The anti-epileptic drug valproic acid causes malformations in the developing craniofacial skeleton of zebrafish larvae.

Valproic acid (VPA) is an anti-epileptic drug known to cause congenital craniofacial abnormalities, including orofacial clefts (OFC). The exact mechanisms by which VPA leads to craniofacial skeletal malformations are poorly understood. In this study, we investigated the effects of VPA on cartilage and bone formation in the zebrafish larval head during 1-13 hpf (early) and 25-37 hpf (late) development in which cranial neural crest cells (CNCCs) arise and then proliferate and differentiate, respectively. Double-staining for cartilage and bone at 5 dpf revealed that VPA reduced cartilage and bone formation in a dose-dependent manner after both early or late exposure. Several different CNCC-derived cartilage and bone elements were affected in both groups. In the early group (100 µM VPA), the posterior head length and the ethmoid plate were reduced in length (both p < 0.01), while mineralization of 4 out of 9 bone elements was often lacking (all p < 0.01). In the late group (100 µM VPA), also the posterior head length was reduced as well as the length of the ceratohyals (both p < 0.01). Similar to early exposure, mineralization of 3 out of 9 bone elements was often lacking (all p < 0.01). These results indicate that both CNCC formation (early) and differentiation (late) are hampered by VPA treatment, of which the consequences for bone and cartilage formation are persistent at 5 dpf. Indeed, we also found that the expression of several genes related to cartilage and bone was upregulated at 5 dpf. These data indicate a compensatory reaction to the lack of cartilage and bone. Altogether, VPA seems to induce craniofacial malformations via disturbed CNCC function leading to defects in cartilage and bone formation.

Fgf8a mutation affects craniofacial development and skeletal gene expression in zebrafish larvae.

Craniofacial development is tightly regulated and therefore highly vulnerable to disturbance by genetic and environmental factors. Fibroblast growth factors (FGFs) direct migration, proliferation and survival of cranial neural crest cells (CNCCs) forming the human face. In this study, we analyzed bone and cartilage formation in the head of five dpf fgf8ati282 zebrafish larvae and assessed gene expression levels for 11 genes involved in these processes. In addition, in situ hybridization was performed on 8 and 24 hours post fertilization (hpf) larvae (fgf8a, dlx2a, runx2a, col2a1a). A significant size reduction of eight out of nine craniofacial cartilage structures was found in homozygous mutant (6-36%, P<0.01) and heterozygous (7-24%, P<0.01) larvae. Also, nine mineralized structures were not observed in all or part of the homozygous (0-71%, P<0.0001) and heterozygous (33-100%, P<0.0001) larvae. In homozygote mutants, runx2a and sp7 expression was upregulated compared to wild type, presumably to compensate for the reduced bone formation. Decreased col9a1b expression may compromise cartilage formation. Upregulated dlx2a in homozygotes indicates impaired CNCC function. Dlx2a expression was reduced in the first and second stream of CNCCs in homozygous mutants at 24 hpf, as shown by in situ hybridization. This indicates an impairment of CNCC migration and survival by fgf8 mutation.

Differences in inhibitory avoidance, cortisol and brain gene expression in TL and AB zebrafish.

Recently, we established an inhibitory avoidance paradigm in Tupfel Long-Fin (TL) zebrafish. Here, we compared task performance of TL fish and fish from the AB strain; another widely used strain and shown to differ genetically and behaviourally from TL fish. Whole-body cortisol and telencephalic gene expression related to stress, anxiety and fear were measured before and 2 h post-task. Inhibitory avoidance was assessed in a 3-day paradigm: fish learn to avoid swimming from a white to a black compartment where a 3V-shock is given: day 1 (first shock), day 2 (second shock) and day 3 (no shock, sampling). Tupfel Long-Fin fish rapidly learned to avoid the black compartment and showed an increase in avoidance-related spatial behaviour in the white compartment across days. In contrast, AB fish showed no inhibitory avoidance learning. AB fish had higher basal cortisol levels and expression levels of stress-axis related genes than TL fish. Tupfel Long-Fin fish showed post-task learning-related changes in cortisol and gene expression levels, but these responses were not seen in AB fish. We conclude that AB fish show higher cortisol levels and no inhibitory avoidance than TL fish. The differential learning responses of these Danio strains may unmask genetically defined risks for stress-related disorders.

