A central question in biology is - how do the different germ layers (endoderm, mesoderm and ectoderm) arise from a small number of embryonic cells that appear similar? In the last few years, the genetic pathways that determine the formation of precursors for endoderm and mesoderm have been revealed in the simple nematode, C. elegans. Dr. Maduro proposes experiments to identify factors that function to determine other tissues in C. elegans, and to examine how these pathways have changed over millions of years. He will employ a combination of molecular and genetic approaches to identify, and evaluate the function of, previously uncharacterized genes important in the early development of C. elegans. He will also perform similar experiments in the related nematode C. briggsae to investigate the evolution of the genetic pathway that specifies endoderm and mesoderm. These studies will differentiate between aspects of these pathways that are constrained in early development, and which are flexible, during evolution. As many of the regulators that function in endomesoderm development have counterparts in other animals, the results will be of broad significance. Dr. Maduro is a junior investigator of Hispanic descent and he will encourage Graduate and Undergraduate students from the ethnically diverse UC Riverside campus to participate in the work. He is a National Academies Fellow in the Life Sciences (2006-2007), and will teach and develop new pedagogy for undergraduate and graduate courses in developmental biology.
When animal embryos develop starting from a single cell, early descendants of that cell while being genetically identical, must establish unique gene expression differences that direct the development of individual tissues and organs. The research focus of this work was to elucidate mechanisms of specification of the endoderm (E) and mesoderm (MS) blastomere precursors in the nematode C. elegans, and to probe evolutionary flexibility in the underlying genetic pathways. In the nematode C.elegans, early cells (blastomeres) arise from a reproducible pattern of cell divisions (Fig. 1A). The MS and E cells are born from the EMS blastomere when it divides. The work performed with this grant advanced our understanding of how MS and E are assigned their fates (Fig. 1B). The PI showed that the MS cell adopts its fate when the MED-1,2 regulators activate TBX-35, which in turn activates CEH-51 to specify the MS (mesoderm) fate. A similar ‘feed-forward loop’ occurs in the E lineage, where MED-1,2 activate END-3, which, along with the POP-1 regulator activates END-1. The combined activities of END-1 and END-3 promote endoderm specification. We showed in this grant that the input of the MED factors is important for making the specification of E robust: Without input from MED-1,2, gut is not specified properly, absent in some embryos and abnormal in others. The role of the conserved factor POP-1 (similar to TCF/LEF factors in other animals, including vertebrates) in the MS cell is to prevent inappropriate expression of END-1 and END-3. In the absence of pop-1 function, embryos inappropriately activate endoderm specification in MS as well as E. Work in this grant showed that aside from this role, POP-1 is dispensable in MS, as triple-mutant pop-1; end-1,3(-) embryos make MS-type tissues. The PI also showed that in the related nematode C. briggsae, the role of POP-1 has changed in MS and E specification. While C. elegans embryos arrest and make excess gut following depletion of maternal pop-1 mRNA, C. briggsae embryos lose all gut (Fig. 2). The phenotype differences revealed by these (and other) knockdown studies that we performed suggests that a change in regulatory logic has occurred in MS and E specification (Fig. 3). Along with these main findings, protocols for detection of transcripts by in situ hybridization were reported, data were presented at regional and International conferences, and various strains were developed and made publicly available, including a difficult-to-make double mutant that abolishes all gut specification. The research outcomes of this work have significance not only to our understanding of how C. elegans develops, but also to how the structure of gene regulatory networks is driven towards the robust, correct outcome of development. There is flexibility in these networks that allows some major regulatory changes to occur, but as long as the output is maintained, there is no ‘optimal’ way to produce a worm. The C. elegans system is a highly tractable platform for the investigation of gene networks in general, and indeed, many other laboratories have built upon the work achieved during this grant. As part of the broader training and educational impacts, three PhD students and two MS students were trained, and more than 10 undergraduates participated in various aspects of the work, some of whom became contributing authors to research publications. In addition to earning tenure at his institution during the course of this award, the PI, himself of Hispanic descent, made advances in teaching at graduate and undergraduate levels on his campus and was honored with a Distinguished Teaching Award in 2012.
