Methods

In situ hybridization and immunohistochemistry

Whole mount in situ hybridization30 and immunohistochemistry10 were performed as previously described. For double in situ hybridization, fluorescein and digoxigenin labeled probes were added simultaneously and sequentially incubated with antibody. Alkaline-phosphatase coupled anti-fluorescein antibody was added first and color was developed by addition of BCIP. After fixation, embryos were incubated with an anti-digoxigenin coupled with alkaline phosphatase and color was developed by addition of NBT/BCIP or BM purple. HNK-1 antibody was obtained from American Type Culture and Pax7 and Pax3 from Developmental Studies Hybridoma Bank. Affinity purified Rabbit anti Brachyury (N terminal 123 aminoacids) polyclonal AB was a kind gift of Dr Susan Mackem, and was pre-absorbed against 4 day old chick embryo heads overnight before use.

Morpholino electroporation

FITC labeled morpholino oligonucleotides were obtained from Gene Tools, LLC. A cPax7 5’UTR antisense morpholino oligonucleotide (5’-TCCGTGCGGAGCGGGTCACCCCC-3’), a cPax3 5’UTR antisense morpholino oligonucleotide (5’-CCAGCGTGGTCATCGCGGCGGCGC-3’) and a cPax7 5’UTR antisense morpholino oligonucleotide carrying 5 mismatches (i.e. 5 changes in comparison to the MoPax7 sequence; 5’-TCgGTcCGGAGccGGTgACaCCC-3’) as a control. Morpholinos were stored at -80°C at a concentration of 1.2µM and diluted 1:1 in 10%sucrose just before injection. St 4 embryos were explanted onto filter paper rings and placed ventral side up on drops of 2% agarose in water and morpholinos were introduced into embryos via electroporation30. They were injected onto the prospective neural crest forming region of the epiblast followed by 2 square electroporation pulses of 5-10 mV at 25 msec. After electroporation, embryos were placed in thin albumin for 1-2 hours to recover and then prepared for dissection or incubation for 8-24 in modified New culture31.

In vitro translation

Full length cPax7, cPax3 or Xenopus EF1 mRNA was transcribed using mMessage mMachine (Ambion). Proteins for each mRNA were translated using Rabbbit Reticulocyte Lysate Nuclease-treated (Promega) according to manufacturer instructions. In each reaction, 2µl of morpholino oligonucleotides of varying concentrations were added and incubated for 15 minutes at room temperature before adding 35S-methionine. Protein samples were stacked in a 4% polyacrylamide gel and separated by 10% polycacrylamide gels. Gels were rinsed in water, fixed for 20’ in 40% methanol/20% glacial acetic acid. After fixing, gels were enhanced in 1M sodium salicylate for 10 minutes. Gels were rinsed in water before drying under vacuum for 30 minutes at 80°C.

Embryo dissection and tissue culture

St 3c-d and st 4 chick embryos were dissected in Ringer’s solution using tungsten or glass needles. The lower layers were removed with careful dissection and the explanted epiblast tissue was placed in PB1 solution for 1-2 hours before embedding it in collagen gels for culture. Collagen gels were prepared by mixing 90µl of rat type I collagen (Collaborative Research, Waltham, Massachusetts) with 10µl of 10X DMEM and 4.5 µl of 7.5% sodium bicarbonate. The collagen gels were covered with 300µl of defined F12/N2 serum free medium and the tissue was cultured in a CO2 incubator at 37oC. To assay for migratory neural crest cells, explants were incubated for 36 – 40 hours.

RT-PCR genes, primers and product sizes:

S17

LEFT AGAAGGCGGCGCGGGTGATCATCG

RIGHT GTTTATTGTAAAAGCAACATAACG

Product size: 409

Slug

LEFT ACCCCATTACTGTGTGGACTACAA

RIGHT TTGGATCTGTCTGCGAAAGCCCTG

PRODUCT SIZE: 520

Brachyury

LEFT ATGCTTGTTTCCTTGCTGCT

RIGHT TTACGGCCTGAGCAAGAGAT

PRODUCT SIZE: 275

Sox2

LEFT AGGCTATGGGATGATGCAAG

RIGHT GTAGGTAGGCGATCCGTTCA

PRODUCT SIZE: 163

Pax7

LEFT ACTGCGACAAGAAGGAGGAA

RIGHT CTCTTCAAAGGCAGGTCTGG

PRODUCT SIZE: 130

PAX7

Forward AACCACATCCGCCATAAGAT

Reverse CCTCGATTTTCTTCTCCACG

PRODUCT SIZE: 201