Goal: Understanding endoderm differentiation and development
Our lab focuses on understanding how the murine definitive endoderm (DE or endoderm), one of the 3 primary germ layers that arise during gastrulation, emerges and differentiates. The endoderm gives rise to the epithelial component of the entire gastrointestinal tract and associated organs including the thyroid, parathyroid, lung liver and pancreas. Understanding how this tissue layer develops normally is key to gaining a deeper understanding of any of the pathological conditions affecting these organs. Furthermore, understanding how mammalian organs form during development in vivo can be used to design more effective protocols aimed at creating endoderm-derived organs or cell types in vitro.
I. Identify the Mechanisms that Support Endoderm Formation and Organogenesis.
To begin to understand how organs emerge from the endoderm, we have used prospective
and retrospective fate mapping of ex vivo and in vivo developing mouse embryos, respectively. We have established conditions that allow us to routinely dissect and manipulate early somite mouse embryos that have yet to initiate endoderm organogenesis (Fig 1; E8.25 embryo) and culture them through the onset of organogenesis (Fig 1; E9.5 embryo). We have used a variety of prospective approaches in this system to create a fate map of the foregut-derived organs, identify the morphological mechanisms used to close the gut tube and to demonstrate that the liver is produced by 2 distinct endoderm progenitor populations that contribute uniquely to the liver (Fig. 1). Recent genetic fate mapping has been used to confirm and establish new foregut organ progenitor relationships and to demonstrate that single hepatoblasts contribute to both hepatocytes and cholangiocytes in vivo.
We hypothesize that the two liver progenitors are supported by different inductive mesenchyme and a future goal of this project is to identify the mesoderm progenitors that support the liver.
II Assessing Heterogeneity in the Nascent Liver.
A goal of our lab is to define the signals and interactions required to initiate and maintain organogenesis from the endoderm. Knowing the answer is critical to the creation of clinically relevant cell types and tissues such as hepatocytes and pancreatic b-cells. While there are many protocols designed to create such cell types in vitro, all are based on cues from normal development. Such in-vitro protocols have failed to re-create the full functionality of the in-vivo derived cell types. We believe that these failures are due in part to the static one-dimensional nature of in vitro protocols while the conditions faced by differentiating cells during development is far more complex, occurring in the context of a dynamic 3-D embryo. To begin to elucidate the role specific pathways play during organogenesis in the context of the embryo, we have manipulated distinct pathways required for early liver and or pancreas development using small molecules. Such an approach has allowed us to uncover a previously unappreciated heterogeneity in the developing liver.
III Annotation of the Mammalian Genome: the Knock-out Mouse Project and Beyond.
Although the mammalian genome has been sequenced for over 15 years, significant gaps remain in the functional annotation of the coding region. These gaps have led to surprising trends in funding and research of human genes: of the estimated 19,000 genes only 2000 are characterized as well studied. Most funded research and drug discovery has been limited to these 2000 genes and their protein product. The reasons for these limitations include when work on the gene was first published (the older the citation, the more likely the gene is well-studied) and if the gene has been studied in an animal model. To overcome these deficits in understanding the mammalian genome, to ultimately widen the drug discovery pipeline and to understand the role particular genes play in human health and development the Knock-Out Mouse Project (KOMP), run by the NIH, is part of an international consortium designed to knock-out each gene in the mouse genome.
Of the 5000+ genes that have been knocked out so far by the consortium, ~25% result in early embryonic lethality. Over the last 5 years I have been an active collaborator on an NIH funded KOMP project designed to phenotypically characterize the homozygous knock-out of understudied genes that result in embryonic loss by E9.5 (PI- Mager lab).
Fig. 3: Our strategy for analysis of 20 novel organogenesis lethal KOMP-generated mouse lines.
Future Directions: Because of our expertise in early mouse development and our success in our collaborative KOMP project, my lab was recently awarded a KOMP grant from NICHD (R01). The goal of this grant is to characterize novel KOMP alleles that exhibit lethality during organogenesis (after E9.5 but prior to E12.5). We will use our expertise in post-gastrulation and early organogenesis to characterize and identify the phenotypic onset of at least 20 KOMP alleles (Fig. 3). We expect that many of the defects will be present at E8.5 and will result in extra-embryonic defects.
Finally we will use KOMP generated conditional ready ES cells to create conditional alleles for up to 5 genes that play putative roles in early embryonic endoderm development, but also play an essential early role in extra-embryonic endoderm development.
Graduate Student - MCB
|tguertin [at] umass.edu
|esisson [at] umass.edu
Viana, Ana Clara Negreiros Parente Capela Sampaio
Graduate Student - ABBS
|aviana [at] umass.edu