Metamorphic ammocoete larva in situ hybridization

An ammocoete larva of the Far Eastern brook lamprey Lethenteron reissneri (syn. Lampetra reissneri) in metamorphosis was acquired from a local supplier in Nagano, Japan, and fixed with 4% paraformaldehyde for 24 h at 4 °C. First and second dorsal fins were dissected, embedded in paraffin wax and sectioned with a thickness of 10 microns. Embryos of the Arctic lamprey Lethenteron camtschaticum (syn. Lethenteron japonicum) were obtained as previously described ³⁸ and staged according to ³⁹. Digoxigenin-labeled riboprobes of the L. camtschaticum HhA, HhB and HhD genes were obtained from Sugahara et al., (2016). 3’ regions of the coding sequence of the L. camtschaticum MyHC1 (myosin heavy chain 1; ⁴⁰) and ColA (collagen A or col2a1a; ⁴¹) genes were amplified by PCR from cDNA prepared from a mix of embryos of L. camtschaticum at different stages. Supplementary Table 1 lists primers used for cloning MyHC1 and ColA. Amplified fragments were cloned into pCRII/TOPO (Life Technologies) and digoxigenin-labeled riboprobes were prepared as described by the manufacturer (DIG RNA Labeling Mix, ROCHE). In situ hybridization experiments on developing dorsal fin sections (10 μm) of L. reissneri and whole-mount embryos of L. camtschaticum were performed according to ³⁸.