The subcellular distributions of zebrafish auxilin family proteins. Spinning disc confocal micrographs of HeLa cells transfected with (A) pCS2-GFP-zGAK and (B) pCS2-GFP-zAux. The cells were also stained for clathrin heavy chain (red). The low GFP-expressing cells are indicated by asterisks. The large GFP- and clathrin-positive aggregates are indicated by arrows. Scale Bar, 10 μm.

Both zGAK and zAux can substitute for Drosophila auxilin. Fluorescent micrographs of Drosophila eye imaginal discs stained with αElav antibody, which labels the nuclei of the neuronal cells (blue). The expressions of zebrafish and Drosophila auxilin genes are shown in green. Homozygous dAux^F956* mutant tissues are indicated by the absence of the membrane-associated mRFP (red). Regions containing excessive Elav-positive cells are indicated by arrows. All the flies carry Act5C-GAL4, UAS-FLP on the second chromosome, and other relevant genotypes include: (A) FRT^5-5Z3515, dAux^F956*/FRT^5-5Z3515, GMR-src-mRFP, (B) UAS-dAux-GFP; FRT^5-5Z3515, dAux^F956*/FRT^5-5Z3515, GMR-src-mRFP, (C) UAS-GFP-zGAK; FRT^5-5Z3515, dAux^F956*/FRT^5-5Z3515, GMR-src-mRFP, (D) UAS-GFP-zAux; FRT^5-5Z3515, dAux^F956*/FRT^5-5Z3515, GMR-src-mRFP, (E) UAS-GFP-zGAK^ΔJ; FRT^5-5Z3515, dAux^F956*/FRT^5-5Z3515, GMR-src-mRFP, and (F) UAS-GFP-zAux^ΔJ; FRT^5-5Z3515, dAux^F956*/FRT^5-5Z3515, GMR-src-mRFP. Scale Bar, 50 μm.

The expression patterns of GAK and auxilin during zebrafish embryonic development. (A, D) Lateral, (B, E) dorsal, and (C, F) anterior views of wild-type embryos at the 15-somite stage. (A-C) zGAK is expressed broadly in the hindbrain (B), forebrain and eyes (C). (D-F) zAux is expressed mostly in neural tissues as described in the main text. (G, H) Dorsal views of 19-somite stage embryos. (G) zGAK is still ubiquitously expressed and (H) zAux remains specific to bilateral stripes of neural cells. (I, K, L, N) Lateral and (J, M) dorsal views of 24 hpf embryos. (I-K) zGAK is seen in the brain, vasculature and otic vesicles. (L-N) zAux remains concentrated in bilateral stripes of neural cells. Panels K and N are close-up views of the posterior regions of the embryos shown in (I) and (L), respectively. In all the images, anterior is to the left, and in all the lateral views, dorsal is up. ey, eye; is, intersomitic vessel; lg, lateral line ganglion; nt, neural tube; ot, otocyte; ov, otic vesicle; sag, statoacoustic ganglion; Scn, Spinal cord neuron; tg, trigeminal ganglion; v, vasculature; vd, ventral diencephalon. Scale Bar, 100 μm.

GAK-MO1 specifically disrupts zGAK splicing. (A) RT-PCR analysis of total RNA extracted from uninjected and GAK-MO1 morphant embryos. From normally spliced zGAK mRNA, a reaction with primers complementary to exon 2 and 4 (2/4) would yield a band of 178 bp, and a reaction with primers complementary to exon 18 and 20 (18/20) would yield a band of 275 bp. While the 275 bp band seemed unaffected by GAK-MO1 injection, the level of 178 bp band was reduced and a new band of 118 bp appeared in GAK morphants. Sequence analysis of this 118 bp band showed that exon 2 was spliced into exon 4, resulting in an in-frame deletion in the kinase domain. (B) The target sequences of GAK-MO1 and GAK-MO1C were placed in front of mCherry and cloned into pCS2. In vitro transcribed mRNAs were co-injected with morpholino into one-cell stage embryos. Bright field and fluorescent images of injected embryos at 6 hpf are shown. Scale Bar, 200 μm.

Cell degeneration phenotype of GAK morphant embryos during development. (A, E) Lateral and (B, F) dorsal views of (A, B) uninjected and (E, F) GAK morphant embryos at 14-somite stage. Visible degeneration in the eyes (E) and the forebrain regions (F) of GAK morphants is indicated by white arrowheads. (C, G) Lateral and (D, H) dorsal views of (C, D) uninjected and (G, H) GAK morphant embryos at 24 hpf. At this stage, the increased cell degeneration within the brain (arrowheads) and thinner yolk extension in GAK morphants is apparent. (I-L) Lateral views of (I, J) uninjected and (K, L) GAK morphant embryos at 36 hpf. In all the lateral views, anterior is to the left and dorsal is up. ey, eye; Hb, hindbrain; nt, neural tube; no, notochord; ov, otic vesicles; ye, yolk extension. Scale Bar, 100 μm.

