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Abrams et al., 2020 - Molecular genetics of maternally-controlled cell divisions. PLoS Genetics   16:e1008652 Full text @ PLoS Genet.

Fig. 1 Maternal-effect cleavage-stage mutants. A. Developmental arrest mutants and wild-type at 2.25 hpf (top row) and at 5.0 hpf (bottom row). For each mutant the phenotype is 100% penetrant within a clutch and across mutant females. At least 50 embryos per mutant female were examined (n = 233 from four srh females, n = 854 from six bmb females, n = 335 from four p10umal females). B. Irregular cleavage mutants and wild type at 0.75 hpf (top row), 1.0 hpf (middle row) and 1.25 hpf (bottom row). Five of 6 mxp females produced embryos shown (n = 459), while one produced embryos with a more mild phenotype (n = 91). C. Wild-type (top row) and cld mutants (bottom row) at 1.0, 1.5, and 4.0 hpf. Penetrance of the cell sloughing phenotype (black arrow) at 4.0 hpf is indicated in lower left corner. Remaining embryos retain relatively normal, but dark, blastoderms and do not survive to 24 hpf.

Fig. 2 Examining nuclear integrity in mixed up and disarray embryos. A. Wild-type (TL), (B) dsy and (C) mxp embryos were fixed at 5-minute intervals spanning 20 minutes (corresponding to the 2 to 4 cell division) and stained with DAPI and phalloidin to mark the DNA and actin at the cell boundaries, respectively. B. Representative embryos (numbers indicated in the lower left corner) from a total of three dsy females. In some cases nuclear divisions were asynchronous (20 min) in embryos from dsy mutant mothers compared to wild type (A). C. Representative embryos (numbers indicated in the lower left corner) from a total of four mxp females. Embryos shown in the upper row underwent cell division timing similar to wild type in (A), whereas the embryos in the lower row were delayed. Scale bars = 200μm.

Fig. 3 Nuclear division is disrupted in p10umal mutants. A. DAPI staining of wild-type and p10umal 8-cell stage embryos (n = 11). Note: only four of the 8-cells are in view. B. Wild-type and C. p10umal fertilization time courses (N = 3 females examined). Embryos were fixed at 16, 22, 25, 28, 31 and 34 mpf. A minimum of five embryos corresponding to each time point were examined (representative images are shown). Pronuclei (16 mpf) and the one-cell zygote (at 22–34 mpf) were stained with DAPI (blue), anti-phospho-histone H3 (red), and anti-PCNA (green). Scale bars = 10μm. The 25 and 28 mpf time points were digitally reduced by 0.5x. D. Schematic representation of the p10umal phenotype at the corresponding time points illustrating the typical DNA bridge between dividing cells. E. Wild type and p10umal mutants at 2.5 and 3.0 hpf stained with DAPI and phalloidin to mark DNA and the cell boundaries, respectively. A minimum of 3 and up to 6 embryos each from 3 different females were examined for each time point (representative images are shown).

Fig. 5 dullahan mutant phenotype. A. Wild-type and (B) dul embryos at 3.5, 5.5, and 24 hpf. Embryos in upper left and center panels are lateral views, and upper right panels are animal pole views. The enlarged cytoplasmic region between the yolk and blastomeres in the dullahan mutant at 3.5 hpf is noted with asterisks. The dorsal shield (arrow) is to the right in the 5.5 hpf wild-type embryo and absent in the mutant. C. Distribution of embryonic phenotypes from five dul females (left) compared to 6 siblings (right): mut #1 (n = 80), mut #2 (n = 71), mut #5 (n = 68), mut #6 (n = 32), mut #7 (n = 105), HET-A (n = 103), HET-B (n = 112), HET-C (n = 76), HET-D (n = 76), HET-A (n = 67), HET-A (n = 118), HET-G (n = 32). In each cross heterozygous or mutant females were crossed to wild-type (TL) males.

Fig. 6 Dorsal markers are reduced in dullahan mutants. chordin mRNA expression in wild-type (A, A’) and dul (B, B’) embryos at 6.0 and 8.0 hpf, respectively. A and A’ are animal views, dorsal to the right. B and B’ are dorsal views. The number of embryos with the shown expression pattern of the total embryos examined is indicated in the upper right or left (inset) corner. goosecoid mRNA expression in wild-type (C, C’) and dul (D, D’) embryos at 6.0 and 8.0 hpf, respectively. The numbers in the lower right corner indicate the number of embryos with any positive goosecoid signal of the total embryos examined.

Fig. 7 p09ajug is an allele of polo-like kinase-1. A. Embryos of p09ajug mutant females exhibit an irregular cleavage phenotype. B. The p09ajug mutation maps to chromosome 1 within a 700 kb interval flanked by SSLP markers, CU467110 and CU104756-4. C. Genomic structure of plk1 indicating the T to G change in the start codon. D. Homozygous p09ajug male sterility can be rescued with Tg(actb2:plk1). Each bar represents a different fish, where M = male and F = female. The first number in the fish name (6-, 4-, 5-) represents a particular fish family, with individual fish identifying information following that number. Total number of embryos scored is indicated at the top of each bar.

Fig. 8 screeching halt encodes SLBP2. A. The srh mutation maps to a 600 kb interval on chromosome 21 flanked by zBX510945 and zBX511168. Recombinants identified between the mutation and the marker per total meiotic events examined is noted below each marker in red. The interval contains 21 predicted ORFs (arrows). The black arrow (on the reverse strand) corresponds to slbp2. The predicted exon-intron structure is indicated below. Note: intron 3 (568 bp) and intron 5 (1961bp) are not drawn to scale (dashed lines) due to size. Mutations corresponding to srhp18ad (T to A, Iln to Asn) and srhsa12562 (C to T, Glu to stop) map to exon 4. B. The sa25162 allele fails to complement srhp18ad. Embryos from sa25162/+ females and ten p18ad/sa25162 trans-heterozygous females, with at least 50 embryos per female (n = 880), shown at 6 hpf. C. RT-PCR of slbp2 (top) and slbp1 (bottom) from wild-type cDNA (ovary, 32-cell and sphere stage). D. SLBP RBDs from zebrafish (zSLBP1 and zSLBP2), Xenopus (xSLBP1 and xSLBP2) and Bovine (bSLBP1 and bSLBP2) were aligned using Clustal Omega [53]. The Iln residue that is mutated to Asn in srhp18ad and the Glu residue that is mutated to a stop codon in slbp2sa12562 are boxed in red. The ‘*’ indicates identical residues, ‘:’ or ‘.’ indicate similar residues.

Fig. 9 Slbp2 is required for histone production during early development. A. Western blot analysis of the four core histones in wild-type and srh embryos. Anti-α-tubulin was used as a loading control. B. The srh developmental arrest phenotype can be rescued by injecting total histone protein into one-cell stage srh embryos. P-values were determined using a Student’s t-test. ****p< 0.0001. srh embryos injected with 5 ng (C-E) or 7.5 ng (F-I) of whole histone. C and F, lateral views imaged at 5 hpf. D and G lateral views imaged at 6 hpf. E and H were imaged at 24 hpf. I was imaged at 48 hpf. Note the head formation and eye pigmentation in H and the presence of melanocytes in I. show less

ZFIN wishes to thank the journal PLoS Genetics for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ PLoS Genet.