1 Program/Abstract # 1 Role of the core promoter in the regulation of gene expression Kadonaga, Jim, UC San Diego, United States The RNA polymerase II core promoter comprises the stretch of DNA that directs the initiation of transcription. This basic definition might suggest that core promoters are simple transcriptional elements that function via common mechanism. Current evidence indicates, however, that there is extraordinary diversity in core promoter structure and activity. For instance, there are a variety of sequence-specific DNA elements in core promoters that include the TATA box, Inr, MTE, DPE, BRE, and TCT motifs. These elements do not act universally – the well-known TATA box is present in only about 15% of human promoters. Instead, specific core promoter elements confer distinct transcriptional activities to core promoters. For example, nearly all of the Drosophila Hox genes have DPE-dependent core promoters, and the Caudal protein, a master regulator of the Hox genes, is an enhancer-binding protein that activates transcription specifically through the DPE motif. Hence, the Hox genes are a DPE-based transcriptional network, and Caudal is a DPE-specific activator. In addition, another core promoter element, the TCT motif, is dedicated to the expression of the network of ribosomal protein genes. These and other findings collectively indicate that the core promoter is a diverse transcriptional regulatory element that is employed for the regulation not only of individual genes, but also of gene networks. Moreover, at a more practical level, optimized versions of the core promoter can be used to achieve the desired specificity and level of expression of transgenes. It is thus evident that a strong understanding of the core promoter is essential for the effective analysis of gene expression. Program/Abstract # 2 Measuring transcriptional dynamics of single cells reveals mechanisms that compensate for the cost of bistability Boettiger, Alistair, Harvard, United States; Bothma, Jacques; Perry, Michael; Levine, Michael, UC Berkeley, United States Cell fate decisions during development produce a discrete set of distinct cell types – rather than a continuum of states. One of the most popular models to explain the formation of distinct states without mixed intermediates envisions development to be comprised of a sequence of binary choices for each cell. However, few examples of such binary fate decisions have been worked out in developing systems and little is known about the molecular mechanisms and functional constraints which enable such decisions in the context of a developing embryo. We present evidence that the mesoderm-ectoderm boundary in the early Drosophila embryo is formed by just such a bistable interaction. Simultaneous expression of competing transcriptional repressors in the ectoderm forces these cells to make a binary choice between a presumptive muscle, or presumptive nerve fate. Genetic perturbation experiments illustrate that the expression of the competing genes is highly constrained by a need to suppress transcriptional noise. Small degrees of expression noise similar to those observed in other systems lead are amplified by the bistable interaction with catastrophic effects on development. To understand how such noise is repressed at the molecular level, we quantify mRNA expression in single-cells throughout early development. We find that redundant repressors, redundant enhancers, and near-saturating transcription levels all contribute ensure unprecedented low levels of noise in expression of the key regulatory gene, snail. Perturbations to any one of these three mechanisms enhance the molecular variations in snail between sister cells and can lead to dramatically increased variation in boundary placement. Program/Abstract # 3 The MED12-MED13 module of Mediator regulates multiple developmental phase transitions during the Arabidopsis life cycle Gillmor, Stewart, Langebio, CINVESTAV-IPN, Mexico; Willmann, Matthew (U Pennsylvania, United States); Silva- Ortega, Claudia (Langebio, CINVESTAV-IPN, Mexico); Poethig, Scott (U Pennsylvania, United States) Temporal coordination of developmental programs is essential for multicellular development, and alteration of developmental timing has been proposed to play an important role in evolution. We have previously shown that the MED12 and MED13 genes CENTER CITY (CCT) and GRAND CENTRAL (GCT) are essential for temporal coordination of pattern formation during Arabidopsis embryogenesis (Gillmor et al., Development, 2010). In the current study, we describe a role for GCT and CCT in global coordination of developmental phase transitions. gct and cct mutations cause embryo-specific genes to be expressed during seedling development, delay vegetative phase