Mary Lou King
Professor of Cell Biology and Anatomy
Ph.D. Indiana University; Postdoctoral training MIT
email: mking@med.miami.edu
Molecular Developmental Biology, Genes Involved in Embryonic Patterning and Germ Cell Specification in Xenopus, mRNA Localization in the Oocyte

RESEARCH INTERESTS

My laboratory studies how spatial patterning and cell fate is determined in the early Xenopus embryo. Our lab and others in the field have shown that the first step in patterning the embryo appears to be the localization of specific mRNAs to the vegetal cortex during oogenesis. Evidence indicates that the proteins encoded by localized mRNAs influence gene expression in a region specific manner, leading to cellular diversification. Our lab has isolated seven vegetally localized mRNAs from Xenopus oocytes. Interestingly, five of these are localized to the germ plasm, a specialized cytoplasmic domain required for the formation of the future germ cells. In contrast, the remaining two RNAs are involved in specifying somatic cell identity. Since only the germ cell lineage has a program of differentiation that maintains totipotency, discovering how the germline is initially specified and develops separate from the somatic lineage is a fundamental problem in developmental biology. Our long-term goals are to 1) understand how precursors to the germ cells retain developmental totipotency within the context of endoderm and 2) characterize the RNA transport systems involved in their localization.

Remarkably, three of these germ plasm RNAs, Xcat-2 (related to nanos), Xdazl (in DAZ family), and DeadSouth (in vasa family) are related to germ cell components in Drosophila. All three of these RNAs encode RNA binding proteins and are translationally regulated. We are interested in identifying the downstream targets of these germ cell components and their function in specifying germ cell development. We have found that anti-sense depletion of Xdazl RNA results in the severe reduction or loss of primordial germ cells because they fail to migrate out of the endoderm.

VegT mRNA, encodes a T-box transcription factor and is a pivotal gene in patterning somatic cells in Xenopus. We have shown that maternal VegT is required for germ layer formation during gastrulation and specifically for endoderm identity. Experimental approaches used in these studies include the creation of dominant negatives, antisense oligos, over-expression, ectopic expression, RT-PCR, immunocytochemistry and in situ hybridization. It will be important to discover the downstream targets of VegT and how the boundaries of endoderm are established.

The mechanism through which the spatial distribution of mRNAs is established and maintained represents a fascinating new area of investigation in both cell and developmental biology that we are actively pursuing. We have found that VegT, and the germ plasm mRNAs localize by at least two different mechanisms and at different times during oogenesis. We have determined the RNA signal required for proper localization of Xcat-2 and VegT and are currently working on isolating the proteins that bind these localization signals. Microinjection studies with mutant constructs of localized RNAs allow us to identify the sequences required for localization and to assess the function of associated proteins. Confocal microscopy of living stage I oocytes injected with fluorescently tagged transcripts permits the study of localization in real time. By studying localized maternal RNAs and their relationship to the cytoskeleton, we hope to understand how molecular polarity is established in the oocyte and how it directs regional specification in the early embryo.

REPRESENTATIVE PUBLICATIONS

Mosquera, L., Forristall, C. Zhou Y., and King, M.L. (1993) A mRNA Localized to the Vegetal Cortex of Xenopus Oocytes Encodes a Protein With a nanos-Like Zinc Finger Domain. Development 117:377-387.

Elinson, R., King, M.L., Forristall, C. (1993) Isolated Vegetal Cortex From Xenopus Oocytes Selectively Retains Localized mRNAs. Developemental Biology 160:554-562.

Forristall, C., Pondel, M., Chen, L. and King, M.L. (1995) Patterns of Localization and Cytoskeletal Association of Two Vegetally Localized RNAs, Vg1 and Xcat-2. Development 121:201-208.

King, M.L. (1995) "mRNA Localization During Frog Oognesis" In: Localized RNAs. pp. 137-148; Molecular Biology Intelligence Unit (ed. H. Lipshitz) R.G. Landes Company of Biomedical Publishers, Austin, TX.

