Figure 5

Hypothetical precursor of the SAN in the lamprey. (A) Nerve fibers of a larval lamprey, Lethenteron japonicum (Tahara's stage 25; see [86]) visualized by whole-mount immunostaining using anti-acetylated tubulin monoclonal antibody (Sigma). See [87] for staining procedure. (B) Illustration of the same specimen as in A to show the locations of SNP, vagus nerve (X) and the hypoglossal nerve (XII). (C) Locations of the spinal motoneurons innervating infraoptic muscles in the ammocoete larva of the lamprey. To label the neurons, rhodamine- and fluorescein-conjugated dextrans (Sigma, St Louis, MO) were injected into the posterior pharyngeal arches and infraoptic muscle, respectively, of a Tahara's stage 25 larva [86], according to the method described by [88]. The injected embryos were incubated at room temperature for 30 minutes before fixation. The fixed specimens were dehydrated and clarified with a 1:2 mixture of benzyl alcohol and benzyl benzoate (BABB). Note that the cell bodies of the SNP are located caudal to those of vagal motoneurons, where the ancestral SAN is expected.