FIGURE SUMMARY
Title

Microarray analysis of zebrafish cloche mutant using amplified cDNA and identification of potential downstream target genes

Authors
Qian, F., Zhen, F., Ong, C., Jin, S.W., Meng Soo, H., Stainier, D.Y., Lin, S., Peng, J., and Wen, Z.
Source
Full text @ Dev. Dyn.

Genotyping of the 18-somite clo homozygous and wild-type/heterozygous sibling embryos by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Equal amount (1/10) of cDNA from the randomly selected single 18-somite embryo was subjected to semiquantitative PCR analysis with specific primers for zebrafish flk1, scl, lmo2, gata1, and e1-globin. actin and elf1a were used as control to indicate the amount of cDNA used. Lanes 4, 7, and 9 are clo mutants, whereas the other lanes (lanes 1, 2, 3, 5, 6, 8, 10, 11, and 12) represent wild-type or heterozygous sibling embryos.

EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Term:
Stage: 14-19 somites

Analysis of amplified cDNA quality. A: DNA electrophoresis analysis. A total of 100 ng of each amplified cDNAs derived from the single 18-somite clo homozygous mutant (lanes 2, 4, and 6) and wild-type or heterozygous sibling embryos (lanes 1, 3 and 5) was separated on 1% agarose gel. Ethidium bromide staining shows an evenly distributed smear of DNA in each lane. B: Virtual Northern analysis. cDNAs of A were transferred to a Hybone-N+ membrane and probed with digoxigenin-labeled flk1, gata1, e1-globin and actin. Whereas elf1a transcript was maintained at a similar level in all the samples, expression of flk1, gata1, and e1-globin were either absent or drastically reduced in the clo mutant embryos (lanes 2, 4, and 6).

EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Term:
Stage: 14-19 somites

Verification of target clones by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). One microgram of total RNA sample (20 embryos each) derived from the wild-type sibling (lanes 1 and 3) and clo mutant embryos (lanes 2 and 4) were reverse transcribed and subjected to PCR analysis with specific primers corresponding to each of the perspective target genes. PCR products were electrophoresed on 1% agarose gel and stained with ethidium bromide. e1-globin and elf1a were used as internal controls.

Temporal and spatial expression of znfl2, cldn g, cpo, cahz, mmp13, ncf1, dusp5, and zvsg1. A: Whole-mount in situ hybridization shows that znfl2 (top panels), cldn g (second panels), cpo (third panels), and cahz (bottom panels) are expressed in erythrocytes. B: Whole-mount in situ hybridization indicates that mmp13 (upper panels) and ncf1 (lower panels) stain myeloid lineage cells. C: Whole-mount in situ hybridization shows that dusp5 (upper panels) and zvsg1 (lower panels) express in the head and trunk vessels. The 3-, 10- and 14-somite stage embryos are dorsal view (3s) and flat-mount in dorsal view (10s and 14s), whereas rests of the embryos are lateral view. In all the panels, the 3-somite stage embryos are oriented anterior to the top and the rest are oriented anterior to the left.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Dev. Dyn.