ZFIN ID: ZDB-FIG-160926-12
Thambyrajah et al., 2016 - A gene trap transposon eliminates haematopoietic expression of zebrafish Gfi1aa, but does not interfere with haematopoiesis. Developmental Biology   417(1):25-39 Full text @ Dev. Biol.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Terms:
Stage Range: 5-9 somites to 26+ somites
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage Range: 5-9 somites to Protruding-mouth

Fig. 5

Gfi1aa expression is lost in primitive erythrocytes of homozygous qmc551 embryos, yet primitive haematopoiesis is unaffected. (A) Genomic organization of the gfi1aaqmc551Gt locus. Oligos I and J were used in the RT-PCR experiments. The sequence complementary to the gfi1aa WISH probe is indicated. The TaqMan probe overlapped with the exon 1/2 boundary and is not shown. (B) Fluorescent images of 20 hpf wt, qmc551 heterozygous and homozygous siblings from an incross of qmc551 heterozygous carriers. (C,H,I,L,M) RNA WISH experiments with indicated probes on 13-20 hpf wt and qmc551 homozygous embryos. Views are posterior with anterior up on all images of 13-16 hpf and lateral with anterior to the left and dorsal up on all images of 18, 20 and 22 hpf embryos. (D) QRT-PCR to determine relative levels of gfi1aa mRNA in 16 hpf whole embryos. EF1α mRNA was used as a loading control. (E) Flow cytometric analysis of embryonic cells at 19 hpf showing green and red fluorescence excited with a 488 nm laser, and detected using the band pass filters 529/28 and 580/23 (central wavelength/width in nm), respectively. (F) Mean green fluorescence observed in GFP+ cells in embryos at 19 hpf. Please note that values are shown relative to heterozygous controls (Two-tailed t-test: p<0.0001). (G) The relative numbers of GFP+ cells in embryos at 19 hpf. (J,Q) Diaminofluorene staining to detect hemoglobin in 3 dpf embryos. (K,R) Images of prRBCs that were isolated from the sinus venosus of terminally anaesthetized 3 day-old embryos and stained with May-Gr├╝nwald and Giemsa. (N) Structure of the gfi1b transcript before and after splicing in the presence and absence of gfi1b morpholinos. In the morphant, exon 3 and 5 sequences are spliced together (dashed line). (O) RT-PCR performed on RNA isolated from uninjected and morpholino-injected wt embryos. (P)gfi1b cDNA sequence representing the alternatively spliced gfi1b mRNA isolated from gfi1b morphant embryos. PCR oligo and divergent C-terminal amino acid sequences are underlined. Sequences derived from different exons are shown in different colors. The numbering of nucleotides corresponds to that of database entry NM_001271841. Encoded amino acids are counted below. Arrows: red - prRBCs, yellow - inner ear hair cells, light blue - anterior lateral mesoderm, orange - hatching gland precursors. Arrowheads: red - prRBCs in heart. Fractions x/y give the number of embryos, x, with depicted phenotype out of all embryos analyzed, y. Embryos are shown with anterior left and dorsal up.

