Construction of chromosome markers from the Lake Victoria cichlid Paralabidochromis chilotes and their application to comparative mapping.

Cichlid fishes in the African Great Lakes are known as a spectacular example of adaptive radiation in vertebrates. Four linkage maps have been constructed to identify the genes responsible for adaptation and speciation, and the genetic linkages of those genes are assumed to play an important role during adaptive evolution. However, it is difficult to analyze such linkages because the linkage groups of one species do not match well with those of the other species. Chromosome markers are a powerful tool for the direct identification of linkage homology between different species. We used information about the linkage map of the Lake Malawi cichlid (Labeotropheus fuelleborni/Metriaclima zebra) to isolate bacterial artificial chromosome (BAC) clones from the BAC library of Paralabidochromis chilotes, Lake Victoria. We identified 18 of 22 P. chilotes chromosomes by single- and multi-color BAC fluorescence in situ hybridization using 19 BAC clones. Comparative mapping with the chromosome markers of P. chilotes in Astatotilapia burtoni (2n = 40) from Lake Tanganyika revealed the chromosome rearrangements that have occurred in this lineage. These chromosome markers will be useful for delineating the process of genome and chromosome evolution in African species.

A primer set to determine sex in the small Indian mongoose, Herpestes auropunctatus.

To enable the accurate sexing of individuals of introduced populations of the small Indian mongoose, Herpestes auropunctatus, we designed a primer set for the amplification of the sex-specific fragments EIF2S3Y and EIF2S3X. Using this primer set, the expected amplification products were obtained for all samples of genomic DNA tested: males yielded two bands and females a single band. Sequencing of each PCR product confirmed that the 769-bp fragment amplified from DNA samples of both sexes was derived from EIF2S3X, whereas the 546-bp fragment amplified only from male DNA samples was derived from EIF2S3Y. The results indicated that this primer set is useful for sex identification in this species.

Chromosome elimination in the interspecific hybrid medaka between Oryzias latipes and O. hubbsi.

An interspecific hybrid medaka (rice fish) between Oryzias latipes and O. hubbsi is embryonically lethal. To gain an insight into the cellular and molecular mechanisms that cause the abnormalities occurring in the hybrid medaka, we investigated the behavior of chromosomes and the expression patterns of proteins responsible for the chromosome behavior. The number of chromosomes in the hybrid embryos gradually decreased to nearly half, since abnormal cell division with lagging chromosomes at anaphase eliminated the chromosomes from the cells. The chromosome lagging occurred at the first cleavage and continued throughout embryogenesis even after the midblastula transition. Fluorescent in-situ hybridization analyses revealed that the chromosomes derived from O. hubbsi are preferentially eliminated in both O. latipes-hubbsi and O. hubbsi-latipes embryos. Whole-mount immunocytochemical analyses using antibodies against alpha-tubulin, gamma-tubulin, inner centromere protein, Cdc20, Mad2, phospho-histone H3 and cohesin subunits (SMC1alpha, SMC3 and Rad21) showed that the expression patterns of these proteins in the hybrid embryos are similar to those in the wild-type embryos, except for phospho-histone H3. Phospho-histone H3 present on chromosomes at metaphase was lost from normally separated chromosomes at anaphase, whereas it still existed on lagging chromosomes at anaphase, indicating that the lagging chromosomes remain in the metaphase state even when the cell has proceeded to the anaphase state. On the basis of these findings, we discuss the cellular and molecular mechanisms of chromosome elimination in the hybrid medaka.

Molecular genetic cascades for external genitalia formation: an emerging organogenesis program.

External genitalia are anatomical structures located at the posterior embryonic region as part of several urogenital/reproductive organs. The embryonic anlage of the external genitalia, the genital tubercle (GT) develops as a bud-shaped structure with an initial urethral plate and later urethra. Embryonic external genitalia are considered to be one of the appendages. Recent experiments suggest that essential regulatory genes possess similar functions for the outgrowth regulation of the GT and limb appendages. The transient embryonic epithelia located in the distal GT are called the distal urethral epithelium (DUE) regulating, at least in part, the (distal) GT development. This review covers the available data about early patterning of GT and discusses the molecular developmental similarities and points of divergence between the different appendages. Development of the male and female external genitalia is also reviewed.

