Developmental and apoptotic defects of udu^tu24 mutants. Morphology of wild-type and udu^tu24 mutants at 20 hpf (A and B) and 2 dpf (C and D). (E and F) WISH for myoD at 14 hpf. myoD is only expressed in the adaxial cells and is not detected in the posterior half of formed somites in udu^tu24 embryos. (G and H) WISH for papc at 18 hpf in dorsal view, with anterior upward. papc is not expressed in the nascent somites of udu^tu24 embryos; indicated by arrows. (I and J) Normaski images of wild-type and udu^tu24 embryos' somites (approximately the 12th to 24th somite region). Somites of wild-type embryos were chevron- shaped with clear boundaries; however, mutant somites were U-shaped and their boundaries were less distinct, indicated by arrowheads. All embryos are represented in lateral view with anterior to the left and dorsal upwards, unless otherwise stated. wt, wild type; yse, yolk sac extension. (K and L) TUNEL assay of whole-mount wild-type and udu^tu24 embryos. Massive amount of apoptotic cells are found throughout udu^tu24 embryos at 22 hpf. (M–R) Examination of sections of TUNEL staining in lateral view and anterior to the left, unless otherwise stated. Midbrain-hindbrain region, dorsal view (M and N), lens and retina (O and P), and tail region (Q and R) of udu^tu24 mutants consisted of many apoptotic cells compared with wild-type embryos. Areas marked by red asterisks denote the otic placode; eyes are removed and mounted in lateral view.

Loss of udu function results in aberrant cell cycle. (A–D) Representative traces from FACS analyses after PI staining of dissociated cells at 22 hpf from wild-type and udu^tu24 embryos. (A and B) Dissociated cells from whole wild-type and udu^tu24 embryos. (C and D) Dissociated cells from wild-type and udu^tu24 embryos' tail region posterior to yolk extension. (E and F) Histograms summarize experimental data from FACS analyses after PI staining of dissociated cells from (E) whole embryos and (F) tail region posterior to yolk extension. The histograms are depicted as average ± SD format from triplicate experiments. udu^tu24 embryos contain fewer cells in the G1 and S phases and have an accumulation of cells in the G2/M phase (asterisks marked those p <0.05, Student's t test; p value for whole embryo: *G1, p = 0.041; S, p = 0.151; *G2/M, p = 0.007; for tail region: G1, p = 0.05; *S, p = 0.035; *G2/M, p = 0.016). (G and H) Projection of the lateral stacks of S phase cells labeled with BrdU at 22 hpf. Images are tail region posterior to yolk extension shown in lateral view with anterior to the left. (I–L) Wild-type and udu^tu24 embryos stained with pH3 antibody (green, arrowheads); nuclei are counterstained with DAPI (blue): eye region (I and J) and hindbrain region (K and L). (M) Percentage of pH3-positive cells in eye and hindbrain regions are obtained by counting pH3-positive cells versus total cells labeled with DAPI and are summarized in histogram, expressing in average ± SD format from four wild-type and four udu^tu24 embryos (p < 0.05, Student's t test).

Udu dynamics and localization during DNA repair and replication. (A and B) Immunohistochemical staining of γ-H2AX in nonirradiated embryos at 22 hpf embryos: γ-H2AX foci in wild-type embryos (A) and udu^tu24 embryos (B). (C–H) Wild-type and udu^tu24 embryos were irradiated with 15 min of UV and stained with γ-H2AX at different time after irradiation. (C and D) γ-H2AX staining after 1 h post-UV treatment. Both UV-treated wild-type and udu^tu24 embryos showed an increased in γ-H2AX foci. Hence, in the presence of extrinsic DNA damage agents, udu^tu24 mutants were able to response accordingly by recruiting DNA repair proteins via the activation of γ-H2AX as indicated by the amplified fluorescence staining. (E and F) γ-H2AX staining of embryos 5 h post-UV irradiation. The level of DNA damage after 5 h of repair were similar in both wild-type and udu^tu24 embryos. (G and H) The number of cells with γ-H2AX signals was significantly reduced in the trunk and tail regions of wild-type and udu^tu24 embryos after 31 h post-UV irradiation. (I and J) γ-H2AX staining of wild-type and udu^tu24 embryos injected with p53-MO. White arrows indicate the γ-H2AX foci. (K) Coimmunoprecipitation of MYC-tagged MCM proteins and FLAG-tagged PAH-L+SANT-L domains. FLAG-tagged PAH-L+SANT-L is immunoprecipitated from the cell lysates and detected with anti-FLAG antibody as a control and with anti-MYC antibody to detect the interacting proteins, MCM3 and MCM4, shown in lanes 4 and 5. (L–O) PAH-L and SANT-L domains are associated with replication foci during S phase. COS7 cells were synchronized at the G1/S border with aphidicolin, released into S phase, labeled with BrdU and fixed at different time points and submitted to laser scanning confocal microscopy for detection: DAPI (blue) (L and P), PAH-L+SANT-L (green) (M and Q), BrdU (red) (N and R), and merged images (yellow) (O and S) show the pattern of replication in early S phase (L–O) and late S phase (P–S). (T–W) Localization of Udu in pericentromeric heterochromatin: DAPI (blue) (T), Udu (green) (U), HP1β (red) (V), and merged image (yellow) (W) shows that Udu is colocalized with HP1β, a protein marker for pericentromeric heterochromatin. IB, immunoblotting; IP, immunoprecipitation.

