Research Interests
We are conducting a whole-genome analysis of gastrulation in the Drosophila embryo. This process is initiated by a maternal transcription factor called Dorsal, which is distributed in a broad concentration gradient across the dorsal-ventral axis of the early embryo. High levels of the gradient initiate the differentiation of the mesoderm, while low levels control the development of the neurogenic ectoderm and dorsal ectoderm. Whole-genome tiling arrays have identified as many as 100 Dorsal target genes, including several microRNA genes. High levels of Dorsal activate target genes required for the invagination and spreading of the mesoderm, such as the FGF receptor Heartless. In contrast, low levels of the gradient activate secreted signaling molecules in the neurogenic ectoderm that control the behavior of the neighboring germ layers.
Tissue-specific enhancers have been identified for ~25 of the Dorsal target genes using a combination of bioinformatics methods and ChIP-chip assays. Coordinately regulated enhancers that respond to similar thresholds of the Dorsal gradient contain shared sequence motifs. Some of these enhancers possess a fixed organization of cis-regulatory elements. Computational modeling methods accurately simulate the type 2 response: activation by intermediate levels of the Dorsal gradient in ventral regions of the neurogenic ectoderm. These studies suggest that slight changes in the spacing between critical binding sites can produce significant variations in gene expression.
The comprehensive analysis of dorsal-ventral patterning led to a surprising result: differential gene activity might be regulated at the level of Pol II elongation. Pol II pausing has been described for several genes in both flies and mammals, including heat shock genes, c-fos, and c-myc. The whole-genome analysis of Pol II profiles suggests that a large fraction of the Dorsal target genes contain paused, or stalled, Pol II. We are currently using a variety of genetic and transgenic methods to determine whether general elongation factors, such as p-TEFb and NELF-E, are essential for the expression of these genes.
The dorsal-ventral patterning network is being used as a foundation to study the embryogenesis of diverse insects, including the malaria mosquito, Anopheles gambiae, the honeybee, Apis mellifera, and the flour beetle, Tribolium castaneum. These studies center on the expansion of the ventral midline in bees and beetles, and the subdivision of the dorsal ectoderm into separate amnion and serosa lineages in the mosquito.
The sea squirt, Ciona intestinalis, is a simple chordate with a small genome that has been recently sequenced and assembled. The Ciona genome represents a simplified version of vertebrate genomes. The Ciona tadpole is initially composed of just ~1,000 cells and there is complete lineage information. It is possible to introduce transgenic DNAs into developing embryos using simple electroporation methods. A provisional circuit diagram is now available for the specification of the major larval tissues during the 32- and 110-cell stages of embryogenesis. This diagram provides a foundation for understanding the differentiation of key organs such as the notochord and heart. We are now using a combination of confocal imaging, cell sorting, and microarray assays to identify the genes and gene interactions responsible for the migration and differentiation of the heart precursor cells.
Publications
Zeitlinger J, Zinzen RP, Stark A, Kellis M, Zhang H, Young RA, Levine M (2007) Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo Genes and Development 21, 385-390.
Zinzen R, Cande JD, Papatsenko D, Levine M (2006) Evolution of the ventral midline in insect embryos Developmental Cell 11, 895-902.
Imai KS, Levine M, Satoh N, Satou Y (2006) Regulatory blueprint for a chordate embryo Science 312(5777):1183-7.
Davidson B, Shi W, Beh J, Christiaen L, Levine M (2006) FGF signaling delineates the cardiac progenitor field in the simple chordate, Ciona intestinalis Genes and Development 20(19):2728-38.
Zinzen RP, Senger K, Levine M, Papatsenko D (2006) Computational models for neurogenic gene expression in the Drosophila embryo Current Biology 16(13):1358-65.
Senger K, Harris K, Levine M (2006) GATA factors participate in tissue-specific immune responses in Drosophila larvae PNAS 103(43):15957-62.
Biemar F, Nix DA, Piel J, Peterson B, Ronshaugen M, Sementchenko V, Bell I, Manak JR, Levine MS (2006) Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array PNAS 103(34):12763-8.
Biemar F, Zinzen R, Ronshaugen M, Sementchenko V, Manak JR, Levine MS (2005) Spatial regulation of microRNA gene expression in the Drosophila embryo Proc Natl Acad Sci USA 102(44): 15907-11.
Ronshaugen M, Biemar F, Piel J, Levine M, Lai EC (2005) The Drosophila microRNA iab-4 causes a dominant homeotic transformation of halteres to wings Genes and Development 19, 2947-2952.
The Center 