X accumulation of LINE-1 retrotransposons in Tokudaia osimensis, a spiny rat with the karyotype XO.

The observation that LINE-1 transposable elements are enriched on the X in comparison to the autosomes led to the hypothesis that LINE-1s play a role in X chromosome inactivation. If this hypothesis is correct, loss of LINE-1 activity would be expected to result in species extinction or in an alternate pathway of dosage compensation. One such alternative pathway would be to evolve a karyotype that does not require dosage compensation between the sexes. Two of the three extant species of the Ryukyu spiny rat Tokudaia have such a karyotype; both males and females are XO. We asked whether this karyotype arose due to loss of LINE-1 activity and thus the loss of a putative component in the X inactivation pathway. Although XO Tokudaia has no need for dosage compensation, LINE-1s have been recently active in Tokudaia osimensis and show higher density on the lone X than on the autosomes.

Biallelic expression of Z-linked genes in male chickens.

In birds, females are heterogametic (ZW), while males are homogametic (ZZ). It has been proposed that there is no dosage compensation for the expression of Z-linked genes in birds. In order to examine if the genes are inactivated on one of the two Z chromosomes, we analyzed the allelic expression of the B4GALT1 and CHD-Z genes on Z chromosomes in male chickens. One base substitution was detected among 15 chicken breeds and lines examined for each gene, and cross mating was made between the breeds or lines with polymorphism. cDNAs were synthesized from cultured cell colonies each derived from a single cell of an F1 male embryo. The allelic expression of the B4GALT1 gene was examined by restriction fragment length polymorphism analysis of the PCR products digested with RSAI, and that of the CHD-Z gene by the single nucleotide primer extension (SNuPE) method. Both of the genes displayed biallelic expression, suggesting that these Z-linked genes were not subject to inactivation in male chickens. Comparison between expression levels in males and females by real-time quantitative PCR suggested that expression was compensated for the CHD-Z gene but not for the B4GALT1 gene.

Chromosome assignment of eight SOX family genes in chicken.

Chromosome locations of the eight SOX family genes, SOX1, SOX2, SOX3, SOX5, SOX9, SOX10, SOX14 and SOX21, were determined in the chicken by fluorescence in situ hybridization. The SOX1 and SOX21 genes were localized to chicken chromosome 1q3.1-->q3.2, SOX5 to chromosome 1p1.6-->p1.4, SOX10 to chromosome 1p1.6, and SOX3 to chromosome 4p1.2-->p1.1. The SOX2 and SOX14 genes were shown to be linked to chromosome 9 using two-colored FISH and chromosome painting, and the SOX9 gene was assigned to a pair of microchromosomes. These results suggest that these SOX genes form at least three clusters on chicken chromosomes. The seven SOX genes, SOX1, SOX2, SOX3, SOX5, SOX10, SOX14 and SOX21 were localized to chromosome segments with homologies to human chromosomes, indicating that the chromosome locations of SOX family genes are highly conserved between chicken and human.

Cloning, genomic structure and chromosomal localization of the gene encoding mouse DNA helicase RECQL5beta.

Five members of the RecQ helicase family, RECQL, WRN, BLM, RTS and RECQL5, have been found in human and three of them (WRN, BLM and RTS) were disclosed to be the genes responsible for Werner, Bloom and Rothmund-Thomson syndromes, respectively. RECQL5 (RecQ helicase protein-like 5) was isolated as the fifth member of the family in humans through a search of homologous expressed sequence tags. The gene is expressed with at least three alternative splicing products, alpha, beta and gamma. Here, we isolated mouse RECQL5 beta and determined the DNA sequence of full-length cDNA as well as the genome organization and chromosome locus. The mouse RECQL5 beta gene consists of 2949 bp coding 982 amino acid residues. Comparison of amino acid sequence among human (Homo sapiens), mouse (Mus musculus), Drosophila melanogaster and Caenorhabditis elegans RECQL5 beta homologs revealed three portions of highly conserved regions in addition to the helicase domain. Nineteen exons are dispersed over 40 kbp in the genome and all of the acceptor and donor sites for the splicing of each exon conform to the GT/AG rule. The gene is localized to the mouse chromosome 11E2, which has a syntenic relation to human 17q25.2-q25.3 where human RECQL5 beta exists. Our genetic characterizations of the mouse RECQL5 beta gene will contribute to functional studies on the RECQL5 beta products.

