Distal limb development and specification of digit identities in tetrapods are under the control of a mesenchymal organizer called the polarizing region. Sonic Hedgehog (SHH) is the morphogenetic signal produced by the polarizing region in the posterior limb bud. Ectopic anterior SHH signaling induces digit duplications and has been suspected as a major cause underlying congenital malformations that result in digit polydactyly. Here, we report that the polydactyly of Gli3-deficient mice arises independently of SHH signaling. Disruption of one or both Gli3 alleles in mouse embryos lacking Shh progressively restores limb distal development and digit formation. Our genetic analysis indicates that SHH signaling counteracts GLI3-mediated repression of key regulator genes, cell survival, and distal progression of limb bud development.

4Corresponding author (Utrecht) Communicated by T.HuntWe have cloned and sequenced the cDNAs encoding Patella vulgata cyclins A and B. The cDNA clones contain an open reading frame of 426 and 408 amino acids respectively, which present similarity with cyclins from other species. Cyclin A and B RNAs are present as polyadenylated and non-polyadenylated RNA in prophase oocytes and are completely polyadenylated in metaphase I. During the first cleavages after fertilization the level of cyclin A and B mRNAs is high and drops when the free swimming stage is reached. Using pl3sucl-Sepharose bead precipitation we demonstrate that cyclin synthesis is triggered during maturation and that inhibition of protein synthesis makes the cyclins disappear rapidly from the metaphase I oocytes, which shift to interphase condition. By microinjecting antisense oligonucleotides into metaphase I oocytes, we demonstrate that in vivo ablation of cyclin A and B messengers together gives the same result, whereas microinjection of only one oligonucleotide does not show any effect.

The actin gene family of the marine molluscPatella vulgata was chosen as a model system to study the regulation of genes expressed during early development in molluscs. Using a hamster actin cDNA clone as a probe, we isolated nine actin cDNA clones from trochophore larvae. The total nucleic acid sequence of three of these clones has been determined. Each clone contains the whole protein encoding region. The deduced amino acid sequences resemble actin proteins from other species to a high extent. The nucleotide sequence from the 3'UTR (UnTranslated Region) and 5'UTR from all nine clones has been resolved. In this way we could identify four different subtypes. Southern blots with genomic DNA were probed with different 3'UTR's corresponding to each subtype to determine the genomic organization. One 3'UTR detected one band probably corresponding with one gene. Another 3'UTR detected one or two genes and the third 3'UTR between two and four genes. Northern blots were used to detect the presence of actin mRNA during different stages of development. In the mature oocyte, actin mRNA is present in low amounts. The level of actin mRNA starts to rise steadily from 8 h after fertilization (88-cell stage) onwards. The level of the different subtype mRNAs, as specified by their 3'UTR rises at different developmental stages and to various extents. This indicates that the expression of each type is regulated independently and in relation to the developmental stage of the embryo.

In this study we show that the onset of embryonic transcription in the marine snail Patella vulgata coincides with the start of the sixth cleavage, when the cell-cycle elongates and divisions become asynchronous. Changes in mRNA content before and after onset of transcription were initially demonstrated by in vitro translation of isolated mRNA from different stages. Before the sixth cleavage, three major mRNAs encoding proteins of 36, 50 and 52 kDa were present. These proteins probably correspond to cyclin A and B and ribonucleotide reductase. After this stage, three major proteins with molecular weights of 36.5, 52.5 and 53 kDa were found after in vitro translation. Via hybrid selected translation and differential screening cDNAs corresponding to the 52.5 and 53 kDa proteins were cloned. The encoded proteins resemble tubulins from other animals to a high extent (between 96.5 and 93.1% identity for α-tubulin and 97.9 and 75.9% for β-tubulin). The 36.5 kDa protein is the previously described actin. Both tubulins were expressed at or shortly after the first asynchronous division after the fifth cleavage.

As the first five cleavages of the Patella vulgata embryo are synchronous, they are well suited to determine the mRNA level of cyclin A and B genes in an embryo. During the third and fourth cleavage cycle the quantity of A and B mRNA is regulated in a cell-cycle-dependent way, reaching a high level between cleavages and a lower level just after mitosis. This implies that transcription of the cyclin genes occurs before the overall transcription increases directly after the fifth cleavage. During the first cleavages cyclin A and B mRNA is localized in distinct parts of the cytoplasm. Between two successive cell devisions it is found as a crescent-shaped domain at the peripheral side of the nucleus. At cytokinesis it is present between two separating nuclei and at newly formed cell membranes. At the fifth cleavage this localization disappears. Changes in the expression pattern of cyclin A and B may be expected after the fifth cleavage, when the first cells become arrested in cell division and differentiate. The mechanism causing cell division arrest of these primary trochoblasts is still unknown. Cell division arrest caused by the absence of cyclin A and/or B mRNA could be conditional for further differentiation. However, a decrease in cyclin A and B mRNA level in the trochoblasts is not detectable until 4 h after their last division. Later in development no cyclin A and B mRNA can be detected in these cells, whereas cyclin A and B mRNA is present in other cells of the embryo. Thus, the absence of cyclin A and B mRNA in primary trochoblasts, and in the later differentiating secondary and accessory trochoblasts is not obligatory for cell division arrest or cell differentiation.