We completed a large insertional mutagenesis screen in zebrafish to identify genes essential for embryonic and early larval development. We isolated 525 mutants, representing lesions in Ϸ390 different genes, and we cloned the majority of these. Here we describe 315 mutants and the corresponding genes. Our data suggest that there are roughly 1,400 embryonic-essential genes in the fish. Thus, we have mutations in Ϸ25% of these genes and have cloned Ϸ22% of them. Re-screens of our collection to identify mutants with specific developmental defects suggest that Ϸ50 genes are essential for the development of some individual organs or cell types. Seventy-two percent of the embryonic-essential fish genes have homologues in yeast, 93% have homologues in invertebrates (fly or worm), and 99% have homologues in human. Yeast and worm orthologues of genes that are essential for early zebrafish development have a strong tendency to be essential for viability in yeast and for embryonic development in the worm. Thus, the trait of being a genetically essential gene is conserved in evolution. This mutant collection should be a valuable resource for diverse studies of cell and developmental biology.

p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. We have explored the interaction of p21 with the currently known Cdks. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5-15 nM) but is much less effective toward Cdc2/cyclin B (Ki approximately 400 nM) and Cdk5/p35 (Ki > 2 microM), and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus, p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes. Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition. With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of exogenous p21. Reconstruction experiments using purified components indicate that multiple molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more than one molecule of p21. Together, these data suggest a model whereby p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression.

Excess retinoids as well as retinoid deprivation cause abnormal development, suggesting that retinoid homeostasis is critical for proper morphogenesis. RALDH-2 and CYP26, two key enzymes that carry out retinoic acid (RA) synthesis and degradation, respectively, were cloned from the chick and show significant homology with their orthologs in other vertebrates. Expression patterns of RALDH-2 and CYP26 genes were determined in the early chick embryo by in situ hybridization. During gastrulation and neurulation RALDH-2 and CYP26 were expressed in nonoverlapping regions, with RALDH-2 transcripts localized to the presumptive presomitic and lateral plate mesoderm and CYP26 mRNA to the presumptive mid- and forebrain. The two domains of expression were separated by an approximately 300-micrometer-wide gap, encompassing the presumptive hindbrain. In the limb region, a similar spatial segregation of RALDH-2 and CYP26 expression was found at stages 14 and 15. Limb region mesoderm expressed RALDH-2, whereas the overlying limb ectoderm expressed CYP26. RA-synthesizing and -degrading enzymatic activities were measured biochemically in regions expressing RALDH-2 or CYP26. Regions expressing RALDH-2 generated RA efficiently from precursor retinal but degraded RA only inefficiently. Conversely, tissue expressing CYP26 efficiently degraded but did not synthesize RA. Localized regions of RA synthesis and degradation mediated by these two enzymes may therefore provide a mechanism to regulate RA homeostasis spatially in vertebrate embryos.

New domains were progressively added to cytoplasmic aminoacyl transfer RNA (tRNA) synthetases during evolution. One example is the UNE-S domain, appended to seryl-tRNA synthetase (SerRS) in species that developed closed circulatory systems. Here we show using solution and crystal structure analyses and in vitro and in vivo functional studies that UNE-S harbours a robust nuclear localization signal (NLS) directing SerRS to the nucleus where it attenuates vascular endothelial growth factor A expression. We also show that SerRS mutants previously linked to vasculature abnormalities either deleted the NLS or have the NLS sequestered in an alternative conformation. A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature. Thus, the essential function of SerRS in vascular development depends on UNE-S. These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates.