In the Wold group we are interested in the composition,evolution and function of regulatory networks that govern how mammalian cell fates are specified and executed during development and during regeneration.This theme extends to related lineages of adult stem cells for our model system and to the way in which cells of this same lineage become tumorgenic. Approaches we are taking to these problems increasingly use genome-wide and proteome-wide assays.To do this some of our efforts now include development of new wet- bench genomic technology and computational methods, the latter developed in an on-going partnership with Professor Eric Mjolsness of JPL/University of California Irvine.

A key challenge is to understand the regulatory events that drive the progression from multipotential precursor cells to determined unipotential progenitors and then to fully differentiated cells.We are currently studying these cell states and transitions using microarray gene expression analysis,global protein:DNA interaction measures,mass spec based proteomics of multiprotein complexes,and comparative genomics.The mouse is our primary experimental animal,and the focal developmental lineage arises from paraxial mesoderm to produce muscle (also bone,skin and fat,among other derivatives). Skeletal myogenesis is governed by both positive-and negative-acting regulatory factors.The MyoD family of four closely related,positive-acting transcription factors are key.Upon transfection each can drive nonmuscle recipient cells into the myogenic pathway.Given their extraordinary power to drive or redirect a cell fate decision,a central goal is to understand how the regulatory network in which they are embedded directs cell fate selection and execution of the differentiation transition.At cellular and molecular levels,it is clear that negative regulators of skeletal myogenesis are probably just as important for regulating the outcome as are the positive regulators.The interaction between positive and negative acting regulators continues to be of particular interest. Multiple negative regulators of skeletal muscle are expressed in multipotential mesodermal precursors and in proliferating muscle precursors (myoblasts).It is generally believed that some of these are important for specifying and/or maintaining precursor cells in an undifferentiated state,though exactly how the system works is unknown.

To define this myogenic regulatory network more comprehensively,we have developed a major collaborative effort with the Deshaies lab here and the John Yates lab at Scripps to modify and apply MudPIT mass spectrometry, coupled with dual affinity epitope tagging,to characterize multiprotein complexes. To define the c is -acting regulatory elements to which these protein complexes bind we have entered into a collaboration with Eric Greene at NIH to isolate and sequence genes from our network from ten vertebrate species each.The computational tools described below are now being used to find candidate conserved regulatory elements,and these are,in turn, being subjected to rapid functional assays via lentiviral mediated transgenesis.These same tools are being used to analyze data from multiple species of worms related,in differing degrees,to C.elegans .This project is in partnership with the Sternberg lab and Hiroke Shizuya here at Caltech,and the DOE Joint Genomics Institute, where large scale DNA sequencing is done.In this project large insert,random shear libraries were made for two new worm species,and genes from several regulatory networks, including the myogenic one are being isolated and sequenced for comparative analysis.In addition to clarifying how many and which worm genomes give us the most leverage for identifying functionally important noncoding elements in the genome,we hope to gain insights into the evolution of myogenic networks across large phylogenetic distances between vertebrates,worms and flies.

Our collaboration with Dr.Timothy Triche and colleagues at Children's Hospital is giving us a picture of how the myogenic developmental pathway relates to cells of the myogenic lineage when they run amok in cancer. Transcriptome analysis of over 100 rhabdomyosarcomas has given us several new insights into the nature of these tumors,plus identification of previously unappreciated signaling pathways that are candidates for causal contributions to tumor properties.Of particular interest was a surprising re-classification of one subgroup of tumors.These are,by histological criteria,of the alveolar class.However,by expression profiling and subsequent computational analysis,these tumors proved to be very different from classic Alveolars and more similar to the other major class (Embyonal).Retrospectively,we were able to relate this to the absence of a chromosomal translocation that characterizes the most alveolar tumors. This analysis also showed,surprisingly,that the genes whose expression best separates conventional alveolar and embryonal types does not correlate with their histological appearance,but rather refers to other molecular differences.We now postulate that these "invisible" differences may have more powerful prognostic capacity than conventional histopathology,since the two tumor classes differ significantly in outcome,than does histopathological classification.This has implications for treatment pathways and prognosis.

Muscle satellite cells are muscle stem cells of the adult,and they are responsible for all muscle regeneration following injury or degenerative insult.We want to understand the lineal origin,cell cycle regulation,and regulation of myogenic status for this self-renewing progenitor cell population.D.Cornelison initiated this work as part of her PhD thesis in the lab by studying in satellite cells the expression and function of a panel of 80 different developmental regulatory genes,growth and trophic factor receptors,cdk/cyclin inhibitory proteins,and cdk/cyclin complexes in the context of mouse muscle regeneration for wild-type and MRF mutant animals. Marie Csete went on to study these stem cells and others from diverse tissues with respect to their responses to major environmental cues,focusing on oxygen availability that is often disrupted by injury.Collaboration with David Anderson's lab on campus and Ron McKay's at the NIH led to the conclusion that oxygen levels normally used in cell culture are both a physiologically high and deleterious. Lower,more physiological levels give dramatic improvement in yields of dopaminergic neurons in both CNS and PNS stem cell cultures.Oxygen levels were also found to influence the selection of cell fate in culture, leading to the provocative possibility that the oxygen microenvironment for a progenitor cell during development in vivo might affect cell fate selection in the brain.There are also intriguing effects of oxygen levels on muscle and fat developmental pathways where aphysiologically high levels of oxygen favor the fat pathway and lower normoxic levels favor accelerated muscle differentiation.The current challenge is to identify the metabolic and regulatory pathways that are responsible for these oxygen effects.

A different bHLH class regulator was discovered by Jeong Yoon in the lab,and it ultimately has made somite formation and vertebrate body segmentation a topic of investigation in the lab.The regulator is called p- Mesogenin1 because it functions early in the specification and subdivision of unsegmented paraxial mesoderm.By analogy with other bHLH regulators,it is likely to function as a sequence specific DNA binding protein.Jeong Yoon in the lab previously showed by gain of function analysis in frog embryos and loss of function studies in a mouse p- Meso knockout that this protein is essential for vertebrate body segmentation (somite formation)and for subsequent cellular survival and maturation of all trunk and tail paraxial mesoderm.Recent work has preliminarily identified regulatory elements responsible for its novel pattern of expression,and it is slated for upcoming multi- genome sequencing and comparative analysis.

An entirely new project for the lab is a collaboration with Dr. Dianne Newman in the Division of Geology and Planetary Science.The topic is environmental bacterial biofilms, and the goal is identify,visualize,and ultimately understand the multiple different metabolic cell states that comprise a biofilm at different stages of its development and under differing environmental stimuli.The degree to which principles and regulatory strategies used by metazoans during development are or are not employed by bacteria in creating biofilm structures is being probed by marking bacteria with multiple GFP derivatives driven by genes that are believed to be differentially expressed within biofilms.