Exogenous retinoic acid during gastrulation induces cartilaginous and other craniofacial defects in Fundulus heteroclitus.

Embryonic levels of retinoic acid (RA) and the response of cells to RA are critical to the normal development of vertebrates. To understand the effects of RA signaling in Fundulus heteroclitus, we exposed embryos to a range of RA concentrations of 2 h during gastrulation. Embryos exposed to low concentrations of RA (10(-10)-10(-7) M) develop normally, whereas those exposed to higher concentrations (5 x 10(-7)-10(-4) M) develop characteristic dose-dependent defects. We describe, in detail, four stages of development that represent morphological effects of RA on (1) cell death and defects in the brain, heart, and eye, (2) relative size and differentiation, (3) duplications of pectoral fins, and (4) deletions in craniofacial cartilage elements. Analysis of cartilaginous skeletal elements demonstrates distinct patterns of deletions in the neurocranium and pharyngeal skeleton in response to increasing concentrations of RA. In F. heteroclitus, RA treatment during gastrulation results in five highly consistent phenotypes, which we have incorporated into an index of embryonic RA defects. This index should be valuable in the genetic analysis of RA pathways and in evaluating chemicals that interfere with embryonic RA signaling.

Fin duplications and deletions induced by disruption of retinoic acid signaling.

Retinoic acid (RA), a derivative of vitamin A, plays a critical role as a signaling molecule in axial patterning of vertebrates. Here we report that RA exposure of zebrafish (Danio rerio) and mummichog (Fundulus heteroclitus) embryos during gastrulation results in homeotic duplications of the pectoral fins in up to 94% of fish. We have observed three to four pairs of fins in an individual fish. Although some duplications are partial, many represent complete axial duplications of the pectoral girdle and fin and include coracoscapulae, proximal radials, and dermal fin elements. Fin duplications are observed only at a defined dose of RA. Inhibition of RA synthesis by exposure to citral during a narrow developmental window leads to fish which lack pectoral fins but can be rescued by addition of exogenous RA, suggesting that RA signaling is critical to fin specification during early development. The ability to consistently induce multiple fins in a large number of vertebrate embryos should contribute to the understanding of genetic regulation of the normal positioning of limbs during embryogenesis.

Characterization of the cassette containing genes for type 3 capsular polysaccharide biosynthesis in Streptococcus pneumoniae.

The capsular polysaccharide is the major virulence factor of Streptococcus pneumoniae. Previously, we identified and cloned a region from the S. pneumoniae chromosome specific for the production of type 3 capsular polysaccharide. Now, by sequencing the region and characterizing mutations genetically and in an in vitro capsule synthesis assay, we have assigned putative functions to the products of the type-specific genes. Using DNA from the right end of the region in mapping studies, we have obtained further evidence indicating that the capsule genes of each serotype are contained in a gene cassette located adjacent to this region. We have cloned the region flanking the left end of the cassette from the type 3 chromosome and have found that it is repeated in the S. pneumoniae chromosome. The DNA sequence and hybridization data suggest a model for recombination of the capsule gene cassettes that not only describes the replacement of capsule genes, but also suggests an explanation for binary capsule type formation, and the creation of novel capsule types.