A set of immediate-early genes that are rapidly activated by serum or purified platelet-derived growth factor in mouse 3T3 fibroblasts has been previously identified. Among these genes, several are related to known or putative transcription factors and growth factors, supporting the notion that some of these genes encode regulatory molecules important to cel growth. We show here that a member of this set of genes, cyr6l (originally identified by its cDNA 3CH61), encodes a 379-amino-acid polypeptide rich in cysteine residues. cyr61 can be induced through protein kinase C-dependent and -independent pathways. Unlike many immediate-early genes that are transiently expressed, the cyr6l mRNA is accumulated from the G/G1 transition through mid-Gl. This expression pattern is due to persistent transcription, while the mRNA is rapidly turned over during the GJ/G1 transition and in mid-G1 at the same rate. In logarithmically growing cels, the cyr61 mRNA level is constant throughout the cel cycle. Cyr6l contains an N-terminal secretory signal sequence; however, it is not detected in the culture medium by immunoprecipitation. Cyr6l is synthesized maximaly at 1 to 2 h after serum stimulation and has a short half-life within the cell.

Generation of left-right asymmetry is an integral partGeneration of left-right asymmetry during development is an integral part of the establishment of the vertebrate body plan (Capdevila et al. 2000;Mercola and Levin 2001;Wright 2001;Yost 2001;Hamada et al. 2002). Specification of the left-right axis requires multiple steps: (1) generation of an initial asymmetric signal in or near the embryonic node, (2) transfer of asymmetric signals from the node to the lateral plate mesoderm (LPM), (3) induction of an evolutionarily conserved cascade of gene expression in the left LPM, and (4) transformation of these left-right asymmetric signals into morphological asymmetries of the visceral organs. In mice, generation of the initial asymmetric signal requires directional fluid flow on the ventral surface of the node (Nonaka et al. 2002). This fluid flow is generated by motile monocilia on cells of the node, and the presence of nodal cilia is conserved in other vertebrates (Essner et al. 2002). However, the mechanism by which directional fluid flow at the node specifies orientation of the left-right axis is controversial (Stern and Wolpert 2002;Tabin and Vogan 2003). In addition, the mechanism for transfer of the initial asymmetric signal from the node to the LPM is unknown.The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism. Mutations in Notch pathway components disrupt embryonic development in diverse multicellular organisms and cause in- Here we demonstrate that the Notch signaling pathway plays a primary role in the establishment of leftright asymmetry in mice by directly regulating expression of the Nodal gene. Embryos mutant for the Notch ligand Dll1 or doubly mutant for the Notch1 and Notch2 receptors exhibit multiple defects in left-right asymmetry. Notably, Dll1 −/− embryos do not express Nodal in the region around the node. Analysis of the enhancer regulating node-specific Nodal expression (termed the NDE) revealed the presence of binding sites for the RBP-J protein. Mutation of these sites destroyed the ability of the NDE to direct node-specific gene expression in transgenic mice. These results demonstrate that Dll1-mediated Notch signaling is essential for generation of leftright asymmetry, and indicate that perinodal expression of the Nodal gene is an essential component of left-right asymmetry determination in mice. Results and Discussion Laterality defects in Dll1 mutant and Notch1/Notch2 double-mutant mouse embryosDuring studies on the role of the Dll1 gene during somitogenesis (Zhang et al. 2002), we observed that some Dll1 −/− embryos (Hrabé de Angelis et al. 1997) exhibited reversed heart looping. We examined this phenotype more closely by performing scanning electron micros-

Evolution of facial morphology arises from variation in the activity of developmental regulatory networks that guide the formation of specific craniofacial elements. Importantly, the acquisition of novel morphology must be integrated with a phylogenetically inherited developmental program. We have identified a unique region of the secondary palate associated with the periodic formation of rugae during the rostral outgrowth of the face. Rugae function as SHH signaling centers to pattern the elongating palatal shelves. We have found that a network of signaling genes and transcription factors is spatially organized relative to palatal rugae. Additionally, the first formed ruga is strategically positioned at the presumptive junction of the future hard and soft palate that defines anterior-posterior differences in regional growth, mesenchymal gene expression and cell fate. We propose a molecular circuit integrating FGF and BMP signaling to control proliferation and differentiation during the sequential formation of rugae and inter-rugae domains in the palatal epithelium. The loss of p63 and Sostdc1 expression and failed rugae differentiation highlight that coordinated epithelial mesenchymal signaling is lost in the Fgf10 mutant palate. Our results establish a genetic program that reiteratively organizes signaling domains to coordinate the growth of the secondary palate with the elongating midfacial complex.

Specific mammalian genes functionally and dynamically associate together within the nucleus. Yet, how an array of many genes along the chromosome sequence can be spatially organized and folded together is unknown. We investigated the 3D structure of a well-annotated, highly conserved 4.3-Mb region on mouse chromosome 14 that contains four clusters of genes separated by gene “deserts.” In nuclei, this region forms multiple, nonrandom “higher order” structures. These structures are based on the gene distribution pattern in primary sequence and are marked by preferential associations among multiple gene clusters. Associating gene clusters represent expressed chromatin, but their aggregation is not simply dependent on ongoing transcription. In chromosomes with aggregated gene clusters, gene deserts preferentially align with the nuclear periphery, providing evidence for chromosomal region architecture by specific associations with functional nuclear domains. Together, these data suggest dynamic, probabilistic 3D folding states for a contiguous megabase-scale chromosomal region, supporting the diverse activities of multiple genes and their conserved primary sequence organization.