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Figure 3. Mapping of ruby mutation. (a) Mapping interval derived from exon capture and BSA. The number of recombinants for each RFLP marker, the genetic distance and the location of the marker in v4.1 and v7.1 genome assemblies are shown. The 220 kb interval contains 7 genes: tmem150a, c2orf68, usp39, sftpb, pax8, psd4 and a portion of il1b. Genes in red have mutations causing amino acid or splice site changes based on the exon capture data. (b) Genomic, cDNA and protein sequences of WT and mutant pax8. The mutation of two contiguous nucleotides (nt) in intron 2 (arrowhead) shifts the acceptor splice site 1 nt, causing the inclusion of an extra G in the transcript (cDNA) (arrowhead). The protein sequence shows a change in frame and shortly after a premature STOP codon (*) abrogating the entire paired box domain. (c) Pax2 expression in wt and mutant ruby embryos by Whole Mount in situ hybridization showing clear impairment of pronephric development (arrowhead). (d) Pax8 morpholino phenocopies ruby phenotype. Pax8 morpholino was injected at one or two-cell stage (single cell injected). Embryos were fixed at st. 36–37 and pax2 expression was used to assess pronephric development. The embryo shown for two-cell stage injection was injected on the right side (based on fluorescent dye tracer [not shown]). Arrowheads indicate abnormal pronephros. Embryos in (c,d) are lateral views with dorsal to the top and anterior to the left (in left columns) or right (in right columns). |
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Display additional annotations [+]
| Gene |
Clone |
Species |
Stages |
Anatomy |
|
rgn
|
|
tropicalis
|
NF stage 35 and 36
to
NF stage 37 and 38
|
pronephric kidney
|
|
pax8
|
|
tropicalis
|
NF stage 35 and 36
to
NF stage 37 and 38
|
hindbrain
,
pronephric nephrostome
,
pronephric duct
,
pronephric tubule
|
|
atp1a1
|
|
tropicalis
|
NF stage 35 and 36
to
NF stage 37 and 38
|
otic vesicle
,
pronephric duct
,
pronephric kidney
,
head
,
pronephric tubule
|
|
slc7a8
|
|
tropicalis
|
NF stage 35 and 36
to
NF stage 37 and 38
|
pronephric kidney
|
|
hnf1b
|
|
tropicalis
|
NF stage 35 and 36
to
NF stage 37 and 38
|
pronephric kidney
,
pronephric tubule
|
|
cdh16
|
|
tropicalis
|
NF stage 35 and 36
to
NF stage 37 and 38
|
pronephric duct
,
pronephric kidney
,
pronephric tubule
|
|
slc5a9
|
|
tropicalis
|
NF stage 35 and 36
to
NF stage 37 and 38
|
pronephric kidney
,
pronephric tubule
|
|
| |
Supplementary Figure 2: Expression of pronephric markers in wt and ruby mutant embryos. Embryos from a heterozygous carrier cross were fixed at 36-37 stage and the expression of pronephric markers: pax8 (paired box 8), pax2 (paired box 2), atp1a1 (ATPase, Na+/K+ transporting, alpha 1 polypeptide) , slc7a8 (solute carrier family 7 (amino acid
transporter light chain, L system), member 8), slc5a9 (solute carrier family 5
(sodium/glucose cotransporter), member 9), smp-30 (senescence marker protein-30), hnf1-b
(HNF1 homeobox b) and cdh16 (cadherin 16), were studied by WMISH. Embryos were genotyped to confirm the mutation after imaging. Arrowheads indicate abnormal pronephros. |
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Supplementary Figure 3: Phenocopy of ruby mutant by pax8 MO injection. Pax8 morpholino was injected at one (a) or two cell stage (single cell injected) (b). Embryos were fixed at st 36-37 and pronephric development was assessed by pax2 expression. Abnormalities in pronephros development were classified: No phenotype; pronephros similar to uninjected controls (UIC); Type I: disrupted and abnormal pronephros similar to ruby mutants on both sides of the embryos. Type II: abnormal pronephros on one side and moderate abnormal pronephros on the other side; Type III: disrupted and abnormal pronephros on the injected side and moderate abnormal phenotype on the uninjected side. Type IV: disrupted and abnormal on the injected side and normal pronephros on the uninjected side. Images of Pax2 WMISH showing each type of phenotype are shown. For Type III and IV, embryos shown were injected on the right side. Arrowheads indicate disrupted and abnormal pronephros.
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