Zhou, Y. and King, M.L. (1996) Localization of Xcat-2 RNA, a Putative Germ Plasm Component, to the Mitochondrial Cloud in Xenopus Stage I Oocytes. Development 122: 2947-2953.

Zhang, J. and King, M.L. (1996) Xenopus VegT RNA is Localized to the Vegetal Cortex During Oogenesis and Encodes a Novel T-box Transcription Factor Involved in Mesodermal Patterning. Development 122:4119-4129.

Zhou, Y. and King, M.L. (1996) RNA Transport to the Vegetal Cortex of Xenopus Oocytes. Developemental Biology 179: 173-183.

Wilkes, C., Buckhaults, P. King, M.L., Moremen, K. and Pierce, M. (1997). Cloning of the Xenopus laevis Oocyte Lectins and Characterization of their mRNA Levels During Early Development. Glycobiology 7:367-372.

Houston, D., Zhang, J., Maines, J., Wasserman, S. and King, M.L. (1998). A Xenopus DAZ-like gene encodes an RNA component of germ plasm and is a functional homologue of Drosophila boule. Development 125: 171-180.

Zhang, J., Houston, D., King, M. L., Payne, C., Wylie, C. and Heasman, J. (1998). The role of maternal VegT in establishing the primary germ layers in Xenopus embryos. Cell 94:515-524.

MacArthur, H., Bubunenko, M., Houston, D. and King, M.L. (1999). Xcat2 RNA is a translationally sequestered germ plasm component in Xenopus. Mech. Develop. 84:75-88.

King, M.L., Zhou, Y., and Bubunenko, M. (1999) "Polarizing Genetic Information in the Egg: RNA localization in the frog oocyte." BioEssay, 21: 546-557.

Zhang, J. and King, M. L. (1999) "PCR based cloning of cortically localized RNAs from Xenopus oocytes" In Methods in Molecular Biology, Vol. 135: Developmental Biology Protocols, Vol. II pp 301-306. (R. S. Tuan and C.W. Lo eds) Humana Press Inc., Totowa, N.J.

MacArthur, H., Houston, D, Bubunenko, M., Mosquera, L. and King, M.L (2000). DeadSouth is a germ plasm specific DEAD-Box RNA helicase in Xenopus related to eIF4A. Mech. Develop. 95:291-295.

Houston, D. W. and King, M.L. (2000). A critical role for Xdazl, a germ plasm-localized RNA, in the differentiation of primordial germ cells in Xenopus. Development 127:447-456.

Houston, D.H. and King, M.L. (2000) "Germ Plasm and Molecular Determinants of Germ Cell Fate". In: Current Topics in Developmental Biology Vol. 50, pp. 155-181. (eds. R.A. Pedersen and G.P. Schatten). Academic Press, N.Y.

Bubunenko, M. and King, M.L. (2001) Biochemical characterization of a cellular structure retaining vegetally localized RNAs in Xenopus late stage oocytes. J Cell Biochem. 80:560-570.

Bubunenko, M., Vempati, U.D., Mowry, K. King, M.L. (2001). Identification of the RNA localization signal for VegT, the endoderm determinant in Xenopus. (In Preparation)


HONORS AND PROFESSIONAL ACTIVITIES

*Damon Runyon-Walter Winchell Fellow
*Basil O-Connor Scholar, March of Dimes
*Editorial Board of Current Topics in Development
*FASEB Board of Directors

Currently Funded Research:

NIH, RO1-GM33932-16 Grant Period: 4/2001-4/2005 "Establishing Germ Cell Fate in
Xenopus" (Received a fundable score in November, pending budget decision)

NSF IBN-96-01209 "RNA Transport" Dates: 3/00-3/03.

Human Frontier Science Program (HFSP) Dates: 11/98-10/01




Mary Lou King, Ph.D.
Department of Cell Biology (R-124)
University of Miami School of Medicine
1011 NW 15th St
Miami, FL 33136
mking@med.miami.edu
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WWW links related to developmental biology and King's research

Society for Developmental Biology
Genetics and Developmental Biology Journals
Xenopus Resource Information
University of Miami School of Medicine

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