A novel member of the F-box/WD40 gene family, encoding dactylin, is disrupted in the mouse dactylaplasia mutant.

Early outgrowth of the vertebrate embryonic limb requires signalling by the apical ectodermal ridge (AER) to the progress zone (PZ), which in response proliferates and lays down the pattern of the presumptive limb in a proximal to distal progression. Signals from the PZ maintain the AER until the anlagen for the distal phalanges have been formed. The semidominant mouse mutant dactylaplasia (Dac) disrupts the maintenance of the AER, leading to truncation of distal structures of the developing footplate, or autopod. Adult Dac homozygotes thus lack hands and feet except for malformed single digits, whereas heterozygotes lack phalanges of the three middle digits. Dac resembles the human autosomal dominant split hand/foot malformation (SHFM) diseases. One of these, SHFM3, maps to chromosome 10q24 (Refs 6,7), which is syntenic to the Dac region on chromosome 19, and may disrupt the orthologue of Dac. We report here the positional cloning of Dac and show that it belongs to the F-box/WD40 gene family, which encodes adapters that target specific proteins for destruction by presenting them to the ubiquitination machinery. In conjuction with recent biochemical studies, this report demonstrates the importance of this gene family in vertebrate embryonic development.

The mouse pudgy mutation disrupts Delta homologue Dll3 and initiation of early somite boundaries.

Pudgy (pu) homozygous mice exhibit clear patterning defects at the earliest stages of somitogenesis, resulting in adult mice with severe vertebral and rib deformities. By positional cloning and complementation, we have determined that the pu phenotype is caused by a mutation in the delta-like 3 gene (Dll3), which is homologous to the Notch-ligand Delta in Drosophila. Histological and molecular marker analyses show that the pu mutation disrupts the proper formation of morphological borders in early somite formation and of rostral-caudal compartment boundaries within somites. Viability analysis also indicates an important role in early development. The results point to a key role for a Notch-signalling pathway in the initiation of patterning of vertebrate paraxial mesoderm.

The cohesion protein MEI-S332 localizes to condensed meiotic and mitotic centromeres until sister chromatids separate.

The Drosophila MEI-S332 protein has been shown to be required for the maintenance of sister-chromatid cohesion in male and female meiosis. The protein localizes to the centromeres during male meiosis when the sister chromatids are attached, and it is no longer detectable after they separate. Drosophila melanogaster male meiosis is atypical in several respects, making it important to define MEI-S332 behavior during female meiosis, which better typifies meiosis in eukaryotes. We find that MEI-S332 localizes to the centromeres of prometaphase I chromosomes in oocytes, remaining there until it is delocalized at anaphase II. By using oocytes we were able to obtain sufficient material to investigate the fate of MEI-S332 after the metaphase II-anaphase II transition. The levels of MEI-S332 protein are unchanged after the completion of meiosis, even when translation is blocked, suggesting that the protein dissociates from the centromeres but is not degraded at the onset of anaphase II. Unexpectedly, MEI-S332 is present during embryogenesis, localizes onto the centromeres of mitotic chromosomes, and is delocalized from anaphase chromosomes. Thus, MEI-S332 associates with the centromeres of both meiotic and mitotic chromosomes and dissociates from them at anaphase.

The vibrator mutation causes neurodegeneration via reduced expression of PITP alpha: positional complementation cloning and extragenic suppression.

The mouse vibrator mutation causes an early-onset progressive action tremor, degeneration of brain stem and spinal cord neurons, and juvenile death. We cloned the vibrator mutation using an in vivo positional complementation approach and complete resequencing of the resulting 76 kb critical region from vibrator and its parental chromosome. The mutation is an intracisternal A particle retroposon insertion in intron 4 of the phosphatidylinositol transfer protein alpha gene, causing a 5-fold reduction in RNA and protein levels. Expression of neurofilament light chain is also reduced in vibrator, suggesting one signaling pathway that may underlie vibrator pathology. The vibrator phenotype is suppressed in one intercross. We performed a complete genome scan and mapped a major suppressor locus (Mvb-1) to proximal chromosome 19.

