Figure S1. Related to Figure 1. MO Validation and knock-âdown of kita, kitb and kitlgb. Figure S2. Related to Figure 2. Identification of osm in zebrafish.
Stem Cell Reports, Volume 10
Supplemental Information
Oncostatin M and Kit-Ligand Control Hematopoietic Stem Cell Fate during Zebrafish Embryogenesis Christopher B. Mahony, Corentin Pasche, and Julien Y. Bertrand
SUPPLEMENTAL MATERIAL Table S1: primers used to clone full-‐length mRNA Table S2: primers used for quantitative RT-‐PCR Table S3: morpholinos used in the study Figure S1. Related to Figure 1. MO Validation and knock-‐down of kita, kitb and kitlgb. Figure S2. Related to Figure 2. Identification of osm in zebrafish. Figure S3. Related to Figures 2 and 3. osm expression is tfec-‐dependent and reduces macrophage differentiation. Figure S4. Related to Figure 3. Characterization of ikaroshigh and ikaroslow populations. Figure S5. Related to Figure 4. Validation of MOs for osmr and osm. Figure S6. Related to Figure 4. osmr morphants have reduced primitive erythropoiesis but normal primitive myelopoiesis. + 2 supplemental videos
Table S1: primers used to clone full-length mRNA si:ch73-47f2.1 (osm)-F
AAAGAATTCCTTCTGATGTGGAGTATTTTAAT
si:ch73-47f2.1 (osm)-R
AAACTCGAGCATAAACTAGCCTTGGGTAAAA
kitlga-F
TCGTTCCATATGAAGAAGTCA
kitlga-R
AAACTCGAGGGGCTGGATTTACACATCCA
kitlgb-F
AAAGAATTCATTCCCATGTTCCACATGAGG
kitlgb-R
AAACTCGAGTTTTTATTAGACCTCTGTGTCTG
Table S2: primers used for quantitative RT-PCR ef1α-F
GAGAAGTTCGAGAAGGAAGC
ef1α-R
CGTAGTATTTGCTGGTCTCG
osm-F
AAACCCCTCATTTCTAAGACCA
osm-R
GTTCTTCAAGTCAAGTTCAGGA
osmr-F
TGGACAGCACAGCAGCTC
osmr-R
CAGCTGCGGGTCACTGC
gp130
GAGCGTCTTCACCATAATGC
(il6st)-F gp130
GTCAAACACGTCCACTTCCA
(il6st)-R il7r-F
TACACCAAACATCCCACA
il7r-R
TCACTCACTGACGCACTT
irf4a-F
TACACATACTCGCCATCAG
irf4a-R
CAGAGACTCACGGTAGAAG
ccr9a-F
ATCATAGAGATCGAGAGGAC
ccr9a-R
CGGTTACATTCATCATGGAT
ikaros-F
GAGGCACAGGAAATGTCCC
ikaros-R
CATCTTGATCCTCTCCGCC
gata3-F
GCGGCCTGTATTACAAATTACAC
gata3-R
GCTGGACATTTTCCTGTTCC
cmyb-F
TGATGCTTCCCAACACAGAG
cmyb-R
TTCAGAGGGAATCGTCTGCT
runx1-F
CGG TGA ACG GTT AAT ATG AC
runx1-R
CTT TTC ATC ACG GTT TAT GC
gata1-F
TGA ATG TGT GAA TTG TGG TG
gata1-R
ATT GCG TCT CCA TAG TGT TG
mpx-F
TGA TGT TTG GTT AGG AGG TG
mpx-R
GAG CTG TTT TCT GTT TGG TG
pu.1-F
AGA GAG GGT AAC CTG GAC TG
pu.1-R
AAG TCC ACT GGA TGA ATG TG
cd41-F
CTG AAG GCA GTA ACG TCA AC
cd41-R
TCC TTC TTC TGA CCA CAC AC
kita-F
CTATGTTGTCAAAGGCAATGCT
kita-R
CCAGACGTCACTCTCAAAGGT
kitb-F
GGATACAGAATGAGTGAGCCTGA
kitb-R
CTCCAGCACCATCTCATCAC
Table S3: morpholinos used in the study Standard control MO
CCTCTTACCTCAGTTACAATTTATA
osmr #1
CCTTTAATGTGAGGAATCACCTGTA
osmr #2
GGCCTTTAGTTTCACCTGTGATGAA
kita
AAAGTTTTCACTTACTGATGACATG
kitb #1
ACCTTTATTTCACAATTCTCACCGT
kitb #2
GGTGTTTGTGTCTAACCGCTTCAGA
kitlgb
GTCTGAATAAAACTCTTACCAGGGT
osm
AGCACTAAAAGCCTAATACTTACGT
Supplemental Figure legends Figure S1. Related to Figure 1. MO validation and knockdown of Kita, Kitb and Kitlgb. (A, B) Schematic of splice blocking MO targeting intron/exon junctions in kita and kitb (schematics are not to scale), along with qPCR analysis of kita and kitb expression after MO injection at different concentrations. Data is mean±SD. cDNA was synthesised from total RNA extracted at 24hpf from a pool of 6-10 embryos. (C) Schematic of second splice blocking MO targeting intron/exon junctions in kitb. (D) ISH to examine runx1 (28hpf) expression. runx1 was reduced in a similar manner as in embryos injected with kitb MO#1. kitb MO#2 was only used here, all other data uses kitb MO#1 (herein referred to as kitb MO). (E) Validation of kitlgb MO that induces exon skipping (confirmed by cDNA synthesis at 24hpf from pools of 6-10 embryos, then sequencing PCR product using reverse primer). (F) kitlgb morphants have reduced runx1 (28hpf) expression, which is rescued by injecting full length mRNA for kitlgb. (G) rag1 expression following MO injection. (H) ISH to examine macrophages
(mfap4
expression)
and
neutrophils
(mpx
expression).
Flk1:eGFP/gata1:DsRed embryos were used to examine blood flow and endothelial cell formation. (I-K) Analysis of the number of pigments on the left side of the yolk sack region in either MO or mRNA injected embryos at 48hpf. NI, non-injected control. +kitlga/+kitlgb, kitlga/kitlgb full length mRNA injected embryos. All data represents mean±SD. Statistical analysis completed using ordinary one-way ANOVA with multiple comparisons. In A, p=0.0008. In D, p=0.0002. In E, p value is less than
