle-même se différencie en épiblaste (Epi) et en endoderme primitif (EPr), caractérisés respectivement par l'expression exclusive de Nanog et de Gata6. La voie FGF/MAPK joue un rôle critique dans l’acquisition de l’identité EPr. J’ai examiné l’expression de pERK, DUSP4 et ETV5 qui permettent de visualiser l'activité des MAPK. Ces analyses ont été effectuées en activant ou inhibant la voie FGF/MAPK, ainsi que dans des embryons mutants pour Nanog et/ou Gata6. Ceci a permis d’observer l’activation de la voie FGF/MAPK dès E3,25. Un autre volet de mon travail a été d'analyser la voie de l’IGF dans les embryons préimplantatoires afin de comprendre l’influence de cette voie dans les différents lignages. J’ai montré que le récepteur activé pIGF1R est exprimé de manière différentielle dans le TE, l’EPr et l’Epi au cours du développement. Une supplémentation d’IGF1 induit une augmentation du nombre de cellules en deux phases, d'abord de l’Epi puis de l’EPr. A l’inverse, une perte de fonction d’IGF1R induit une diminution du nombre de cellules entre E3,75 et E4,25. / During preimplantation, mouse embryo produces two cellular lineages, the trophectoderm (TE), and the inner cell mass (ICM), which differentiates in epiblast (Epi) and primitive endoderm (PrE), characterized respectively by the complementary expression of Nanog and Gata6. FGF/MAPK pathway plays a critical role in the acquisition of a PrE identity. I examined the expression of the markers of MAPK activity pERK, DUSP4 and ETV5. The analyze was performed with activation or inhibition of FGF/MAPK pathway and in mutant embryos for Nanog or Gata6. This showed that FGF/MAPK pathway is activated as soon as E3,25. I have also analyzed the IGF pathway in preimplantation embryos in order to understand the role of this pathway in embryonic lineages. I showed that active receptor pIGF1R is differentially expressed in TE, PrE and Epi during embryonic development. Supplementation with IGF1 induces an increase in cell number in two phases, first in Epi then in PrE. Conversely, loss of function of IGF1R induces a decrease in cell number between E3,75 and E4,25.

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Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 144-160.