Fgf/MAPK signalling is a crucial positional cue in somite boundary formation.

The temporal and spatial regulation of somitogenesis requires a molecular oscillator, the segmentation clock. Through Notch signalling, the oscillation in cells is coordinated and translated into a cyclic wave of expression of hairy-related and other genes. The wave sweeps caudorostrally through the presomitic mesoderm (PSM) and finally arrests at the future segmentation point in the anterior PSM. By experimental manipulation and analyses in zebrafish somitogenesis mutants, we have found a novel component involved in this process. We report that the level of Fgf/MAPK activation (highest in the posterior PSM) serves as a positional cue within the PSM that regulates progression of the cyclic wave and thereby governs the positions of somite boundary formation.

Two mouse piwi-related genes: miwi and mili.

Genes belonging to the piwi family are required for stem cell self-renewal in diverse organisms. We cloned mouse homologues of piwi by RT-PCR using degenerative primers. The deduced amino acid sequences of mouse homologues MIWI and MILI showed that each contains a well-conserved C-terminal PIWI domain and that each shares significant homology with PIWI and their human counterparts HIWI. Both miwi and mili were found in germ cells of adult testis by in situ hybridization, suggesting that these genes may function in spermatogenesis. Furthermore, mili was expressed in primordial germ cells (PGCs) of developing mouse embryos and may therefore play a role during germ cell formation. MIWI may be involved in RNA processing or translational regulation, since MIWI was found to possess RNA binding activity. Our data suggest that miwi and mili regulate spermatogenesis and primordial germ cell production.

Fgf signalling through MAPK cascade is required for development of the subpallial telencephalon in zebrafish embryos.

The telencephalon is formed in the most anterior part of the central nervous system (CNS) and is organised into ventral subpallial and dorsal pallial domains. In mice, it has been demonstrated that Fgf signalling has an important role in induction and patterning of the telencephalon. However, the precise role of Fgf signalling is still unclear, owing to overlapping functions of Fgf family genes. To address this, we have examined, in zebrafish embryos, the activation of Ras/mitogen-activated protein kinase (MAPK), one of the major downstream targets of Fgf signalling. Immunohistochemical analysis reveals that an extracellular signal-regulated kinase (ERK), a vertebrate MAPK is activated in the anterior neural boundary (ANB) of the developing CNS at early segmentation stages. Experiments with Fgf inhibitors reveal that ERK activation at this stage is totally dependent on Fgf signalling. Interestingly, a substantial amount of ERK activation is observed in ace mutants in which fgf8 gene is mutated. We then examine the function of Fgf signalling in telencephalic development by use of several inhibitors to Fgf signalling cascade, including dominant-negative forms of Ras (Ras(N17)) and the Fgf receptor (Fgfr), and a chemical inhibitor of Fgfr, SU5402. In treated embryos, the induction of telencephalic territory normally proceeded but the development of the subpallial telencephalon was suppressed, indicating that Fgf signalling is required for the regionalisation within the telencephalon. Finally, antisense experiments with morpholino-modified oligonucleotides suggest that zebrafish fgf3, which is also expressed in the ANB, co-operates with fgf8 in subpallial development.

The mouse Hoxd13(spdh) mutation, a polyalanine expansion similar to human type II synpolydactyly (SPD), disrupts the function but not the expression of other Hoxd genes.

Polyalanine expansion in the human HOXD13 gene induces synpolydactyly (SPD), an inherited congenital limb malformation. A mouse model was isolated, which showed a spontaneous alanine expansion due to a 21-bp duplication at the corresponding place in the mouse gene. This mutation (synpolydactyly homolog, spdh), when homozygous, causes malformations in mice similar to those seen in affected human patients. We have studied the genetics of this condition, by using several engineered Hoxd alleles, as well as by looking at the expression of Hox and other marker genes. We show that the mutated SPDH protein induces a gain-of-function phenotype, likely by behaving as a dominant negative over other Hox genes. The mutation, however, seems to act independently from Hoxa13 and doesn't appear to affect Hox gene expression, except for a slight reduction of the HOXD13 protein itself. Developmental studies indicate that the morphological effect is mostly due to a severe retardation in the growth and ossification of the bony elements, in agreement with a general impairment in the function of posterior Hoxd genes.

