embryos. Asterisk represents loss of or reduced expression. Brackets represent

Transcription

1 Supplemental Figures Supplemental Figure 1. tfec expression is highly enriched in tail endothelial cells (A- B) ISH of tfec at 15 and 16hpf in WT embryos. (C- D) ISH of tfec at 36 and 38hpf in WT embryos. Asterisk represents loss of or reduced expression. Brackets represent maintained expression. Supplemental Figure 2. Specific tfec splice variants are expressed in caudal endothelial cells (A) As described previously, zebrafish tfec (ztfec) is regulated by different promoters which gives several transcripts which contain unique first exons (either exon 1 (pink), exon 1a (orange) or exon 1b (green). Each transcripts retains a specific 5 UTR sequence. (B) Schematic diagrams of translated proteins from several MITF family members within zebrafish, human (longest variant) and mouse. This diagram additionally includes the translated proteins from injected tfec mrna used within this study (hl- ztfec, second from bottom) and the dominant negative form of tfec mrna also injected in this study (DN- tfec, bottom schematic). Each schematic highlights the specific domains within each protein and length in amino acids. QR represent glutamine rich domain, MAPK: consensus MAP kinase phosphorylation site, AD: transcriptional activation domain, AD?: possible additional transcriptional activation domain, bhlh: beta helix- loop- helix DNA binding domain, PKA: camp- dependent phosphorylation site, N: N terminal domain, C: C terminal domain, aa: amino acids. Schematics in A and B are not to scale. (C- F) ISH at 24hpf in the tail of tfec using probes that recognize all transcripts (C), tfec- P1 only (D) or tfec- P2 only (E). Brackets indicate maintained

2 expression, asterisk indicates loss of expression. (F) QPCR data examining tfec expression (using primers that recognize all transcripts ( common tfec ) or specific transcripts (either tfec- P1 or tfec- P2) in FACS sorted cells. Fold enrichment shown for each set of primers was calculated from expression within caudal endothelial cells compared to egfp negative cells from the whole embryo at 26hpf. Data represents mean ± S.E.M. Supplemental Figure 3. tfec overexpression does not affect HSC emergence and differentiation or vascular formation and augments markers of HSPC in an HSC dependent manner (A- B) ISH for runx1 following no injection (NI) or hl- ztfec injection (+tfec) at 26hpf. (C- D) ISH for rag1 following NI or +tfec at 4dpf. White dotted line represents outline of thymus. (E- F) ISH for flk1 following NI or +tfec 24hpf. (G- J) ISH of cmyb in embryos from in crossed runx1 +/- parents, resulting in siblings with WT phenotype (G and H) or runx1 - /- embryos (I and J). Embryos were either kept as NI control or +tfec. (K- N) ISH of ikaros in embryos from in crossed cmyb +/- parents, resulting in siblings with WT phenotype (K and L) or cmyb - /- embryos (M and N). Supplemental Figure 4. tfec overexpression increases the number of HSPCs present and retained in the tail niche (A) Schematic indicating approximate imaging area in the tail, as indicated by the black box. (B- C) Confocal imaging in CHT of double positive flk1:mcherry/cymb:egpf embryos (NI) to characterize cuddled HSPCs at 3 and 4dpf. (D) Schematic indicating

3 approximate imaging area in the tail, as indicated by the black box. (E- F) Confocal imaging in CHT of double positive flk1:egpf/flk1:cre/βactinswitch:dsred embryos (NI) to characterize cuddled HSPCs at 3 and 4dpf. White arrow heads indicate HSPCs closely associated to endothelial cells. (G) Confocal imaging in the CHT of double positive flk1:mcherry/cd41:gpf embryos in NI or following hl- ztfec (+tfec) injection at 3 and 4dpf. (I) Analysis of small GFP low cells at 3dpf and 4dpf. (J- P) Anti- GFP and ph3 immunostaining at 3 or 4dpf of either non- injected (NI) or hl- ztfec injected (+tfec) cd41:gpf embryos. (Q) Quantification of the number of the ph3 + HSPCs in NI or hl- ztfec injected embryos at 3 or 4dpf. Data represents mean ± S.E.M. NS, not significant, *: p<0.05, ***: p< Scale bar represents 25µm. Supplemental Figure 5. CRISPR/Cas9 generation of tfec - /- mutants (A- C) Schematic outlying a - 32bp CRISPR/Cas9 mediated mutation (tfec - /- ), that results in a premature STOP codon and severely truncated protein. Schematic is not to scale, AA represent amino acid sequence, * represents premature STOP codon, PAM represents protospacer adjacent motif. (D- E) ISH for mpx and mfap4 at 4dpf in sibling and tfec - /- mutants. (F- I) ISH for runx1 (26 hpf) and rag1 (4dpf) in sibling and tfec - /- mutants. (J) ISH for cmyb in sibling (Sib,) or tfec - /- embryos at 4dpf following no injection (NI) or following injection of hl- ztfec (+tfec). (K) Analysis of cmyb expression. Supplemental Figure 6. Caudal endothelial cells are enriched in niche cytokines (A) Experimental outline. (B- D) QPCR expression of hematopoietic niche cytokines in FACS sorted cells outlined in A, data represents biological triplicates. Statistical analyses