Isolation and characterization of ribosomal DNA variants from Sciara coprophila.

The ribosomal RNA multigene family in the fungus fly Sciara coprophila contains a total of only 65 to 70 repeat units. We explored the types and frequencies of variant repeats in this small multigene family by characterizing different cloned rDNA variants from Sciara. Although we did not observe any intergenic spacer length variants in Sciara, we found a variant due to the insertion of a putative mobile element (lambda Bc11), and variants containing ribosomal insertion elements. By DNA sequence analysis of rDNA/non-rDNA junctions, there are three distinct types of ribosomal insertion elements found in Sciara rDNA: two correspond to the R1 and R2 insertion elements found in other dipterans (clones lambda Bc5 and pBc1L1, respectively), and one is a novel class of ribosomal insertion elements (R3, exemplified by clone pBc6D6) which so far is unique to Sciara. Together, the several different rDNA variants make up from 12 to 20% of the rDNA in Sciara. These results are discussed in the context of evolution of the ribosomal RNA multigene family.

The Drosophila mei-S332 gene promotes sister-chromatid cohesion in meiosis following kinetochore differentiation.

The Drosophila mei-S332 gene acts to maintain sister-chromatid cohesion before anaphase II of meiosis in both males and females. By isolating and analyzing seven new alleles and a deficiency uncovering the mei-S332 gene we have demonstrated that the onset of the requirement for mei-S332 is not until late anaphase I. All of our alleles result primarily in equational (meiosis II) nondisjunction with low amounts of reductional (meiosis I) nondisjunction. Cytological analysis revealed that sister chromatids frequently separate in late anaphase I in these mutants. Since the sister chromatids remain associated until late in the first division, chromosomes segregate normally during meiosis I, and the genetic consequences of premature sister-chromatid dissociation are seen as nondisjunction in meiosis II. The late onset of mei-S332 action demonstrated by the mutations was not a consequence of residual gene function because two strong, and possibly null, alleles give predominantly equational nondisjunction both as homozygotes and in trans to a deficiency. mei-S332 is not required until after metaphase I, when the kinetochore differentiates from a single hemispherical kinetochore jointly organized by the sister chromatids into two distinct sister kinetochores. Therefore, we propose that the mei-S322 product acts to hold the doubled kinetochore together until anaphase II. All of the alleles are fully viable when in trans to a deficiency, thus mei-S332 is not essential for mitosis. Four of the alleles show an unexpected sex specificity.

Identification of the Finnish founder mutation for diastrophic dysplasia (DTD).

Diastrophic dysplasia (DTD) is especially prevalent in Finland and the existence of a founder mutation has been previously inferred from the fact that 95% of Finnish DTD chromosomes have a rare ancestral haplotype found in only 4% of Finnish control chromosomes. Here we report the identification of the Finnish founder mutation as a GT-> GC transition (c.-26 + 2T > C) in the splice donor site of a previously undescribed 5'-untranslated exon of the diastrophic dysplasia sulfate transporter gene (DTDST); the mutation acts by severely reducing mRNA levels. Among 84 DTD families in Finland, patients carried two copies of the mutation in 69 families, one copy in 14 families, and no copies in one family. Roughly 90% of Finnish DTD chromosomes thus carry the splice-site mutation, which we have designated DTDST(Fin). Unexpectedly, we found that nine of the DTD chromosomes having the apparently ancestral haplotype did not carry DTDST(Fin), but rather two other mutations. Eight such chromosomes had an R279W mutation and one had a V340del deletion. We consider the possible implications of presence of multiple DTD mutations on this rare haplotype.

Mei-S332, a Drosophila protein required for sister-chromatid cohesion, can localize to meiotic centromere regions.