An AFLP-based genome-wide mapping strategy.

To efficiently determine the chromosomal location of phenotypic mutants, we designed a genome-wide mapping strategy that can be used in any crop for which a dense AFLP (Amplified Fragment Length Polymorphism) map is available or can be made. The AFLP technique is particularly suitable to initiate map-based cloning projects because it detects many markers per reaction. First a standard set of AFLP primer combinations that results in a framework of AFLP markers well dispersed over the genome is selected. These primer combinations are applied to a limited number of mutant individuals from a segregating population to register linkage and non-linkage of the AFLP markers to the gene-of-interest. Further delineation of the area of interest is accomplished by analyzing the remaining recombinants and additional mutant individuals with AFLP markers that lie within the identified region. We illustrate the usefulness of the method by mapping three rotunda ( ron) leaf-form mutant loci of Arabidopsis thaliana and show that in the initial phase of map-based cloning projects a 400-600 kb interval can be identified for the average mutant locus within a few weeks. Once such an area is identified and before initiating the more time-consuming fine-mapping procedure, it is essential to examine publicly available databases for candidate genes and known mutants in the identified region. The 390-kb interval on chromosome 4 that harbors the ron2 mutation, also carries a known flower mutant, leunig ( lug); upon crossing, the two mutants appeared to be allelic. When no such candidates are found, the mapping procedure should be continued. We present a strategy to efficiently select recombinants that can be used for fine mapping.

AFLP maps of Petunia hybrida: building maps when markers cluster.

AFLP mapping in Petunia hybrida was undertaken with the intention of building a high-density genetic map suitable for applications such as map-based gene cloning. In total five maps were constructed from two mapping populations, with placement of more than 800 markers. Despite the large number of markers the resulting map is roughly ten-fold smaller than those of other plant species, including the closely related tomato. Low levels of recombination are reflected in clusters of tightly linked markers, both AFLPs and RFLPs, in all the maps. Clustering patterns vary between mapping populations, however, such that loci tightly linked in one population may be separable in another. Combined with earlier reports of aberrant meiotic pairing and recombination, our results suggest that, for species like petunia, map-based cloning may be more complex than in model species such as arabidopsis and tomato.

A physical amplified fragment-length polymorphism map of Arabidopsis.

We have positioned amplified fragment-length polymorphism (AFLP) markers directly on the genome sequence of a complex organism, Arabidopsis, by combining gel-based AFLP analysis with in silico restriction fragment analysis using the published genome sequence. For placement of the markers, we used information on restriction fragment size, four selective nucleotides, and the rough genetic position of the markers as deduced from the analysis of a limited number of Columbia (Col)/Landsberg (Ler) recombinant inbred lines. This approach allows for exact physical positioning of markers as opposed to the statistical localization resulting from traditional genetic mapping procedures. In addition, it is fast because no extensive segregation analysis is needed. In principle, the method can be applied to all organisms for which a complete or nearly complete genome sequence is available. We have located 1,267 AFLP Col/Ler markers resulting from 256 SacI+2, MseI+2 primer combinations to a physical position on the Arabidopsis genome. The positioning was verified by sequence analysis of 70 markers and by segregation analysis of two leaf-form mutants. Approximately 50% of the mapped Col/Ler AFLP markers can be used for segregation analysis in Col/C24, Col/Wassilewskija, or Col/Cape Verde Islands crosses. We present data on one such cross: the localization of a viviparous-like mutant segregating in a Col/C24 cross.

Analysis by Transposon Display of the behavior of the dTph1 element family during ontogeny and inbreeding of Petunia hybrida.