4 was completed using an un- paired Student s T- test, comparing cytokine expression to whole zebrafish. Data represents mean ± S.E.M. NS, no significance, *: p<0.05, **: p<0.01. Supplemental Figure 7. tfec controls tail niche cytokine production (A) Experimental outline. (B- D) QPCR data showing gene expression in FACS sorted tail ECs (outlined in a) following NI (n=5 for all, except cxcl12a where n=3), hl- ztfec (+tfec) (n=5 for all, except cxcl12a where n=3) or DN- tfec (+DN) (n=3 for all) injection. Each n number represents an average of biological triplicates, experiments were repeated at least three times. Statistical analyses was completed using an un- paired Student s T- test, comparing NI to either hl- ztfec (+tfec) injected or DN- tfec injected. (E- F) Neutrophil and macrophage number were analyzed by ISH for mpx and mfap4 respectively and quantified by counting cell number within the tail in either NI, hl- ztfec injected (+tfec) or DN- tfec injected (+DN- tfec) at 4dpf. (G) Erythropoiesis was analyzed by performing ISH for gata1 and comparing the percentage of embryos with a v.high, high or normal phenotype in the NI, hl- ztfec injected (+tfec) or DN- tfec injected at 4dpf. Statistical analysis was completed using an un- paired Student s t- test, comparing NI to either hl- ztfec (+tfec) injected or DN- tfec injected. Data represents mean ± S.E.M. NS, no significance, *: p<0.05 **: p<0.01, ***:p<0.005.

5 Supplemental Movie 1 Bright field recording of live blood flow in tfec mutant siblings at 8dpf in the tail region. Supplemental Movie 2 Bright field recording of live blood flow in tfec - /- mutants at 8dpf in the tail region Supplemental Movie 3 Fluorescent recording of gata1:dsred embryos at 8dpf in the tail region (Upper embryo is sibling control, lower embryo is tfec - /- ).

6 Gene Forward Reverse tfec common (ISH) GGCGGCGATACAACATCAAC TGAGGGTGCTGCAGAAGAAG tfec- P1 (ISH) GTAATCTCTCCACTTGAGGG CTGGGAGTGGATGGGCAACA tfec- P2 (ISH) ATGAGCCGCTGCTGTGTGGT GAGGTACTGTTTGACCTGCT DN tfec (mrna injection) hl- ztfec (mrna AAAGAATTCATGGCTGATCAAGA ACATGACAC AAACTCGAGCTATGACAGTTCGACTGTGCCC injection) AAATCATGCGCAATGGACACATG AAACTCGAGGTCCAGTCAGAGATCGTCTG Kitlgb (mrna injection) AAAGAATTCATTCCCATGTTCCAC ATGAGG AAACTCGAGTTTTTATTAGACCTCTGTGTCT G Supplemental Table 1. Primers used for ISH probe cloning and mrna synthesis

7 Gene Forward primer Reverse primer ef1a GAGAAGTTCGAGAAGGAAGC CGTAGTATTTGCTGGTCTCG csf3a AACTACATCTGAACCTCCTG GACTGCTCTTCTGATGTCTG csf3b GGAGCTCTGCGCACCCAACA GGCAGGGCTCCAGCAGCTTC csf1a CACTACTGAACCCCTCATCCA GCTCACTGTTGGACAAATGC csf1b CAGCACTGCGTACCTTCATT TTGCGTCTCGAATGCTACAG kitlga GTTTGCAATCGCAGTGGTGG ACAGAGCGGGACCTTCTTCT kitlgb ATGGCATGAACTCTGCGGTT CGCATTCTTGCTCCACAACG epo GCTGGATTCTGAGATGCAGATA GCGTAATGGGGAGGACAG thpo CCTGCATGTAGTCAGACTGCTC ACCCAGACACCTCTCTACCG cxcl12a CGTAGTAGTCGCTCTGATGG TGGGACTGTGTTGACTGTGGAA cxcl12b GGAGCATCCGAGAGATCAAG TGTTCTTCAGCTTGGCAATG tfec common CCAGGCATCATAATGCAAAA TGATCAGCACCGTACACCTC tfec- P1 GGGATCCTACTTTCGTGGACC CGTCTGGTTGAGGTGGTACT tfec- P2 CGCTGCTGTGTGGTTAAAGTC CTAGCGAGTTTGCTGCCCAA Supplemental Table 2. List of QPCR primers used within this study

8 Gene Forward Reverse UAS AAAACTAGTCGTGTGGAGGAGCTCAAAG AAAGCGGCCGCGGGATCACGCGGCCATCA Gal4 GCTACTGTCTTCTATCGAACAAGC TGCTGTCTCAATGTTAGAGGC Tfec CRISPR CCAAGAGTGAGCTATAATCA GACATGAACTTGGCTTATAC Supplemental Table 3. Primers used for cloning and/or genotyping

9 Supplementary Figure 1

10 Supplementary Figure 2

11 Supplementary Figure 3

12 Supplementary Figure 4

13 Supplementary Figure 5

14 Supplementary Figure 6

15 Supplementary Figure 7