Gene Expression Details
Gene Antibody Fish Conditions Stage Qualifier Anatomy Assay
EGFP gfi1aaqmc551Gt/+ control 20-25 somites posterior lateral mesoderm erythroid lineage cell IFL
gfi1aaqmc551Gt/qmc551Gt control 20-25 somites posterior lateral mesoderm erythroid lineage cell IFL
gata1a WT control 26+ somites erythroblast ISH
gfi1aaqmc551Gt/qmc551Gt control 26+ somites erythroblast ISH
gfi1aa WT control 5-9 somites posterior lateral mesoderm erythroid lineage cell ISH
10-13 somites posterior lateral mesoderm erythroid lineage cell ISH
14-19 somites otic vesicle ISH
14-19 somites posterior lateral mesoderm erythroid lineage cell ISH
20-25 somites inner ear ISH
20-25 somites posterior lateral mesoderm erythroid lineage cell ISH
gfi1aaqmc551Gt/qmc551Gt control 5-9 somites Not Detected posterior lateral mesoderm erythroid lineage cell ISH
10-13 somites Not Detected posterior lateral mesoderm erythroid lineage cell ISH
14-19 somites Not Detected posterior lateral mesoderm erythroid lineage cell ISH
14-19 somites whole organism RTPCR
20-25 somites inner ear ISH
20-25 somites Not Detected posterior lateral mesoderm erythroid lineage cell ISH
gfi1ab WT control 5-9 somites anterior lateral mesoderm ISH
5-9 somites Not Detected erythroblast ISH
5-9 somites otic placode ISH
10-13 somites Not Detected erythroblast ISH
10-13 somites otic placode ISH
14-19 somites Not Detected erythroblast ISH
14-19 somites otic placode ISH
20-25 somites Not Detected erythroblast ISH
20-25 somites inner ear ISH
gfi1aaqmc551Gt/qmc551Gt control 5-9 somites anterior lateral mesoderm ISH
5-9 somites Not Detected erythroblast ISH
5-9 somites otic placode ISH
10-13 somites Not Detected erythroblast ISH
10-13 somites otic placode ISH
14-19 somites Not Detected erythroblast ISH
14-19 somites otic placode ISH
20-25 somites Not Detected erythroblast ISH
20-25 somites inner ear ISH
gfi1b WT control 5-9 somites otic placode ISH
10-13 somites erythroblast ISH
10-13 somites posterior lateral mesoderm erythroid lineage cell ISH
14-19 somites erythroblast ISH
14-19 somites otic vesicle ISH
14-19 somites posterior lateral mesoderm erythroid lineage cell ISH
20-25 somites erythroblast ISH
20-25 somites posterior lateral mesoderm erythroid lineage cell ISH
gfi1aaqmc551Gt/qmc551Gt control 5-9 somites otic placode ISH
10-13 somites posterior lateral mesoderm erythroid lineage cell ISH
14-19 somites erythroblast ISH
14-19 somites otic vesicle ISH
14-19 somites posterior lateral mesoderm erythroid lineage cell ISH
20-25 somites erythroblast ISH
20-25 somites posterior lateral mesoderm erythroid lineage cell ISH
hbbe1.1 WT control 26+ somites erythroblast ISH
gfi1aaqmc551Gt/qmc551Gt control 26+ somites erythroblast ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
WT + MO4-gfi1b + MO4-tp53 + MO5-gfi1b control Protruding-mouth erythroblast decreased amount, abnormal
gfi1aaqmc551Gt/qmc551Gt control 5-9 somites posterior lateral mesoderm erythroid lineage cell gfi1aa expression absent, abnormal
10-13 somites posterior lateral mesoderm erythroid lineage cell gfi1aa expression absent, abnormal
14-19 somites posterior lateral mesoderm erythroid lineage cell gfi1aa expression absent, abnormal
14-19 somites whole organism gfi1aa expression decreased amount, abnormal
20-25 somites posterior lateral mesoderm erythroid lineage cell gfi1aa expression absent, abnormal
gfi1aaqmc551Gt/qmc551Gt + MO4-gfi1b + MO4-tp53 + MO5-gfi1b control Protruding-mouth erythroblast increased size, abnormal
Protruding-mouth erythroblast nucleus structure, abnormal
Protruding-mouth erythrocyte maturation decreased occurrence, abnormal
Acknowledgments:
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Reprinted from Developmental Biology, 417(1), Thambyrajah, R., Ucanok, D., Jalali, M., Hough, Y., Wilkinson, R.N., McMahon, K., Moore, C., Gering, M., A gene trap transposon eliminates haematopoietic expression of zebrafish Gfi1aa, but does not interfere with haematopoiesis, 25-39, Copyright (2016) with permission from Elsevier. Full text @ Dev. Biol.