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First published online 5 October 2005
doi: 10.1242/dev.02049

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POPK-1/Sad-1 kinase is required for the proper translocation of maternal mRNAs and putative germ plasm at the posterior pole of the ascidian embryo

¹ Department of Biological Sciences, Graduate School of Science, Osaka University, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan
² Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuda, Midori-ku, Yokohama 226-8501, Japan
³ Faculty of Integrated Arts and Sciences, Tokushima University, 1-1 Minami-Josanjima, Tokushima 770-8502, Japan

View larger version (17K): [in a new window]   Fig. 1. Comparisons of Hr-POPK-1 with homologs in other animals. Hr-POPK-1 shows high similarity to Cs-SAD-1 of C. elegans (Accession No. AB014885), Mm-SAD-A of mouse (Accession No. AY533671), Hs-SAD1 of human (Accession No. BC016681) and CG6144 of Drosophila melanogaster (Accession No. NM135570). Humans have at least three other homologs in the genome. These proteins share an N-terminal kinase domain (orange). The amino acid identity compared with Hr-POPK-1 is indicated in the diagrams. They had two other conserved domains (blue), whose function is unknown. Hr-POPK-1 and PAR-1 of C. elegans show lower similarity in their kinase domains, and PAR-1 has no similarity outside the kinase domain. Numbers on the right side indicate the number of total amino acid residues.

View larger version (74K): [in a new window]   Fig. 2. Tissue formation in embryos injected with POPK-1 MO. (A-C,E,G-M) Uninjected controls. (A'-C',E',G'-M') POPK-1 MO was injected into fertilized eggs after the completion of ooplasmic segregation. (D,F,I,L) Diagram of the vegetal hemisphere at the 110-cell stage, showing presumptive muscle, mesenchyme, notochord and endoderm blastomeres, respectively. Anterior is up. In (L), endoderm precursors are yellow, trunk lateral cell precursors are blue. B7.5 blastomeres give rise to muscle (red) and trunk ventral cells (purple). (A,A') Morphology. (B,B') Expression of muscle-specific acetylcholinesterase (AChE) in tailbud embryos. (C,C') Expression of myosin protein (Mu-2 antigen) in larvae. (E,E') Expression of myosin in embryos whose cleavages were arrested at the 110-cell stage. (G,G') Mesenchyme-specific Mch-3 antigen in cleavage-arrested embryos. (H,H') Notochord-specific Not-1 antigen. (J,J') Not-1 antigen in cleavage-arrested embryos. (K,K') Expression of alkaline phosphatase (ALP). (M,M') ALP in cleavage-arrested embryos. Ectopic ALP activity is observed in the posteriormost (B7.5) cells (arrowhead) in (M'). Scale bars: 100 µm.

View larger version (33K): [in a new window]   Fig. 3. Hr-POPK-1 acts upstream of macho-1. (A) Normal uninjected larva. (A') Larva injected with POPK-1 mRNA. Head with pigment cells and kinked tail are recognizable. (B,B') Expression of AChE in uninjected embryo (B), and in embryo injected with POPK-1 mRNA (B'). Cleavage was arrested at the 110-cell stage. No ectopic muscle formed. (C-E) Expression of AChE in uninjected tailbud embryos (C), in embryos injected with macho-1 MO (D) and in embryos co-injected with macho-1 MO and POPK-1 mRNA (E). Loss of muscle cell was not rescued by co-injection of the mRNA. (F) Expression of AChE in embryos injected with control MO (left), in embryos co-injected with POPK-1 MO and macho-1 mRNA (center) (excess amounts of muscle cells are formed), and in embryos injected with POPK-1 MO (right) (no muscle is formed). Scale bars: 100 µm.

