DEV023614 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Multiple sequence alignment of part of chick element C with different vertebrate species. Alignment was performed using ClustalW software on a 321 bp chick sequence corresponding to the fragment carrying both HP1 and HP2 Hox/Pbx sites used in the gel retardation experiments in Fig. 1. The HP1 and HP2 sites and the different Meis (M1, M2, M3)-binding sites are indicated with the blue arrows. The oligonucleotides carrying the HP1 and Meis sites or the HP2 site used in gel retardation experiments are also indicated. Strictly conserved nucleotides are boxed in black and highly conserved ones are boxed in grey. Dashes indicate gaps in the alignment.

• Supplemental Figure S2 -

  Fig. S2. Expression patterns of mouse Meis and Prep family members during hindbrain segmentation. In situ hybridizations for Meis1 (A-E), Meis2 (F-J), Meis3 (K-O) and Prep2 (P-T) were performed using purple/brown staining at the indicated somite stage (ss). In B,D,G,I,L,N,Q,S, co-hybridization for Krox20 was added, using red staining to allow positioning of the prospective r3. (A) At the 1 somite stage (ss), Meis1 is expressed in the caudal neural plate, up to the posterior part of the prospective hindbrain. (B-E) Later on, expression extends anteriorly to approach the prospective r3/r4 boundary at 3 ss and to include the prospective r3 at 5-6 ss. (F-I) At 2 ss, Meis2 is expressed with an anterior limit rostral to prospective r3, which may correspond to the r1/r2 boundary at 5 ss. (J) Later on, Meis2 expression is maintained in r2 and r3, but is progressively downregulated posteriorly in the hindbrain. (K,L) At 1 ss, Meis3 is expressed caudally up to the prospective r3/r4 boundary approximately. (M,N) At 5-6 ss, high relative levels of expression are observed in r4 and the spinal cord, and low levels in r3, r5 and r6. (O) At later stages, Meis3 expression is downregulated, except in r4 and at the rhombomere boundaries. No expression of Prep1 was detected between 1 and 10 ss (data not shown). (P,Q) By contrast, at 2 ss, Prep2 is expressed in the entire neural plate, except in the prospective r3 and r4. (R-T) Absence of expression in r3 is maintained up to the 10 ss.

• Supplemental Figure S3 -

  Fig. S3. Hoxa2 binds to element C in vivo. Chromatin extracted from zebrafish embryos co-injected with meis1.1 and Myc-tagged hoxa2 RNAs was subjected to the ChIP procedure in the absence (first lane) or presence of anti-Myc antibody (second lane). Immunoprecipitated DNA was analyzed by PCR using primers designed to amplify the core of element C (297 bp). The third lane shows the PCR products obtained from the input chromatin.

• Supplemental Figure S4 -

  Fig. S4. An oligomere of the HP1 site drives reporter expression with a Hoxb1-like pattern in transgenic embryos. (A,B) Oligomers of wild type (A, 3-mer) and mutated (B, 5-mer) HP1 sites were inserted into the lacZ reporter construct and electroporated into the chick neural tube. Whereas the mutated version is inactive, the wild-type oligomerized site drives specific reporter expression in r4, the posterior hindbrain and the spinal cord.