Mutations in the Drosophila mei-S332 gene cause premature separation of the sister chromatids in late anaphase of meiosis I. Therefore, the mei-S332 protein was postulated to hold the centromere regions of sister chromatids together until anaphase II. The mei-S332 gene encodes a novel 44 kDa protein. Mutations in mei-S332 that differentially affect function in males or females map to distinct domains of the protein. A fusion of mei-S332 to the green fluorescent protein (GFP) is fully functional and localizes specifically to the centromere region of meiotic chromosomes. When sister chromatids separate at anaphase II, mei-S332-GFP disappears from the chromosomes, suggesting that the destruction or release of this protein is required for sister-chromatid separation.

Maintenance of the DNA puff expanded state is independent of active replication and transcription.

The maintenance of the expanded state of DNA puffs II/2B and II/9A in polytene chromosomes from stage 14 x 7 Sciara coprophila salivary glands was assayed after inhibition of RNA synthesis, DNA synthesis, or both processes together. Heat shock conditions were established in order to inhibit transcription. Polypeptides of Mr 72,000 and 36,000 were produced in Sciara after heat shock. The gene encoding the Mr 72,000 polypeptide, the homolog of Drosophila hsp70, was cloned. In situ hybridization detected Sciara hsp70 at bands 4A and 17C on chromosome IV. Sciara hsp70 encodes a 2.3 kb heat shock mRNA. DNA puffs (e.g., DNA puffs 2B and 9A on chromosome II) remained fully expanded even after inhibition of transcription by heat shock or actinomycin D, or after inhibition of DNA replication by aphidicolin, or inhibition of both RNA synthesis and DNA synthesis together by actinomycin D plus aphidicolin. Therefore, maintenance of the DNA puff expanded state in Sciara does not require ongoing transcription and/or replication. Mechanisms for initiation and for maintenance of puffs (open chromatin structure) are discussed.

Serrate2 is disrupted in the mouse limb-development mutant syndactylism.

The mouse syndactylism (sm) mutation impairs some of the earliest aspects of limb development and leads to subsequent abnormalities in digit formation. In sm homozygotes, the apical ectodermal ridge (AER) is hyperplastic by embryonic day 10.5, leading to abnormal dorsoventral thickening of the limb bud, subsequent merging of the skeletal condensations that give rise to cartilage and bone in the digits, and eventual fusion of digits. The AER hyperplasia and its effect on early digital patterning distinguish sm from many other syndactylies that result from later failure of cell death in the interdigital areas. Here we use positional cloning to show that the gene mutated in sm mice encodes the putative Notch ligand Serrate. The results provide direct evidence that a Notch signalling pathway is involved in the earliest stages of limb-bud patterning and support the idea that an ancient genetic mechanism underlies both AER formation in vertebrates and wing-margin formation in flies. In addition to cloning the sm gene, we have mapped three modifiers of sm, for which we suggest possible candidate genes.

Disruption of the nuclear hormone receptor RORalpha in staggerer mice.

Homozygous staggerer (sg) mice show a characteristic severe cerebellar ataxia due to a cell-autonomous defect in the development of Purkinje cells. These cells show immature morphology, synaptic arrangement, biochemical properties and gene expression, and are reduced in numbers. In addition, sg heterozygotes show accelerated dendritic atrophy and cell loss, suggesting that sg has a role in mature Purkinje cells. Effects of this mutation on cerebellar development have been studied for 25 years, but its molecular basis has remained unknown. We have genetically mapped staggerer to an interval of 160 kilobases on mouse chromosome 9 which was found to contain the gene encoding RORalpha, a member of the nuclear hormone-receptor superfamily. Staggerer mice were found to carry a deletion within the RORalpha gene that prevents translation of the ligand-binding homology domain. We propose a model based on these results, in which RORalpha interacts with the thyroid hormone signalling pathway to induce Purkinje-cell maturation.