Hgf (Hepatocyte growth factor) is a pleiotropic growth factor that signals through its membrane bound tyrosine kinase receptor Met (proto-oncogene associated with cellular metastatic cancer; (Bottaro et al

Hgf (Hepatocyte growth factor) is a pleiotropic growth factor that signals through its membrane bound tyrosine kinase receptor Met (proto-oncogene associated with cellular metastatic cancer; (Bottaro et al., 1991; Rubin et al., 1993)). cerebellar region, with being expressed medially in the ventricular zone region and being expressed dorsally in the upper rhombic JAK1-IN-4 lip region. Single hybridizations with (A), (B), and (C) also show the complementary expression domains of the and genes. Anterior is usually to the left and dorsal (transverse sections) is usually up in all supplemental figures. midbrain, mb; cerebellum ,cb; ventricular zone, VZ; upper rhombic lip (URL). Scale bar, 50m. Supplemental Physique 2: Expression of gene in the GFP-positive cells derived from the VZ of hybridization with probe (B), and merged (C). transcript is usually expressed in the GFP-positive cells derived from the (A-C) and (D-F) in the VZ-derived cells of the cerebellum (cb). expression is usually downregulated in morphants compared to controls, while expression is usually unchanged. Scale bar, 50m. Supplemental Physique 4: Expression of URL markers (and hybridization of (A,B) and (C-F) in control embryos (A,C,E,E,E) and Met morphants (B,D,F,F,F). There is no significant change in expression of and in Met morphants compared to control embryos. Scale bar, 50m. Supplemental Physique 5: Neurons derived from URL are unaltered in Met signaling morphants Lateral view (A-F) of expression in the locus coeruleus neurons (indicated by arrows) in control (A,D), Met morphants (B,E), and Hgf morphants (C,F) of 24hpf (A-C) and 48hpf (D-F) embryos. expression SMOC1 in neurons derived from the URL is usually unchanged in morphants compared to controls at 24hpf, but there is a slight delay in the dorsal to JAK1-IN-4 ventral migration of these neurons. At 48hpf, these neurons have completed their migration to the ventral hindbrain region in both controls and morphants. Scale bar, 50m. Supplemental Physique 6: Met signaling plays a role in neuronal survival Dorsal view (A-C) of control (A), Met morphants (B), and Hgf morphants (C) at 24hpf showing Acridine Orange staining in live embryos to detect cell death. Anterior to the left. There is an increase in cell death in both Met and Hgf morphants, not only in the cerebellar region but also throughout the rest of the hindbrain (indicated in brackets) and other CNS regions. Scale bar, 50m. Supplemental Table 1: Concentration dependent phenotypic responses to Met and Hgf morpholino microinjections mg/ml: concentration of each morpholino injection and n: number of morpholino-injected Islet1-GFP transgenic embryos immunostained with GFP and EphA4 antibodies, scored at 48hpf by fluorescence microscopy according to the following criteria. r4/r5/r6: number of embryos with partially migrated facial motor neurons; r6: number of embryos whose facial motor neurons have completed their migration. Conditions that have been marked in bold were used further to assess the migration phenotype of the embryos showing the r4/r5/r6 (incomplete migration) in more detail (Fig 7G). UTR: Met morpholino blocking untranslated region; SB-E8I8: Met morpholino blocking the exon 8 – intron 8 splice donor site; SB-E6I6: Hgf1 morpholino blocking the exon 6 – intron 6 splice donor site; SB-E4I4: Hgf2 morpholino blocking the exon 4 – intron 4 splice donor site. Supplemental Table 2: Phenotypic responses to Met and Hgf morpholino microinjections mg/ml: concentration of each morpholino injection and n: number of morpholino-injected Islet1-GFP transgenic embryos immunostained with GFP and EphA4 antibodies, scored at 48hpf by fluorescence microscopy according to the following criteria. r4/r5/r6: number of embryos with partially migrated facial motor neurons; number (%) of neurons in r4: number of FMNs failing to migrate out of r4; ; number (%) of neurons in r5: JAK1-IN-4 number (%) of FMNs failing to migrate out JAK1-IN-4 of r5; number (%) of neurons in r6: number (%) of FMNs that have completed their migration to r6. The distribution of FMNs (% of neurons within r4,r5,r6) is usually illustrated in Fig. 7G. Abstract During development, Met signaling regulates a range of cellular processes including growth, differentiation, survival and migration. The gene encodes a tyrosine kinase receptor, which is usually activated by Hgf (hepatocyte growth factor) ligand. Altered regulation of human MET expression has been implicated in autism. In mouse, Met signaling has been shown to regulate cerebellum development. Since abnormalities in cerebellar structure have been reported in some autistic patients, we have used the zebrafish to address the role of Met signaling during cerebellar development and thus further our understanding of the molecular basis of autism. We find that zebrafish is usually expressed in the cerebellar primordium, later localizing to the ventricular zone (VZ), with the and ligand genes expressed in surrounding tissues. Morpholino knockdown of either Met or its Hgf ligands leads to a significant reduction in the size of the cerebellum, primarily as a consequence of reduced proliferation. Met signaling knockdown disrupts specification of JAK1-IN-4 VZ-derived cell types, and also reduces granule cell numbers, due to an early effect on cerebellar proliferation and/or.