Development • Supplementary information
Development 143: doi:10.1242/dev.127506: Supplementary information
Development 143: doi:10.1242/dev.127506: Supplementary information
Fig. S1. The expression of Ring1A and RING1B in wild type and Ring1A/B-dKO
forelimb buds and forelimb development in Ring1A-KO mice. (A) The expression of
Ring1A in E10.5 and E12.5 forelimb buds revealed by ISH analysis. Ring1A transcript is
uniformly detected in forelimb bud at E10.5 and preferentially in the proximal region at E12.5
by antisense (AS) probes. Sense probe was used as a negative control. (B) The expression of
RING1B in E10.5 and E12.5 forelimb buds revealed by immunofluorescence analysis.
RING1B is equally expressed in the mesenchymal cells (indicated as “mesenchyme” at the
bottom) in proximal, middle and distal regions and ectodermal cells (indicated as “ectoderm”)
of forelimb bud at E10.5 and E12.5. Scale bar, 6 µm. (C) External morphology and skeletal
pattern of forelimb at E17.5 in Ring1A-/-;Ring1Bfl/fl (Ring1A-KO). sc, scapula; S, stylopod; Z,
zeugopod; A, autopod. (D) Loss of RING1B expression in Ring1A/B-dKO forelimb buds at
E10.5. RING1B is present in all cells in the control (Ring1A-KO) forelimb bud at E10.5 (left).
In Prx1-Cre;Ring1A-/-;Ring1Bfl/fl (Ring1A/B-dKO) forelimb bud (right), RING1B expression
was lost in a majority of mesenchymal cells (indicated as “mesenchyme”) where the Prx1-Cre
“ectoderm”). Scale bars, 10 µm.
Development • Supplementary information
transgene is active. RING1B was still expressed in the ectodermal cells (indicated as
Fig. S2. Phenotypes and molecular changes in Ring1A/B-dKO forelimb buds at E10.5.
(A) Normal frequency of apoptotic and proliferative cells in Ring1A/B-dKO forelimb buds at
E10.5. TUNEL assay and Ki67 immunostaining did not reveal apparent changes in cell
apoptosis or proliferation, respectively, between control (Ring1A-KO) and Ring1A/B-dKO
Development • Supplementary information
Development 143: doi:10.1242/dev.127506: Supplementary information
Development 143: doi:10.1242/dev.127506: Supplementary information
forelimb buds at E10.5. DAPI was used for counter-staining. Dotted lines in TUNEL panels
indicate the shape of forelimb bud. Enlarged views for regions indicated by dotted rectangles
in upper panels of Ki67 staining are shown at the bottom. Scale bar, 100 µm. (B) The
expression of genes annotated as “limb development” or “limb morphogenesis” by Gene
Ontology (GO) in the proximal and distal region of the forelimb buds. (left) The 112 genes in
these categories were aligned by hierarchical clustering based on their relative expression
levels in proximal or distal domain of E10.5 forelimb buds. A gene exhibiting the most
distally skewed expression (Fgf4) is placed at the top. Relative expression level of each gene
revealed by microarray analysis is shown by using color codes. (right) Other GO properties
(“Transcription factor”, “signal transduction”, “cell surface”, and “extracellular”) given to the
112 genes are also shown. Genes identified as “distal genes” and “proximal genes” are
showing in blue and red fonts, respectively. The genes at which the expression was confirmed
by ISH or/and RT-qPCR in this study were indicated by arrow. (C) Hierarchical clustering of
the 111 genes based on their expression levels in the proximal and distal region of the
dataset is depicted by a dendrogram at the bottom. (D) Correlation coefficient among the four
datasets analyzed in (C). Note that correlation between the distal region of Ring1A/B-dKO
forelimb buds and the proximal forelimb bud of control is stronger than the control distal
region. (E) The expression of Shh, Grem1, Hoxa11 and Rarb in control (Ring1A-KO) and
Ring1A/B-dKO forelimb buds at E10.5 revealed by ISH (left) and RT-qPCR (right) analyses.
In RT-qPCR analysis, the expression level of Rarb in forelimb buds at E10.5 was normalized
to Gapdh and is depicted as fold change relative to the proximal region of the control
Development • Supplementary information
Ring1A-KO (control) and Ring1A/B-dKO (dKO) forelimb buds. Correlation among each
Development 143: doi:10.1242/dev.127506: Supplementary information
forelimb buds. Although the Rarb expression was increased by approximately 15-fold in the
distal region of Ring1A/B-dKO forelimb buds as compared to the control by RT-qPCR, this
difference was not considerably reflected on results by ISH. This differential outcome
between RT-qPCR and ISH is likely due to difference in respective detection dynamic range.
Development • Supplementary information
Error bars indicate the s.d. of two biological replicates.
