Research & Scholarship
Current Research and Scholarly Interests
With the complete sequence of the human and mouse genomes on the horizon, the critical next step in genomic analysis is to provide functional criteria for the newly discovered proteins. Understanding the diverse protein functions present within complex genomes must encompass many different and unique approaches. These functional approaches currently include two-hybrid screens for interacting proteins, microarray techniques for visualizing expression complexity, and other assays designed to elucidate specialized functions. We have developed one such functional-based assay that we are utilizing to identify potentially hundreds of molecules that can alter specific cell-fate responses.
Our main focus is to understand the signals necessary for patterning and specifying diverse cellular fates during gastrulation in the mouse. Mouse gastrulation, even more so than amphibian and teleost gastrulation, is a period of vast differentiation and growth. During this stage, the mouse embryo transitions from having only two cell types, to having hundreds. Although an incredibly rich source of cell signaling, the mouse gastrula has not been used by molecular biologists to mine for molecules. This is mainly due to the size (100mm) and inaccessibility of the mouse gastrula, which therefore precludes the effective use of biochemistry, embryology and molecular assays in general.
We have devised a screen that taps the identity of molecules involved in cell-fate specification during mouse gastrulation. This approach delivers random combinations of cDNAs from mouse gastrula libraries into the more tractable Xenopus embryo. We then observe these embryos for changes in specific marker gene expression, indicating changes positive or negative in cell-fate. We are particularly interested in the alteration of mesodermal, endodermal, neural, endothelial and somitic cell-fate decisions. By proceeding with a trial run of the screen, we have already identified 17 molecules, 8 of which have no previously understood function.
Of the 8 unexplored molecules identified, we are currently characterizing 4 in-depth. One of these inhibits vasculogenesis and causes the ectopic formation of neurons. Another is an endogenous inhibitor of MAP Kinase signaling and is required for the formation of mesodermal cells. Two others can induce the formation of endoderm. Studies on these proteins, and others like them, are on going in our laboratory.
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Evolutionary perspectives into placental biology and disease
Applied & Translational Genomics
2013; 2: 64-69
In all mammals including humans, development takes place within the protective environment of the maternal womb. Throughout gestation, nutrients and waste products are continuously exchanged between mother and fetus through the placenta. Despite the clear importance of the placenta to successful pregnancy and the health of both mother and offspring, relatively little is understood about the biology of the placenta and its role in pregnancy-related diseases. Given that pre- and peri-natal diseases involving the placenta affect millions of women and their newborns worldwide, there is an urgent need to understand placenta biology and development. Here, we suggest that the placenta is an organ under unique selective pressures that have driven its rapid diversification throughout mammalian evolution. The high divergence of the placenta complicates the use of non-human animal models and necessitates an evolutionary perspective when studying its biology and role in disease. We suggest that diversifying evolution of the placenta is primarily driven by intraspecies evolutionary conflict between mother and fetus, and that many pregnancy diseases are a consequence of this evolutionary force. Understanding how maternal-fetal conflict shapes both basic placental and reproductive biology - in all species - will provide key insights into diseases of pregnancy.
View details for DOI 10.1016/j.atg.2013.07.001
View details for PubMedCentralID PMC5121266
The E3 ubiquitin ligase GREUL1 anteriorizes ectoderm during Xenopus development
2002; 251 (2): 395-408
We have identified a family of RING finger proteins that are orthologous to Drosophila Goliath (G1, Gol). One of the members, GREUL1 (Goliath Related E3 Ubiquitin Ligase 1), can convert Xenopus ectoderm into XAG-1- and Otx2-expressing cells in the absence of both neural tissue and muscle. This activity, combined with the finding that XGREUL1 is expressed within the cement gland, suggests a role for GREUL1 in the generation of anterior ectoderm. Although GREUL1 is not a direct inducer of neural tissue, it can activate the formation of ectopic neural cells within the epidermis of intact embryos. This suggests that GREUL1 can sensitize ectoderm to neuralizing signals. In this paper, we provide evidence that GREUL1 is an E3 ubiquitin ligase. Using a biochemical assay, we show that GREUL1 catalyzes the addition of polyubiquitin chains. These events are mediated by the RING domain since a mutation in two of the cysteines abolishes ligase activity. Mutation of these cysteines also compromises GREUL1's ability to induce cement gland. Thus, GREUL1's RING domain is necessary for both the ubiquitination of substrates and for the conversion of ectoderm to an anterior fate.