Programmed cell death in wild-type and GAK morphant embryos during development. (A, C) Lateral and (B, D) dorsal views of TUNEL-stained (A, B) wild-type and (C, D) GAK morphant embryos at the 14-somite stage. At this stage, the control embryos display no detectable apoptosis, whereas GAK morphants have a low level of programmed cell death (indicated by black arrowheads). (E, G) Lateral and (F, H) dorsal views of TUNEL-stained (E, F) wild-type and (G, H) GAK morphants at 24 hpf. While the control animal exhibits a low level of apoptosis in anterior brain and the posterior body regions (arrowheads), GAK morphants exhibit a high level of apoptotic cell death in the brain, as well as in the neural tube (arrowheads). However, as compared to the control wild-type embryo, no obvious increase in apoptosis was observed in the posterior region of GAK morphants (indicated by arrows). In all the lateral views, anterior is to the left and dorsal is up. ey, eye; Hb, hindbrain; MHB, mid-hindbrain boundary; nt, neural tube; s, somites; tb, tailbud. Scale Bar, 100 m.

Expression patterns of regional brain markers in wild-type and GAK morphant embryos. (A-D) krox20 expression in (A, C) uninjected and (B, D) GAK morphant embryos at (A, B) 12- to 14-somite stage and (C, D) 24 hpf. At 12- to 14-somite stage, krox20 expression in the rhombomeres 3 (R3) and 5 (R5) of the control and injected embryos appears comparable. However, at 24 hpf, the spacing between R3 and R5 is dramatically reduced in GAK morphants (white arrowhead). (E, F) Lateral and (G, H) dorsal views of fgf8 expression in (E, G) wild-type and (F, H) GAK morphant embryos at 18- to 20-somite stage. The domains of fgf8 expression in the forebrain and mid-hindbrain boundary (MHB) are up-regulated in GAK morphants (black arrows). (I, J) shh expression in (I) wild-type and (J) GAK morphants at the 18-somite stage. In all the lateral views, anterior is to the left and dorsal is up. d, ventral diencephalon; f, floor plate; os, optic stalk; s, somite; t, telencephalon; dd, dorsal diencephalon; tb, tailbud. Scale Bar, 100 μm.

Expression patterns of HuC and Her4 in wild-type and GAK morphant embryos. (A, B) Close-up dorsal views of HuC expression in (A) wild-type and (B) GAK morphant embryos at the 8-somite stage. Micrographs of a lower magnification are shown in the insets. At this stage, more cells appeared to express HuC in GAK morphant embryos, suggesting the presence of more neural progenitor cells. (C-F) Lateral views of HuC expression in the brain regions of (C, E) wild-type and (D, F) GAK morphant embryos at 24 to 28 hpf. A comparison of (C) and (D) shows a reduction in HuC-positive cells in the forebrain and midbrain in GAK morphants. Similarly, a comparison of (E) and (F) shows the disorganization and reduction of HuC-positive cells in the hindbrain in GAK morphants. (G-J) Lateral views of Her4 expression patterns in (G, I) wild-type and (H, J) GAK morphants at the (G, H) 8-somite stage or at (I, J) 24 to 28 hpf. At the 8-somite stage, Her4 expression in GAK morphant embryos was significantly reduced, as compared to the wild type. In contrast, the expression of Her4 in the brain of wild-type and GAK morphant embryos at 24 to 28 hpf was comparable. In all the panels, anterior is to the left, and in all the lateral views, dorsal is up. Fb, forebrain; Hb, hindbrain; MHB, mid-hindbrain boundary; Mb, midbrain; mbp, midbrain basal plate; R, rhombomeres; tb, tailbud; te, telencephalon; vd, ventral diencephalon. Scale Bar, 100 μm.

The phenotypes of GAK-MO2 morphants are similar to those of GAK-MO1 morphants. (A) RT-PCR analysis of the total RNA from embryos injected with GAK-MO2. Using the RNA prepared from uninjected embryos, a 2/4 reaction (using primers complementary to exon 2 and 4) yielded a band of 178 bp, and an 18/20 reaction yielded a band of 275 bp and a slightly smaller non-specific band. On the other hand, using the RNA prepared from GAK-MO2 morphants, the 178 bp band was unaffected. However, the 275 bp band was absent (the non-specific band was unaffected) and a new band of 359 bp appeared. Sequence analysis of this 359 bp band revealed that the injection of GAK-MO2 caused the retention of intron 18, resulting in a frame-shift truncation in the PTEN region (immediately after Lys⁶⁷⁹). (B, C) Lateral views of uninjected and GAK-MO2 morphants at 36 hpf. In (B), anterior is to the left and dorsal is up, and in (C), anterior is up and dorsal is to the right. ey, eye; Hb, hindbrain; ov, otic vesicles; ye, yolk extension. Scale Bar, 100 μm.

Expression patterns of HuC and Her4 in wild-type and GAK morphant embryos at the 10-somite stage. (A, B) Lateral views of HuC expression in (A) wild-type and (B) GAK morphant embryos at the 10-somite stage. Similar to the 8-somite stage, more cells appeared to express HuC in GAK morphant embryos at the 10-somite stage, suggesting the presence of more neuronal cells. (C-D) Close-up top views of HuC expression in the brain regions (indicated by brackets in A&B) of (C) wild-type and (D) GAK morphant embryos. (E, F) Lateral views of Her4 expression patterns in (E) wild-type and (F) GAK morphants at the 10-somite stage. At this stage, Her4 expression in GAK morphant embryos appeared reduced, as compared to the wild type. (G-H) Close-up top views of Her4 expression in the brain regions (indicated by brackets in E&F) of (G) wild-type and (H) GAK morphant embryos. In all the panels, anterior is to the left, and in all the lateral views, dorsal is up. Fb, forebrain; Hb, hindbrain; Mb, midbrain; mes, mesencephalon; te, telencephalon; Tg, Trigeminal ganglion; tb, tailbud. Scale Bar, 100 μm.

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