Expression of mid1ip1l and gene phylogeny. (A) RT-PCR analysis over developmental time (h or days post fertilization) of mid1ip1l RNA, co-amplifying β-actin RNA as an internal control. mid1ip1l RNA is maternally expressed, with levels subsiding at later stages. (B-G) In situ hybridization analysis. mid1ip1l antisense probes label early cleavage stage embryos (B, 25/25; E,F), with a reduction by dome stage (20/20; G). Levels and distribution of mid1ip1l RNA are similar in aura mutants (from aurt9792/t9792 females, D). Control sense RNA (C). (H-J) Mid1ip1l antibodies label within cortical F-actin in wild type (26/26; H). Reduced labeling is observed in aurt9792 embryos (20/20; I) and no localization in mid1ip1l uw39 embryos (21/21; J). (K) Higher resolution imaging of wild type (from H) shows Mid1ip1l protein localizing as puncta within cortical F-actin aggregates (8/8). Insert (K) shows a 2× magnification of the boxed region. In wild type, Mid1ip1l protein becomes enriched at the forming furrow (4/4; L) and induced wounds (4/4; M). (H-L) 45 mpf; (M) 15mpf. (N) RT-PCR analysis of mid1ip1a, mid1ip1b and mid1ip1l. RT-PCR data (A,N) are representative of two trials. (O) Phylogenetic tree for Mid1ip1 protein shows zebrafish mid1ip1b as most closely related to other vertebrate mid1ip1 genes, and mid1ip1a and mid1ip1l as products of gene duplication. Ixodes scapularis (deer tick) is an outgroup. Numbers in red indicate branch support values; number in black indicates branch scale bar. Scale bars: 10µm in H-J,L,M; 5µm in K.
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