View details for DOI 10.1006/dbio.2002.0814
View details for Web of Science ID 000179377900015
View details for PubMedID 12435366
Wnt signaling in Xenopus embryos inhibits Bmp4 expression and activates neural development
GENES & DEVELOPMENT
1999; 13 (23): 3149-3159
We report a new role for Wnt signaling in the vertebrate embryo: the induction of neural tissue from ectoderm. Early expression of mouse wnt8, Xwnt8, beta-catenin, or dominant-negative GSK3 induces the expression of neural-specific markers and inhibits the expression of Bmp4 in Xenopus ectoderm. We show that Wnt8, but not the BMP antagonist Noggin, can inhibit Bmp4 expression at early gastrula stages. Furthermore, inhibition of beta-catenin activity in the neural ectoderm of whole embryos by a truncated TCF results in a decrease in neural development. Therefore, we suggest that a cleavage-stage Wnt signal normally contributes to an early repression of Bmp4 on the dorsal side of the embryo and sensitizes the ectoderm to respond to neural inducing signals from the organizer. The Wnt targets Xnr3 and siamois have been shown previously to have neuralizing activity when overexpressed. However, antagonists of Wnt signaling, dnXwnt8 and Nxfrz8, inhibit Wnt-mediated Xnr3 and siamois induction, but not neural induction, suggesting an alternative mechanism for Bmp repression and neuralization. Conversely, dnTCF blocks both Wnt-mediated Xnr3 and neural induction, suggesting that both pathways require this transcription factor.
View details for Web of Science ID 000084287900011
View details for PubMedID 10601040
From receptor to nucleus: the Smad pathway
CURRENT OPINION IN GENETICS & DEVELOPMENT
1997; 7 (4): 467-473
The transforming growth factor-beta (TGF-beta) superfamily plays a central role in the specification and patterning of cells in the early embryo. Several years ago, the TGF-beta s were shown to signal through serine/threonine receptor kinases. Now, with the identification of Smad proteins, we can trace the TGF-beta signal transduction pathway from the receptors into the nucleus.
View details for Web of Science ID A1997XU02500004
View details for PubMedID 9309176
A novel mesoderm inducer, Madr2 functions in the activin signal transduction pathway
GENES & DEVELOPMENT
1996; 10 (15): 1880-1889
A functional assay to clone mouse mesoderm inducers has identified the mouse gene Mad related 2 (Madr2). Madr2 induces dorsal mesoderm from Xenopus ectoderm and can mimic the organizer in recruiting neighboring cells into a second axis. By analyzing the expression of a lacZ/Madr2 fusion protein, we find Madr2 confined to the nucleus in the deep, anterior cells of the second axis, whereas in epidermal and more posterior cells the protein is cytoplasmically localized. This context-dependent nuclear localization suggests that in certain regions of the embryo, Madr2 responds to a localized signal and amplifies this signal to form the second axis. Furthermore, although Madr2 remains unlocalized in ectodermal explants, addition of activin enhances the concentration of Madr2 in the nucleus. Significantly, a functional lacZ fusion to a carboxy-terminal portion of Madr2 is nuclear localized even in the absence of activin. This indicates that Madr2 contains a domain that can activate downstream components and a repressive domain that anchors the protein in the cytoplasm. Nuclear localization of Madr2 in response to activin, and the activin-like phenotypes induced by overexpression of Madr2, indicate that Madr2 is a signal transduction component that mediates the activity of activin.
View details for Web of Science ID A1996VB85000004
View details for PubMedID 8756346
- A human Mad protein acting as a BMP-regulated transcriptional activator Nature 1996; 381