Cloning, chromosomal mapping, and characteristic 5'-UTR sequence of murine cytosolic sialidase.

We have totally sequenced a cytosolic sialidase [EC 3.2.1.18] by RT-PCR from the murine thymus (murine thymic sialidase, MTS) which has a 1844-base length (encoding 385 amino acids including two sialidase motifs) and is the longest cytosolic sialidase ever reported. MTS has high and relatively low homologies with those of mammalian cytosolic sialidases from the mouse brain (99%), rat (91%), and human skeletal muscle (75%), and those of the mouse lysosomal (47%) and membrane-bound (51%) sialidases, respectively. Chromosomal mapping, being the first report of mouse cytosolic sialidase gene, showed that the MTS gene is localized to the distal part of mouse chromosome 1D and to rat chromosome 9q36. RT-PCR with the site-specific primers revealed that the coding region was expressed in all organs tested, but expressions including the 5'-UTR were barely detectable except for in the upper-thymic fraction. Also, soluble sialidase activity in the thymus was the highest of these organs. There were mRNA instability signals and AT-rich regions in 143 bp of MTS 5'-end.

Efficient chromosomal transposition of a Tc1/mariner- like transposon Sleeping Beauty in mice.

The presence of mouse embryonic stem (ES) cells makes the mouse a powerful model organism for reverse genetics, gene function study through mutagenesis of specific genes. In contrast, forward genetics, identification of mutated genes responsible for specific phenotypes, has an advantage to uncover novel pathways and unknown genes because no a priori assumptions are made about the mutated genes. However, it has been hampered in mice because of the lack of a system in which a large-scale mutagenesis and subsequent isolation of mutated genes can be performed efficiently. Here, we demonstrate the efficient chromosomal transposition of a Tc1/mariner-like transposon, Sleeping Beauty, in mice. This system allows germ-line mutagenesis in vivo and will facilitate certain aspects of phenotype-driven genetic screening in mice.

A novel sox gene, 226D7, acts downstream of Nodal signaling to specify endoderm precursors in zebrafish.

Vertebrate endoderm development has recently become the focus of intense investigation. We have identified a novel sox gene, 226D7, which is important in zebrafish endoderm development. 226D7 was isolated by an in situ hybridization screening for genes expressed in the yolk syncytial layer (YSL) at the blastula stage. 226D7 is expressed mainly in the YSL at this stage and, during gastrulation, its expression is also detected in the forerunner cells and endodermal precursor cells. The expression of 226D7 is positively regulated by Nodal signaling. The knockdown of 226D7 using morpholino antisense oligonucleotides results in a lack of sox17-expressing endodermal precursor cells during gastrulation, and, consequently, lacks endodermal derivatives such as gut tissue. The effect is strictly restricted to the endodermal lineage, while the mesoderm is normally formed, a phenotype that is nearly identical to that of the casanova mutant (Dev. Biol. 215 (1999) 343). We further demonstrate that overexpression of 226D7 increases the number of sox17-expressing endodermal progenitor cells without upregulating the expression of the Nodal genes, cyclops and squint. Region-specific knockdown and overexpression of 226D7 by injection into the YSL suggest that 226D7 in the YSL is not involved in endoderm formation and 226D7 in the endoderm progenitor cells is important for endoderm development. Taken together, our data demonstrate that 226D7 is a downstream target of Nodal signal and a critical transcriptional regulator of early endoderm formation.

Comparative FISH mapping of human cDNA clones to chromosomes of the musk shrew (Suncus murinus, Insectivora).

