Dorsoventral Patterning of the Zebrafish Embryo
93
of dorsoventral patterning. However, it can be ruled out as a Spemann
organizer signal of the zebrafish embryo.
6.7 dino, swirl, and snailhouse Are chordino, bmp2b,
andbmp7
As a first step of the candidate testing approach, we mapped the loci of
candidate genes and of the dorsoventral mutations, or carried out a
direct linkage analysis to test whether genes and mutations co segregate
during meiosis. For this purpose, a polymorphism in the candidate gene
has to be identified which allows the gene of the mutant line to be
distinguished from that of a wild-type reference line the mutant is
crossed into. In the case of chardina and dina, linkage analysis was
carried out using an EcoR1 restriction fragment length polymorphism
(RFLP) present between the dina mutant line and the wik-reference line
(Schulte-Merker et al. 1997). FI hybrids from a cross of a dina carrier
and a wik fish were raised, dina carriers identified and set up with each
other. The resulting F2 embryos were sorted into wild-type and mutant
pools which were analyzed for the chardina RFLP in genomic Southern
blots. Analysis of over 200 mutant embryos revealed complete absence
of the wik-specific chardina allele, indicating that the chardina gene
and the dina mutation are linked and located within 0.25 cM from each
other (no recombination in 400 meioses). Cloning of the chardina
cDNA from one of the dina alleles, dintt250, revealed a 104 bp deletion
at the 5' end of the coding region, which only leaves the first 40 amino
acids of the original protein. This deletion causes a frameshift which
leads to a premature termination of the protein after an additional 50
amino acids. Since these alterations occur upstream of the first functional, cysteine-rich domain, dintt250 Chordino is most likely a functional null mutant.
Similar analyses, in this case using a single strand conformational
polymorphism (SSCP) approach, revealed that the swirl mutation is
linked to the zebrafish bmp2b gene (Kishimoto et al. 1997). Here, two
alleles of similar strength were sequenced. In one of them, sw,.tc300, a
conserved cysteine residue normally involved in the formation of intramolecular disulfide bonds, is replaced by a tryptophane, while in the
93
of dorsoventral patterning. However, it can be ruled out as a Spemann
organizer signal of the zebrafish embryo.
6.7 dino, swirl, and snailhouse Are chordino, bmp2b,
andbmp7
As a first step of the candidate testing approach, we mapped the loci of
candidate genes and of the dorsoventral mutations, or carried out a
direct linkage analysis to test whether genes and mutations co segregate
during meiosis. For this purpose, a polymorphism in the candidate gene
has to be identified which allows the gene of the mutant line to be
distinguished from that of a wild-type reference line the mutant is
crossed into. In the case of chardina and dina, linkage analysis was
carried out using an EcoR1 restriction fragment length polymorphism
(RFLP) present between the dina mutant line and the wik-reference line
(Schulte-Merker et al. 1997). FI hybrids from a cross of a dina carrier
and a wik fish were raised, dina carriers identified and set up with each
other. The resulting F2 embryos were sorted into wild-type and mutant
pools which were analyzed for the chardina RFLP in genomic Southern
blots. Analysis of over 200 mutant embryos revealed complete absence
of the wik-specific chardina allele, indicating that the chardina gene
and the dina mutation are linked and located within 0.25 cM from each
other (no recombination in 400 meioses). Cloning of the chardina
cDNA from one of the dina alleles, dintt250, revealed a 104 bp deletion
at the 5' end of the coding region, which only leaves the first 40 amino
acids of the original protein. This deletion causes a frameshift which
leads to a premature termination of the protein after an additional 50
amino acids. Since these alterations occur upstream of the first functional, cysteine-rich domain, dintt250 Chordino is most likely a functional null mutant.
Similar analyses, in this case using a single strand conformational
polymorphism (SSCP) approach, revealed that the swirl mutation is
linked to the zebrafish bmp2b gene (Kishimoto et al. 1997). Here, two
alleles of similar strength were sequenced. In one of them, sw,.tc300, a
conserved cysteine residue normally involved in the formation of intramolecular disulfide bonds, is replaced by a tryptophane, while in the