View larger version (53K): [in a new window]   Fig. 4. Localization of postplasmic/PEM mRNAs in embryos injected with Hr-POPK-1 MO. (A,B,C, left columns) Embryo injected with control MO. (A,B,C, right columns) Embryo injected with POPK-1 MO. (A) macho-1 localization at the early 8-cell stage (animal views), and late 8-cell stage (posterior views). (B) macho-1 localization at the 2-cell stage (posterior views), 4-cell stage (animal views), 16-cell stage (animal views), and 32-cell stage (animal views). White arrowheads indicate macho-1 mRNA localization. (C) Localization of various Type I postplasmic/PEM RNAs. The probes are indicated on the left side. Scale bars: 100 µm. (D) Semi-quantitative RT-PCR analysis of macho-1, Hr-POPK-1, Hr-ZF1 and Hr-Notch mRNA using ten uninjected embryos (left) and ten embryos injected with POPK-1 MO (right) at the late 8-cell stage. RT+ and RT–, with and without reverse transcriptase.

View larger version (39K): [in a new window]   Fig. 5. CAB formation. (A,B) The CAB in extracted 8-cell embryo injected with control MO (A) and in embryo injected with POPK-1 MO (B). Bilateral CABs are indicated by white arrowheads. Animal views. (C,D) The CAB at the 4-cell stage. (C) Control MO. (D) POPK-1 MO. The CAB precursors are more scattered than control. (E,F) Closer views of A and C. White arrowheads indicate each CAB. Anterior is up. Scale bars: 25 µm.

View larger version (99K): [in a new window]   Fig. 6. Unequal cleavages in MO-injected 16-cell embryos. (A) In control embryo, B4.1 blastomeres divide into larger B5.1 and smaller B5.2 blastomeres, as indicated by red bar. (B) Normal CAB in extracted embryo. (C) In most cases, POPK-1 MO-injected embryos also showed normal cleavage pattern. (D) Occasionally, unequal cleavage failed. (E) Unequally cleaved POPK-1 MO-injected embryo had small CAB. (F) Even if POPK-1 MO-injected embryos failed to divide unequally, there was always a small CAB. The shape and size of the CAB was indistinguishable from those in embryos showing unequal cleavage. Scale bar: 100 µm.

View larger version (59K): [in a new window]   Fig. 7. Type I postplasmic/PEM RNA localization on isolated cortices of eggs and 8-cell embryos. cER was stained with DiIC18 (3) and emits red fluorescence. Particle with intense red fluorescence in left panels are micelles of DiIC18 (3). In-situ hybridization signals of Hr-POPK-1, Hr-ZF1 and Hr-Wnt-5 are green. (A-D) Fluorescent images of the cortices isolated from unfertilized eggs. (A) cER (left) and Hr-POPK-1 mRNA (middle). The right panel shows a merged image. (B) cER (left), Hr-ZF1 (middle) and a merged image (right). (C) cER (left), Hr-Wnt-5 (middle) and a merged image (right). (D) cER (left), macho-1 (middle) and a merged image (right). White arrowheads in A-C indicate small granules with bright green fluorescence. (E-G) Confocal images of the CAB domain in the posterior cortices isolated from 8-cell stage embryo. (E) Hr-POPK-1 mRNA. (F) Hr-ZF1 mRNA. (G) Hr-Wnt-5 mRNA. Scale bars: 5 µm.

View larger version (116K): [in a new window]   Fig. 8. Confocal images of the cER/mRNA domain in the posterior cortices in uninjected embryo and embryo injected with POPK-1 MO. cER was stained with DiIC18 (3) and emits red fluorescence. In-situ hybridization signal of Hr-PEM1 is green. (A) cER (left) and Hr-PEM1 (middle) in cortex that was isolated from an uninjected embryo at 8-cell stage. The right panel shows a merged image. (B) cER-rich domain and Hr-PEM1-rich domain overlap in cortices isolated from embryo injected with POPK-1 MO. White arrowheads indicate the cER-rich region. cER/mRNA domain is smaller and rounded in embryos injected with POPK-1 MO. (C-E) Live cortices isolated from 2-, 4-, and 8-cell embryos, respectively. (F) cER and Hr-PEM1 mRNA in posterior cortices isolated from 4-cell stage embryo. Hr-PEM1-rich domain overlaps with the cER-rich domain, as shown by the merged image (right). Scale bars: 10 µm.