change, and delay flowering. Genetic analysis indicates that GCT and CCT regulate the progression of vegetative growth and flowering in parallel with the phytohormone GA, and the chromatin remodeling protein PICKLE. We demonstrate that the delay in the onset of adult vegetative traits is due to upregulation of miR156 expression in gct and cct mutants, and that the delay in flowering is due in part to upregulation of the floral repressor FLC. Thus, GCT and CCT regulate vegetative and floral transitions by repressing the repressors miR156 and FLC. 2 Program/Abstract # 4 RA-FGF antagonism during vertebrate body axis extension: feedback signaling from stem cell progeny to niche Duester, Gregg L.; Cunningham, Thomas; Brade, Thomas, Sanford-Burnham, La Jolla, United States; Trainor, Paul, Stowers, Kansas City, United States; Sandell, Lisa (University of Louisville, United States) Vertebrate embryos develop in a head to tail fashion from a caudal progenitor zone. During body axis extension, bipotential neuromesodermal stem cells in the caudal progenitor zone generate progeny that contribute to both neuroectoderm (hindbrain-spinal cord) and paraxial mesoderm (somites). Presomitic mesoderm exits the caudal progenitor zone and undergoes segmentation into somites anterior to a wavefront of Fgf8 activity generated in caudal progenitors. A major transition in mouse development occurs at E7.5 when presomitic mesoderm begins generating retinoic acid (RA) via Rdh10 and Raldh2 enzymes that sequentially metabolize retinol to retinaldehyde and then to RA which acts as a diffusible signal. Embryos deficient in RA synthesis exhibit small somites and limb defects that we have hypothesized are the result of expanded Fgf8 expression, but it is unclear how RA regulates mesoderm development. Here, we show that reduction of FGF signaling in Raldh2-/- embryos lacking RA synthesis is sufficient to rescue somitogenesis. Rdh10 mutants initially lack RA synthesis and exhibit expanded Fgf8 expression and small somites as well as stunted forelimbs, but later recover normal caudal somites and hind limbs due to late-appearing RA activity that arises at the neuroectoderm/epiblast junction; a RA-reporter transgene sensitive to 0.25 nM RA demonstrates that RA activity does not act in presomitic mesoderm for somitogenesis and that RA is not required for limb patterning. Our findings demonstrate that progeny of the caudal progenitor zone produce RA that controls axial development by feedback signaling to the border of the stem cell niche designed to repress Fgf8 in neuromesodermal stem cells at the neuroectoderm/epiblast junction. Program/Abstract # 5 Muscle satellite cells are primed for myogenesis, but maintain quiescence with sequestration of Myf5 mRNA targeted by microRNA-31 in mRNP granules Crist, Colin G., McGill University, Montreal, Canada; Montarras, Didier; Buckingham, Margaret (Institut Pasteur, Paris, France) Regeneration of adult tissues depends on stem cells that are primed to enter a differentiation programme, while remaining quiescent. How these two characteristics can be reconciled is exemplified by skeletal muscle where the majority of quiescent satellite cells transcribe the myogenic determination gene Myf5, without activating the myogenic programme. We show that Myf5 mRNA, together with microRNA-31, which regulates its translation, are sequestered in mRNP granules present in the quiescent satellite cell. In activated satellite cells, mRNP granules are dissociated, relative levels of miR-31 are reduced, and Myf5 protein accumulates, which initially requires translation, but not transcription. Conditions that promote the continued presence of mRNP granules delay the onset of myogenesis. Manipulation of miR-31 levels affects satellite cell differentiation ex vivo and muscle regeneration in vivo. We therefore propose a model in which post- transcriptional mechanisms hold quiescent stem cells poised to enter a tissue specific differentiation programme. Program/Abstract # 6 Predominant role of Hoxa5 gene during mouse lung development Jeannotte, Lucie; Boucherat, Olivier; Montaron, Séverine; Aubin, Josée (University of Laval, Canada); Philippidou, Polyxeni; Dasen, Jeremy (NYU School of Medicine, United States) Lung development depends on reciprocal interactions between the epithelium and the surrounding mesenchyme. The mesenchyme can instruct epithelial differentiation but the nature of the mesenchymal factors involved still remains elusive. Hox genes encode transcription factors specifying regional identity along the body axes and in regulating morphogenesis during development. In mammals, 39 Hox genes are organized in 4 clusters and classified in 13 paralog groups. Several Hox genes are expressed in a distinct spatio-temporal fashion during