Forty-one cDNA clones of human functional genes were newly mapped to chromosomes of the musk shrew (Suncus murinus, Insectivora) by fluorescence in situ hybridization, and a comparative cytogenetic map of 51 genes, including 10 genes reported in our previous study, was constructed between human (HSA) and musk shrew (SMU) chromosomes. In this comparative map, the 51 genes localized to human autosomes, except HSA 8, 16, and 20, were mapped to 15 shrew autosomes, except SMU 4, 16, 17 and 18. Twelve conserved segments were identified between human and shrew chromosomes, and six segments among the musk shrew, human, and mouse. Our results defined the presence of at least one inversion and several interchromosomal rearrangements that occurred during evolution after the two species diverged from a common ancestor. Localization of three major histocompatibility complex (MHC) genes to shrew chromosome 3 suggested that the MHC genes of the musk shrew are located in a cluster on chromosome 3. The cytogenetic map constructed in this study is the first cytogenetic map with many functional genes in insectivore species. This approach provides clues for clarifying the chromosomal evolution in this order.

Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration.

The analysis of newt lens regeneration has been an important subject in developmental biology. Recently, it has been reported that the genes involved in the normal eye development are also expressed in the regenerative process of lens regeneration in the adult newt. However, functional analysis of these genes has not been possible, because there is no system to introduce genes efficiently into the cells involved in the regeneration. In the present study, lipofection was used as the method for gene transfer in cultured pigmented iris cells that can transdifferentiate into lens cells in newt lens regeneration. Positive expression of a reporter gene was obtained in more than 70% of cells. In addition, the aggregate derived from gene-transfected cells maintained its expression at a high level for a long time within the host tissue. To verify the effectiveness of this model system with a reporter gene in lens regeneration, Pax6, which is suggested to be involved in normal eye development and lens regeneration, was transfected. Ectopic expression of lens-specific crystallins was obtained in cells that show no such activity in normal lens regeneration. These results made it possible for the first time to analyze the molecular mechanism of lens regeneration in the adult newt.

Expression and chromosome location of hamster Ku70 and Ku80.

Ku proteins play an important role in DNA double-strand break (DSB) repair, chromosome maintenance, and growth regulation. To understand the fundamental characteristics of Ku proteins, we examined the electrophoretic mobility and expression of hamster Ku70 and Ku80 and determined the chromosome locations of their genes. The electrophoretic mobility of hamster Ku proteins are different from that of human Ku proteins. No significant changes in the quantity of Ku proteins were observed in CHO-K1 cells treated with 10 Gy of ionizing radiation, suggesting that both proteins are expressed constitutively in amounts adequate to repair DNA DSBs. The chromosome locations of the Ku genes were determined by direct R-banding fluorescence in situ hybridization. The Ku70 gene was localized to Syrian hamster chromosome 4qa4.1--> qa4.2 and Chinese hamster chromosome 2p3.1, and the Ku80 gene was localized to Syrian hamster chromosome 4qb5--> qb6.1 and Chinese hamster chromosome 2p3.5-->p3.6. These results provide clues to the biological functions of Ku, as well as useful information for constructing comparative chromosome maps between hamsters and other mammalian species, including human, mouse, and rat.

Sequence polymorphisms, allelic expression status and chromosome locations of the chicken IGF2 and MPR1 genes.

By screening 26 chicken breeds and lines, DNA polymorphisms were identified in the IGF2 and MPR1 genes, of which mammalian homologues are parentally imprinted, and the GAPD gene, a housekeeping control. Using the polymorphisms as genetic markers, we found that all three genes are expressed biallelically in embryonic tissues. IGF2 and MPR1 were mapped on chicken chromosomes 5 and 3, respectively, by fluorescence in situ hybridization, demonstrating conserved linkage homology between mammals and birds.

Cloning and chromosome mapping of human and chicken Iroquois (IRX) genes.

Three highly homologous homeobox genes (caupolican, araucan and mirror) have been identified in Drosophila. These genes belong to the novel Iroquois complex, which acts as a pre-pattern molecule in Drosophila neurogenesis. Recently several vertebrate Iroquois homologues (Irx) were isolated and found to be involved in pattern formation of various tissues. Here we report cytogenetic mapping of four human and five chicken Iroquois genes by FISH. Our findings revealed that vertebrate Irx genes are clustered at two different loci.