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The stem-loop binding protein CDL-1 is required for chromosome condensation, progression of cell death and morphogenesis in Caenorhabditis elegans

Yuki Kodama1, Joel H. Rothman2, Asako Sugimoto1,3,4,* and Masayuki Yamamoto1

1 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo, 113-0032, Japan
2 Neuroscience Research Institute, University of California-Santa Barbara, Santa Barbara, CA 93106, USA
3 PRESTO, Japan Science and Technology Corporation, Kawaguchi, 332-0012, Japan
4 Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, Kobe, 650-0047 Japan



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  		Fig. 1. cdl-1 gross phenotypes. A wild-type N2 embryo at the pretzel stage (A) and a cdl-1(e2510) embryo (B) are shown. Terminally arrested cdl-1 embryos show excess cell corpses, variable defects in body elongation, and a failure in attachment of the pharynx to the buccal opening. Scale bar, 10 µm.

 


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  		Fig. 2. Quantitation of the number of cell corpses during embryogenesis. (A) Wild-type N2. (B) cdl-1(e2510). (C) cdl-1(e2501). (D) cdl-1(w37). (E) mnDf90/cdl-1(e2510). The y axis represents the average number of cell corpses visible in embryos of each genotype at each stage. Stages are defined as follows: 220~310 minutes (I), 320~410 minutes (II), 420~510 minutes (III), 520~610 minutes (IV), 620~710 minutes (V), and 720~810 minutes (VI) after first cleavage. Error bars indicate s.d. Numbers of embryos examined were (A) 17, (B) 23, (C) 11, (D) 8 and (E) 32, respectively. Asterisks (*) indicate that the difference from the wild type at the same stage is significant (P<0.01).

 


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  		Fig. 3. Duration of visible cell corpses. (A) Wild-type N2. (B) cdl-1(e2510). (C) cdl-1(RNAi). Each panel illustrates the time of appearance and disappearance of all corpses detected in 4D images of a single embryo. (A typical result is displayed here for each genotype.) Each horizontal bar represents a corpse, and its length indicates duration of the corpse. The corpses are ordered according to the time of their appearance in the embryo. Scoring of corpses in N2 (A) was discontinued at ~400 minutes, because the twitching movement made observation of corpses difficult. For B and C, cell corpses were scored until the end of the time-lapse recording at 668 minutes (B) and 797 minutes (C) after first cleavage, respectively.

 


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  		Fig. 4. Defects in pharynx morphogenesis in cdl-1 mutant embryos. (A) Movement of the pharynx primordium toward the anterior of the head. Each picture was taken from a 4D recording of the same embryo. (a-d) Wild type and (e-h) cdl-1(e2501). Arrowheads indicate the anterior tips of the pharyngeal basement membrane. The buccal capsule is indicated in d and h (arrows). Time from first cleavage: (a) 390 minutes, (b) 435 minutes, (c) 480 minutes, (d) 610 minutes, (e) 390 minutes, (f) 480 minutes, (g) 600 minutes, and (h) 1140 minutes. (B, C) Merged images of immunostaining with a pharynx-specific antibody, 3NB12 (red), and Nomarski microscopy. A wild-type N2 L1 larva (B) and a cdl-1(e2501) embryo (C) that has elongated over 3-fold are shown. The buccal capsule (arrow) and the anterior tip of the pharynx (arrowhead) are indicated.

 


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  		Fig. 5. The cdl-1 gene. (A) Domain structures of CDL-1 and related proteins. The RNA stem-loop binding domain (striped boxes) are shared by all four proteins. Other domains conserved in the vertebrate proteins are not found in CDL-1. (B) Sequence alignment of the RNA binding domain of CDL-1 with human (hSLBP), mouse (mSLBP), Xenopus (xSLBP1) and Drosophila (dSLBP) SLBPs. The mutated residues in e2510 (P-to-S missense mutation) and e2501 (nonsense mutation) are indicated. (C) Structure of the cdl-1 ORF. The mutation sites in e2510 and e2501 and the deleted region in w37 are indicated. The neighboring predicted ORFs are also shown.

 


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  		Fig. 6. CDL-1 interacts with the stem-loop structure in the 3'-UTR of core-histone mRNA. (A) The yeast three-hybrid system (SenGupta et al., 1996) was used to test the RNA binding specificity of CDL-1. The assay is schematically shown. (B) Structure of the two stem-loop RNAs used in the experiments. wtRNA: the conserved stem-loop sequence in the 3'-UTR of C. elegans core histone mRNA. rvRNA: the sequence with a reversed stem-loop. (C) RNA-binding activity of CDL-1 protein as assayed by ß-galactosidase activity. IRE (Iron Response Element) and IRP (Iron Response Protein) were used as a pair of positive controls (SenGupta et al., 1996). (D) Activation of HIS3. Transformants indicated in C were streaked on –His plates containing 5 mM 3-aminotriazol.

 


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  		Fig. 7. RNAi phenotypes of cdl-1 and core histone genes. (A,D) The predominant RNAi phenotypes for cdl-1 (A) and the histone H4 gene (D). Embryos arrest with around 50 cells in both cases. (B,C,E,F) Embryos showing later embryonic RNAi phenotypes, which are similar to those of the cdl-1 mutants. (B,C) cdl-1, (E,F) the histone H2B gene. Arrows indicate cell corpses. Arrowheads indicate the anterior tip of the pharynx. Scale bar, 10 µm.

 


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  		Fig. 8. RNAi of either cdl-1 or the histone genes resuls in defects in chromosome condensation and segregation. Embryos co-stained with the anti-alpha tubulin antibody (red) and DAPI (green) are shown. (A,B) Wild type. (C,D) cdl-1(RNAi). (E,F) RNAi of the histone H2B gene. In wild type, chromosomes are properly condensed at metaphase (B right) and segregated at anaphase (B left) through telophase (A). In embryos in which CDL-1 or histones are depleted, chromosome bridges (C,D,F) and decondensed chromosomes at metaphase and anaphase (D,E) can be often observed. Scale bar, 10 µm.

 

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© The Company of Biologists Ltd 2002