Fig. S3. Identification of RING1 target genes to mediate PD specification of forelimb
buds. (A) The RING1B expression in E10.5 forelimb buds upon excess RA signaling.
RING1B expression in the proximal and distal regions of control and RA-treated wild type
forelimb buds at E10.5 did not show obvious differences by immunofluorescence analysis.
Scale bar, 10 µm. (B) Venn diagram representation for the overlaps among genes occupied by
RING1B and H3K27me3 in mouse ES cells (ESCs) and forelimb buds at E10.5 (Limb bud).
The number of genes in each fraction is indicated. (C) Comparative representation for
RING1B binding level around TSS between the proximal (blue bars) and distal (yellow bars)
Development • Supplementary information
Development 143: doi:10.1242/dev.127506: Supplementary information
Development 143: doi:10.1242/dev.127506: Supplementary information
regions of forelimb buds at E11.5 at the 27 transcription factor genes, which are bound and
repressed by RING1B, expressed in the proximal region and activated by excess RA signaling.
Sequence tags allocated around ±4kb of TSS regions were counted and normalized against
total mapped reads. (D) The distal-to-proximal ratios of RING1B binding around TSS of
respective genes shown in (C). P-values were calculated using paired t-test of normalized read
counts on 100-bp windows between the proximal and distal. (E) RING1B-binding at the Meis1
locus in E11.5 proximal and distal forelimb buds revealed by ChIP-seq analysis. Right panels
show the binding around the TSS. Intensive RING1B enrichment was observed around
the TSS and RBS (RING1B-Binding Site at the 3’ region of Meis1). Note the more RING1B
binding in the distal than the proximal. (F) Intensive accumulation of RING1B and
H3K27me3 around TSS and RBS of the Meis1 in distal region of forelimb buds at E10.5
revealed by ChIP-qPCR analysis. Bindings of RING1B and H3K27me3 at P-I and P-II (left)
and RBS (right) were compared between proximal and distal regions of E10.5 forelimb buds.
Development • Supplementary information
Error bars indicate s.e.m. of three biological replicates.
Development 143: doi:10.1242/dev.127506: Supplementary information
Fig. S4. Generation of the conditional Meis2flox allele.
and open triangles represent loxP and FRT sites, respectively. In the parental Meis2neo allele,
the Neo cassette was removed to generate the Meis2flox conditional allele by mating with FLP
deleter line (CAG-FLP). The Meis2 deleted allele (Meis2del ) was generated by crossing mice
carrying Meis2flox allele with a Cre-deleter strain. (B) Southern blot analysis showing
wild-type (WT), flox (flox) and deleted (KO) alleles after mating with Cre deleter mice.
SphI-BamHI digested genomic DNA was analyzed by hybridization with the 3’ probe shown
in (A). The positions of the wild type (WT), Meis2flox and Meis2KO bands are indicated. (C)
PCR analysis for the wild type (WT) and Meis2del (KO) alleles.
Development • Supplementary information
(A) Schematic representation of the strategy used to generate Meis2 mutant alleles. Closed
Development 143: doi:10.1242/dev.127506: Supplementary information
Table S1. List of RING1B-bound genes in the forelimb buds at E10.5
Click here to Download Table S1
Table S2. List of primers
Development • Supplementary information
Click here to Download Table S2
Development 143: doi:10.1242/dev.127506: Supplementary information
Supplementary Materials and Methods
Immunostaining and Apoptosis Detection
Embryos at E10.5 were fixed overnight at 4°C in 4% paraformaldehyde/PBS and processed
for paraffin wax embedding. Paraffin sections (5 µm) were deparaffinized and washed in an
ethanol series. For immunostaining, sections were permeabilized with HistoVT One (nacalai
tesque) according to the manufacturer’s protocol, blocked for 30 minutes in 3% skim
milk/PBS and incubated with RING1B (1:100, clone #3) (Atsuta et al., 2001) and Ki67 (1:200,
AB9260 Millipore) in 3% bovine serum albumin/PBS overnight at 4°C. After washing,
sections were incubated with secondary antibodies for 3 hours, counterstained with DAPI and
mounted in PermaFluor Aqueous Mounting Medium (Thermo Fisher Scientific). For
apoptosis detection, after washing in an ethanol series, sections were treated with 3%
hydrogen peroxide in water for 30 minutes, followed by permeabilization with HistoVT One.
The assay was performed with ApopTag In Situ Detection Kit (CHEMICON) according to the
References
Atsuta, T., Fujimura, Y., Moriya, H., Vidal, M., Akasaka, T. and
Koseki,
H.
(2001). Production of monoclonal antibodies against
mammalian Ring1B proteins. Hybridoma 20, 43-46.
Development • Supplementary information
manufacturer’s protocol.