Hyperglycemia delays rostral initiation sites during neural tube closure.

Neural tube defects contribute greatly to perinatal loss, physical handicap, mental retardation and other developmental defects, yet the mechanisms through which they occur are poorly understood. One hindrance to the study of these defects at the cellular and molecular levels is the low frequency with which they arise in susceptible animals. The present study utilizes a culture system for the study of rodent exencephaly, an animal model of human anencephaly, in which a high frequency of affected animals are obtained by culture in hyperglycemic rat serum. Rat embryos were dissected at day 9.5 from timed-pregnant Sprague-Dawley dams and cultured under standard conditions developed by New [Biol. Rev. (1978) 53,81-122]. Embryos cultured under elevated glucose conditions are able to close the caudal neural tube with the failure of neural tube closure limited to the rostral neuralepithelium. In this report we present the novel finding that, although at the end of culture frequently only the hindbrain region remains open, the normal sequence of events expected during rostral closure anterior to the hindbrain is markedly delayed. In embryos cultured in hyperglycemic serum, both rostral initiation sites II and III are significantly delayed. The degree of delay increases with increasing glucose concentration in the culture medium. These studies support the use of this defined in vitro model of anencephaly for studies of the molecular and cellular bases underlying the failure of hindbrain closure and demonstrate that sufficient numbers of affected animals can be produced to obtain significant results.(ABSTRACT TRUNCATED AT 250 WORDS)

Prepupal differentiation in Drosophila: distinct cell types elaborate a shared structure, the pupal cuticle, but accumulate transcripts in unique patterns.

The components of the pupal cuticle are the main differentiation products synthesized by both the larval and adult epidermis during the prepupal period of Drosophila development. The pupal cuticle is formed in vitro by imaginal discs in response to a 6 h pulse of 20-hydroxyecdysone (20-HE). We previously described the isolation and initial characterization of four ecdysone-dependent genes (EDGs) whose expression in imaginal discs occurs only in response to a pulse of 20-HE. In this report, we demonstrate that the pattern of temporal and tissue-specific expression of these EDGs in vivo is like that expected for genes that encode pupal cuticle proteins. Transcripts of these genes are detected in prepupae only in the epidermis and only when cuticle components are synthesized and secreted. Nonetheless, their temporal and spatial patterns of accumulation differ. EDG-84A-1 transcripts accumulate only in prepupae and only in imaginal cells. EDG-78E and EDG-64CD transcripts accumulate at the same time in both larval and imaginal cells. EDG42-A transcripts appear first in prepupae in imaginal cells and then, after a 2-4 h lag, in larval cells. It is evident that some genes are not restricted in their expression to only larval or imaginal epidermis.

Molecular genetics of Delta, a locus required for ectodermal differentiation in Drosophila.

Delta (Dl) is one of the six known zygotic neurogenic genes, each of which is essential for proper segregation of the embryonic ectoderm into neural and epidermal lineages. Molecular analysis of Dl reveals that it is a transcriptionally complex locus that yields multiple maternal and zygotic transcripts. DNA sequence analysis suggests that the predominant product of the locus is a putative transmembrane protein exhibiting homology to blood coagulation factors and epidermal growth factor of vertebrates. The structure of this product is consistent with the hypothesis that Dl participates in cell-cell interactions that are central to establishment of the epidermal lineage within the developing ectoderm. Genetic analyses demonstrate that Dl mutations can modify the imaginal phenotypes that result from heterozygosity for Notch (N) mutations as well as the interaction between particular alleles of Notch (N) and Enhancer of split [E(spl)], two other members of the neurogenic gene set. Vital interactions also occur between Dl and N. Given the structures of products encoded by N, Dl, and E(spl), we suggest that the synergistic phenotypic interactions observed among mutations in these three loci result from physical, as opposed to regulatory, interactions.

Delta, a Drosophila neurogenic gene, is transcriptionally complex and encodes a protein related to blood coagulation factors and epidermal growth factor of vertebrates.

