Supplemental Data

A bruno-like Gene Is Required for

Stem Cell Maintenance in Planarians

Tingxia Guo, Antoine H.F.M. Peters, and Phillip A. Newmark

A

B

C

Figure S1. Structure of the Smed-bruli Gene and Protein

(A) Exon/intron map of Smed-bruli. The seven exons are depicted by rectangles (ORF=shaded;

UTRs=unshaded); the six small introns are indicated by thin lines, with the size of the intron (in bases)

listed below. Alternative splicing leads to the six-nucleotide insertion shown at the beginning of exon 5.

Three poly(A) sites have been observed in different EST clones.

(B) Smed Bruli protein domains. The two RNA recognition motifs (RRMs) are separated by a non-

conserved linker domain. For comparison, zebrafish Etr-1 is used to show the three RRM domains

typically observed in members of the Bruno-like family. The positions of peptide sequences used for

antibody production are indicated.

(C) Alignment of C-terminal sequences (containing RRM3) of selected Bruno-like family members. The

two RNP motifs (RNP1 and RNP2) are indicated. GenBank accession numbers.: dmBru-3

(NP_729923); hBRUNOL5 (NP_068757.2); zfEtr-1 (BAA95118); Unc-75 (NP_492958.3);

mBRUNOL6 (Q8BJ35); dmBruno (AAB58464.1).

Figure S2

Figure S2. Additional Characterization of SMEDWI-1-Positive Cells by Double Staining

(A-C) Distribution of Smedwi-1-positive cells. Double immunofluorescent staining of (A) phospho-

tyrosine and (B) SMEDWI-1 shows posterior branches of the digestive system and neoblasts,

respectively. Overlay image (C) reveals SMEDWI-1-positive cells surrounding the two posterior gut

branches.

(D-L) Comparison of SMEDWI-1 protein and transcript distribution. Whole-mount

immunofluorescence of SMEDWI-1 and fluorescent in situ hybridization (FISH) of smedwi-1. (D)

Immunostaining with SMEDWI-1 antibody shows positive cells in the mesenchyme. (E) smedwi-1 FISH

detects a similar distribution of smedwi-1 transcripts as that of SMEDWI-1 protein. (F) Overlay of (D)

and (E) shows many cells labeled with both smedwi-1 and SMEDWI-1 signals. (G) The expression

pattern of SMEDWI-1 protein and (H) smedwi-1 transcripts in more superficial section compared to (D-

F). (I) Overlay image of (G) and (H) shows many SMEDWI-1-positive cells are weakly/not labeled with

Smedwi-1 transcripts. (J-L) Immunofluorescence of SMEDWI-1 (J), and FISH of smedwi-1 (K) detect

SMEDWI-1 protein but not smedwi-1 transcripts in cells in front of the photoreceptors. (L) Overlay of

DIC image with (J) and (K).

(M-O) High magnification view of double immunofluorescence of SMEDWI-1 and H3-S10P. The

colocalization of SMEDWI-1 and H3-S10P signals indicates the small population of SMEDWI-1-

positive cells progressing through mitosis at any time. Panels J-L show anterior region, all other panels

show tail regions; anterior to the upper left. Images in panels A-C and J-L are maximum projections;

images in panels D-I and M-O are single confocal sections. Yellow stars, photoreceptors. Scale bars: in

L, 50 µm in A-C, 33.3 µm in D-I, 100 µm in J-L; in O, 10 µm in M-O.

Figure S3

I

Figure S3. Specificity of Anti-Histone H4 Monomethyl-K20 Antibodies

(A, C, E, G) Whole-mount immunofluorescence with anti-SMEDWI-1 antibodies shows neoblast

distribution in the samples.

(B, D, F, H) Whole-mount immunofluorescence with anti-H4-K20me1 antibodies (B) without peptide

incubation, or pre-incubated with different peptides: (D) H4-K20me1 peptide; (F) unmodified histone

H4 peptide; (H) H4-K20(me)2 peptide. Sections shown in B, D, F, H are the same sections shown in A,

C, E, G, respectively. All panels show tail regions; anterior to the upper left. Scale bar (shown in H)

equals 50 µm in all panels.

(I). Dot blot against indicated amounts of various unmodified, mono-, di-, and tri-methylated Histone

peptides. Note the specificity of anti-H4-K20me1 for the monomethylated form.