p53 and its downstream target genes induce apoptosis in udu^tu24 embryos. (A and B) WISH of p53 in the zebrafish embryos at 22 hpf displays a higher level of expression in many parts of udu^tu24 mutants. Arrowheads indicate pronephron. (C–E) Developmental and apoptotic defects in udu^tu24 embryos can be rescued by inactivating p53 function with MO. (E) TUNEL staining of udu^tu24 embryos injected with 1.77 pmol of p53-MO. (F and G) WISH of myoD at 14 hpf. Note the myoD-striped pattern in the somites of injected embryos, whereas myoD-expressing cells were only found in the adaxial cells of udu^tu24 embryos. (H and I) Myotome boundaries of udu^tu24 embryos can be rescued with p53-MO injection as shown by titin at 25 hpf. (J) Real-time PCR of relative mRNA levels of 22 hpf wild-type and udu^tu24 embryos. elf1a is the endogenous control for the experiment. The histogram is depicted as average ± SD format from two independent duplicated experiments. Homozygous udu^tu24 embryos have elevated levels of p53, mdm2, p21^WAF/CIP1, caspase 8, bax, gadd45αl, gadd45β1, and gadd45β2 and barely reduced level of cyclin D1 compared with wild-type embryos. All embryos are showed in anterior to the left and dorsal upward.

Activation of Atm-Chk2 pathway in udu^tu24 mutants. (A) Real-time PCR of atm, atr, chk1 and chk2 mRNA levels extracted from 22 hpf embryos. The histogram is depicted as average ± SD format from three independent experiments (p < 0.05, Student's t test). The transcript level of atm and chk2 are significantly up-regulated in udu^tu24 mutants. (B and C) Western analyses showed increased levels of Chk2 and phospho-Chk2 (Ser33) in udu^tu24 embryos compared with wild-type embryos at 22 hpf. (D–H) Neutral single-cell electrophoresis or comet assay of untreated wild-type cells (D), untreated udu^tu24 cells (E), cells from p53-MO–injected udu^tu24 embryos (F), cells from p53-MO–injected wild-type embryos (G), and wild-type cells (H) isolated from embryos irradiated with UV for 1 h. The head is composed of intact DNA, whereas the tail consists of DNA with DSB. (I–K) TUNEL staining of udu^tu24 embryos treated with DMSO (I), 15 μM KU55933 (J), and 200 μM CGK733 (K) at 26 hpf. The number of TUNEL-positive cells is significantly reduced in embryos treated with KU55933 (62.5%; n =5/8) and CGK733 (80%; n = 8/10). (L–N) Phenotypes of chk2 morphants at 21 hpf. (N) Homozygous udu^tu24 embryos injected with chk2-MO have tail-tips that were not round-shaped but tapered (arrowhead). Somite structures are restored in 2.08 pmol chk2-MO–injected udu^tu24 embryos, compared with (M) noninjected udu^tu24 embryos. (O–Q) TUNEL staining of embryos at 21 hpf. (Q) The number of TUNEL-positive cells is significantly reduced in chk2-MO–injected udu^tu24 embryos, compared with (P) noninjected udu^tu24 embryos. (L and O) Wild-type embryos as the positive control for phenotypic observations and TUNEL assay, respectively.

Model of Udu function in DNA damage response. Udu may be required for DNA replication by interacting with MCM3 and MCM4. Without Udu, particularly its PAH-L and SANT-L domains, replication cannot proceed properly and DNA damage response is activated via Atm-Chk2-p53 pathway. However, the molecular mechanism leading to DNA damage response is not clear. Furthermore, it is unknown whether Udu has a more direct role in regulating p53 transcriptionally. The cell cycle defects found in udu mutants could be replication-linked, in addition to the effect of p53.