Delta (D1) is required for normal segregation of the embryonic ectoderm into neural and epidermal cell lineages in Drosophila melanogaster. Loss-of-function mutations in D1 and other zygotic neurogenic loci lead to expansion of the neuroblast population at the expense of the dermoblast population within the ectoderm. Characterization of the transcriptional organization and maternal/embryonic expression within the chromosomal interval corresponding to D1 reveals that the locus encodes multiple transcripts: a minimum of two maternal transcripts, approximately 4.5 and 3.6 kb in length, and four zygotic transcripts, approximately 5.4 (two distinct species), 3.5, and 2.8 kb in length. These transcripts differ on the bases of differential splicing and differential polyadenylation site choice. The DNA sequence of a cDNA clone representing the predominant transcripts of the locus indicates that D1 encodes a transmembrane protein homologous to blood coagulation factors and epidermal growth factor. The relationship between coding sequences and transcript-specific exons within the locus suggests that D1 encodes multiple translational products.

Prepupal differentiation of Drosophila imaginal discs: identification of four genes whose transcripts accumulate in response to a pulse of 20-hydroxyecdysone.

We have isolated and initially characterized a novel set of four genes expressed during the prepupal differentiation of imaginal discs of Drosophila melanogaster. These four ecdysone-dependent genes are named EDG-42A, EDG-64CD, EDG-78E and EDG-84A-1 based on their respective chromosomal locations. Their expression is like that expected for genes encoding proteins that participate in the formation of the pupal cuticle. Transcripts complementary to these genes accumulate in imaginal discs during an 18-hr in vitro culture period that begins with a 6-hr pulse of 20-hydroxyecdysone (20-HE). Transcripts for three of these genes were not detected in imaginal discs following culture in the absence or the continuous presence of 20-HE (1 microgram/ml). Transcripts corresponding to EDG-64CD exhibit delayed accumulation in the continuous presence of 20-HE. Transcripts corresponding to three of the genes are only detected in the prepupal stage of development. Only EDG-64CD is complementary to transcripts present at other stages of development. One of the genes, EDG-78E, encodes a pupal cuticle protein. This is the first reported isolation of a set of steroid hormone-responsive genes that require first the presence, then removal of hormone for transcript accumulation.

Cytogenetic definition and morphogenetic analysis of Delta, a gene affecting neurogenesis in Drosophila melanogaster.

We have conducted a genetic analysis of a small interval of the third chromosome known to include Delta (Dl), a locus that affects the segregation of the ectoderm into neural and epidermal lineages during embryogenesis and the morphogenesis of some ectodermally derived structures, in Drosophila melanogaster. This analysis has led to the definition of seven independent complementation groups, one of which is Delta, within the interval extending from 91F6-13 to 92A2. Among the extant mutations in these seven loci, only mutations in Dl lead to the so-called neurogenic phenotype: hypertrophy of the nervous system and reduction of the epidermis. Combined cytogenetic and genetic analyses allow us to define absolute proximal (91F5-92A1) and distal (92A2) cytogenetic limits for the Dl locus. We have isolated hypomorphic and amorphic alleles of Dl and find that, for any given allele, there is an inverse correlation between neural hypertrophy and epidermal reduction in embryos and a direct correlation between the severity of embryonic phenotypes in mutant homozygotes and hemizygotes and the imaginal phenotype in heterozygous adults.

Gene within a gene: nested Drosophila genes encode unrelated proteins on opposite DNA strands.

A pupal cuticle protein gene has been found within an intron of a Drosophila gene that encodes three purine pathway enzymatic activities. The intronic gene is encoded on the DNA strand opposite the purine pathway gene and is itself interrupted by an intron. Whereas the purine pathway gene is active throughout development, the intronic cuticle protein gene is expressed primarily over a 3 hr period in the abdominal epidermal cells of prepupae that secrete the pupal cuticle. Therefore, a housekeeping gene and a developmentally regulated gene function in a nested arrangement.