DEV02485 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

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• Supplemental Figure S1 -

  Fig. S1. Most hematopoietic and vascular markers assessed are expressed normally in nz171 mutants. (A-D) Expression of hematopoietic markers in whole-mount embryos as detected by in situ hybridization. Anterior to the left (except B, top panels: posterior view). (A) scl expression in wild-type and nz171 mutant embryos, as indicated on the panels. At 30 h.p.f., expression levels of scl in nz171 embryos are similar to those in wild type, and at 48 h.p.f. scl expression is still observed in the ICM region, suggesting that it has not been downregulated as observed in wild-type embryos. (B) gata2 expression in 6-somite, 18 h.p.f. and 24 h.p.f. wild-type and nz171 embryos as indicated on the panels. gata2 transcript levels in nz171 mutants are similar to wild type levels. (C) Expression of various hematopoietic genes in wild-type and nz171 mutant embryos, as indicated on the panels. The presence of myeloid precursors in the ALM of nz171 embryos is indicated by the normal expression of cebpa and c-myb in this area. (D) Vascular markers fli1 and flk1 are expressed relatively normally in nz171 mutants at 24 h.p.f. (E) Schematic illustrating the expression status of hematopoietic genes in nz171 mutants according to when they are expressed developmentally. Green, gene expression not affected; orange, reduced; red, completely absent.

• Supplemental Figure S2A
• Supplemental Figure S2B
• Supplemental Figure S2C -

  Fig. S2. cDNA and protein sequence of Rad21nz171 and previously characterized alleles. The rad21 transcript contains 2615 nt and encodes a protein of 643 residues (NCBI). There are two chromosome 16- associated rad21 sequences available at NCBI: NM_199595/BC045311 and AY423040/Ensembl Transcript ENSDART00000005927. An alignment of proteins encoded by these two transcripts revealed a polymorphism at position 592, where glycine is substituted for serine in NM_199595/BC045311. (A) Annotated sequence of the cDNA synthesized from rad21nz171 homozygotes. The sequence is 2428 bp and incorporates the entire open reading frame. We designed antisense morpholino oligonucleotides directed against the rad21 transcript: two targeting the start and 5′ regions, and one targeting the 5′ donor of exon 3. Morpholino binding sites are indicated by highlighting: UTR_MO, yellow; ATG_MO, green; SPLX3_MO, pink. The start codon is boxed and the stop codon is indicated in bold. The G->T mutation in the rad21nz171 allele is boxed and highlighted in red. (B) Comparison of the protein sequence of two Rad21 sequences deposited in GenBank, NM_199595 and AY423040, with the Rad21nz171 protein sequence (Rad21 17.1). The stop codon at position 277 created by the mutation is indicated by an asterisk. The 276 amino acid truncated protein resulting from this mutation is predicted to be non-functional, as the C-terminal winged helix domain important for contacting the Smc1 head (Nasmyth and Haering, 2005) is deleted. Rad21nz171 and AY423040 both have serine at position 592, whereas NM_199595 has glycine. The hydrophilic nature of this region is conserved. (C) Top, immunoblot showing lack of Rad21 protein in rad21 morphants, indicating correct targeting of rad21 mRNA by morpholinos. The Rad21 antibody (AB3233, Chemicon) is directed to the C-terminus of the protein and is not expected to recognize truncated products. HT29 is a human colon carcinoma cell line; 5 μg total protein was loaded as a positive control for the anti-Rad21 antibody. ‘Control’ refers to non-injected wild-type embryos. Embryos were harvested at 48 hpf, and 20 μg total protein was loaded per lane. Bottom, anti-α-tubulin immunoblot as a loading control.

• Supplemental Figure S3 -

  Fig. S3. gata1 expression levels are reduced in rad21 morphants and mutants during early embryogenesis. Whole-mount embryos stained for gata1 expression. Upper panels are wild type; lower panels are rad21ATGMO morphants (MO) or rad21nz171 homozygotes as indicated. (A) Posterior views of embryos, stages as indicated. Onset of expression of gata1 is slightly delayed in rad21 morphants, as evidenced by gata1 expression in the 6s morphant appearing similar to its expression in the 4s wild-type embryo. All rad21 morpholino-injected embryos (rad21ATGMO; 0.5 pmol) at 4s (n=29/29) and 6s (n=20/20) had reduced/delayed expression of gata1. On close inspection of rad21nz171 mutants, gata1 expression appears to be ‘patchy’ in intensity within the PLM stripes. (B) Lateral views, anterior to the left, stages as indicated. gata1 expression is noticeably reduced in the PLM/ICM of rad21nz171 mutants.

• Supplemental Figure S4 -

  Fig. S4. Targeted knockdown of the smc3 gene. Antisense morpholinos were designed to target the 5′ UTR (smc3UTRMO), the ATG start codon (smc3ATGMO), the splice site of exon 1, 3′ donor (smc3Splx1MO) and the splice site of exon 5, 3′ donor (smc3Splx5MO) of the smc3 mRNA. Ensembl locus ENSDARG00000019000 and sequence BC044408 were used to inform morpholino oligonucleotide design. Embryos were able to develop to 48 h.p.f. when given a 0.25-0.5 pmol dose of morpholino targeting smc3. Higher doses of 2-3 pmol caused arrest in early blastula stages. Total knockdown of Smc3 function should prevent cell division altogether. Therefore, post-blastula embryos are considered to be smc3 hypomorphs rather than smc3 nulls. (A) Representative gross morphology of Smc3 morphants at 48 hpf generated by a 0.5 pmol dose of smc3Splx1MO, compared with a wild-type embryo of the same stage (top). All morpholinos targeting smc3 generated a similar phenotype. (B) Representative anti-Smc3 immunoblot of protein obtained from embryos injected with 3 pmol smc3UTRMO. Individual embryos that had arrested at the early blastula stage were lysed and the entire sample was used for immunoblotting along with stage-matched controls treated in the same manner. The blot was cut in two and treated with anti-Smc3 antibody (Chemicon, 1:1000) or anti-α-tubulin (Sigma, 1:2000) to provide a loading control. 5 μg of protein from HT29 colon carcinoma cells was used as a positive control for the antibodies. (C) Splice-site morpholinos smc3Splx1MO and smc3Splx5MO are effective in blocking splicing of the smc3 mRNA, and show partial activity in smc3 hypomorph morphants. Agarose gel electrophoresis of PCR products obtained by RT-PCR of cDNA obtained from 24-hpf embryos. M, DNA size marker with number of base pairs indicated at left. RT-, control, morphant embryos processed without reverse transcriptase; MO, embryos injected with 2 pmol of smc3 morpholino (identity indicated at side); WT, wild-type embryos; nz171, rad21nz171 mutant embryos. White asterisks indicate bands of expected size should the intron involved at each splice junction remain unspliced. Double asterisk, band of unpredicted size. Primer sequences for RT-PCR: smc3Splx1 forward, 5′-CTGAGGAGTGTTTTGTGC-3′ and reverse, 5′-GATGACATTGTGTTTTGAGC-3′; smc3Splx5 forward, 5′-CGAGTCATTTCAGCCTTCG-3′ and reverse, 5′-TTACTGCGGGAGAATCCAG-3′.