Transposon Display is a high-resolution method that was used here to visualize simultaneously individual members of the dTph1 transposable element family of Petunia hybrida. The method provides a tool for accurate analyses of copy numbers and insertion frequencies, and a means to study the behavior of a family of elements as a whole. Somatic insertion events can be identified and insertion events in a cell of the L2 apical lineage can be distinguished unequivocally from those in a cell outside this lineage. In sublines of the high-copy-number line W137, an average insertion rate equivalent to transposition of 10% of the total number of element copies in each generation was measured, copy number increases of over 20% in four generations were recorded, and element position turnover was analyzed. Insertion events are detected essentially randomly both in time and space. The general applicability of the technique for the analysis of the transpositional behavior of element systems is discussed.

Petunia Ap2-like genes and their role in flower and seed development.

We have isolated three Apetala2 (Ap2)-like genes from petunia and studied their expression patterns by in situ hybridization. PhAp2A has a high sequence similarity to the A function gene Ap2 from Arabidopsis and a similar expression pattern during flower development, suggesting that they are cognate orthologs. PhAp2B and PhAp2C encode for AP2-like proteins that belong to a different subgroup of the AP2 family of transcription factors and exhibit divergent, nearly complementary expression patterns during flower development compared with PhAp2A. In contrast, all three PhAp2 genes are strongly expressed in endosperm. The phenotype of the petunia A-type mutant blind cannot be attributed to mutations in the petunia Ap2 homologs identified in this study, and reverse genetics strategies applied to identify phap2a mutants indicate that PhAp2A might not be essential for normal perianth development in petunia. Nevertheless, we show that PhAp2A is capable of restoring the homeotic transformations observed in flowers and seed of the ap2-1 mutant of Arabidopsis. Although the interspecific complementation proves that PhAp2A encodes a genuine Ap2 ortholog from petunia, additional factors may be involved in the control of perianth identity in this species.

Transposon Display identifies individual transposable elements in high copy number lines.

The dTph1 transposable element family of Petunia hybrida line W138 consists of between 100 and 200 members. A strategy that allows simultaneous detection of individual elements is described. Sequences flanking dTph1 elements are amplified by means of a ligation-mediated PCR. The resulting fragments are locus-specific and can be analysed by polyacrylamide gel electrophoresis. One of the applications of Transposon Display is the isolation of dTph1-tagged genes. Fragments that co-segregate with a mutant phenotype can be extracted from the gel and reamplified, providing access to tagged genes, as demonstrated in a reconstruction experiment. Data on the molecular identification of a phenotypic mutant, isolated in a random tagging experiment is also presented. Upon sequencing, the obtained candidate fragment was found to be identical to part of the previously identified Fbp1 gene.

Transcriptional and post-transcriptional regulation of chloroplast gene expression in Petunia hybrida.

To study the control of differential gene expression during plastid biogenesis in Petunia hybrida, we have investigated the in vivo translation and transcription of the rbc L gene, coding for the large subunit of ribulose bisphosphate carboxylase (LSU), and the psa A gene, coding for P700 chlorophyll-a apoprotein (AP700). Differential expression of these plastid-encoded genes was studied in two developmentally different plastid systems, proplastid-like organelles from the green cell suspension AK2401 and mature chloroplasts from green leaves. In vivo translation of rbc L and psa A transcripts was analysed using specific antibodies. Specific transcript levels were analysed using internal fragments of the rbc L and psa A genes. A standardization procedure was used so that a direct correlation could be made between the amount of products and gene copy number. In Petunia hybrida the amount of LSU polypeptides present in both plastid types does not correspond to the amount of specific mRNA for the gene. Although the rbc L transcripts are present in both plastid types, the LSU protein is only present in green leaf plastids and not in cell culture plastids. In vitro translation of isolated rbc L transcripts give similar results, thereby suggesting that differences in the primary structure of the transcripts are responsible for the observed discrepancy. In contrast to this, the amount of AP700 polypeptides does correspond to the amount of the psa A transcripts. Therefore, our results indicate that the expression of chloroplast genes during plastid biogenesis takes place on at least two different levels: expression of the rbc L gene is regulated post-transcriptionally while expression of the psa A gene is regulated at the transcriptional level.