Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on July 29, 2003

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Human Molecular Genetics, 2003, Vol. 12, No. 18 2293-2299
DOI: 10.1093/hmg/ddg254
© 2003 Oxford University Press

Growth retardation and skin abnormalities of the Recql4-deficient mouse

Yuko Hoki^1,, Ryoko Araki^1,, Akira Fujimori^1,, Tatsuya Ohhata^1,, Haruhiko Koseki², Ryutaro Fukumura¹, Miki Nakamura¹, Hirokazu Takahashi¹, Yuko Noda¹, Seiji Kito¹ and Masumi Abe^1,*

¹National Institute of Radiological Sciences, Chiba, Japan and ²Department of Molecular Embryology, Graduate School of Medicine, Chiba University, Chiba, Japan

Received April 16, 2003; Revised July 8, 2003; Accepted July 20, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Mutations in the Recql4 gene are very likely responsible for a subset of Rothmund–Thomson syndrome (RTS) cases, but until now there has been no animal model to confirm this. Knockout mice in which the Recql4 gene is disrupted at exons 5–8 exhibit embryonic lethality at embryonic day 3.5–6.5. We generated a helicase activity-inhibited mouse by deleting exon 13 of Recql4, which is one of the coding exons of the consensus RecQ-helicase domain. This domain is the primary site of mutations that have been identified in RTS patients. The exon 13-deleted Recql4-deficient mice are viable, but exhibit severe growth retardation and abnormalities in several tissues, and embryonic fibroblasts show a defect in cell proliferation. Abnormalities in the Recql4-deficient mice are similar to those in RTS patients, suggesting that defects in the Recql4 gene may indeed be responsible for RTS. We speculate that the loss of Recql4 helicase activity results in the prematurely aged appearance observed in some RecQ helicase diseases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Rothmund identified a condition characterized by poikiloderma and juvenile cataracts, and Thomson a condition characterized by genetic bony abnormalities and poikiloderma (1,2). These were later found to be part of the same syndrome, designated Rothmund–Thomson Syndrome (RTS). This is a severe autosomal recessive disorder characterized by growth retardation, poikiloderma, hair loss, cataracts, bony malformations and a high incidence of malignancies, especially osteosarcomas (3–5). These characteristics also suggest that RTS is a premature aging syndrome (6). Premature aging symptoms have been extensively described in connection with the Werner syndrome, which is caused by mutations in the WRN gene, and also observed in the Bloom syndrome, caused by mutations in the BLM gene (6). Both genes encode proteins that belong to the RecQ helicase family. Other abnormal phenotypes observed in RTS patients are also similar to those of the Werner and Bloom syndromes. A part of RECQL4 has been identified in the EST database as paralogous to WRN and BLM (7). This suggests a link between the RECQL4 gene and RTS and, indeed, mutations were found in the RECQL4 gene in RTS patients, most of which were identified in the helicase domain (8–11) (Fig. 1A). To confirm whether RECQL4 is related to RTS and to prepare an animal model for RTS, Recql4 gene disruption was attempted in mice. Ichikawa et al. reported that Recql4 knockout mice in which exons 5–8 were replaced with a knockout vector bearing LacZ and PGKneo genes died between embryonic day 3.5–6.5 (3). They also found that the growth rate of both the inner cell mass and trophoblast cells of the blastocysts of the Recql4^-/- mice markedly decreased. They proposed a role for RECQL4 during mouse early development. No data about RNA and its products in the knockout mice were included.

View larger version (41K): [in this window] [in a new window]   Figure 1. Creation of a Recql4-deficient mouse. (A) Mutations identified in the RECQL4 gene of RTS patients are indicated by arrows (8–11). Boxes indicate exons, and shaded boxes demonstrate exons coding the RecQ-helicase domain. (B) The mouse Recql4 locus, targeting vector and targeted locus. TK and Neor indicate the thymidine kinase gene and neomycin-resistance gene, respectively. ‘S’ and ‘X’ indicate the recognition sites of SacI and XbaI, respectively. ‘5' probe’ and ‘3' probe’ show the regions used as probes for Southern hybridization. The arrows indicate transcription direction. Arrowheads indicate primers used for RT–PCR. (C) Southern blot showing the presence of the 7 kb SacI and the 8 kb XbaI genomic fragment in wild-type mice and the 9 kb and the 6 kb fragments in the Recql4-deficient mice, respectively. MEFs were used for the analysis. (D) The sequence of the exon 13-deleted transcript. (E) Quantitative PCR showing the presence and expression level of the exon 13-deleted transcript in the testis of the Recql4-deficient mice. RT–PCR was performed using total RNA fractions prepared from the testis of wild-type mice and Recql4-deficient mice. To measure the amounts of wild-type transcript and the exon 13-deleted transcript, we prepared various amounts (0–0.1 µg) of total RNA from wild-type mice, which were added to 1.0 µg of total RNA from Recql4-deficient mice, and used this RNA mixture as template for RT–PCR.

 
Here we generated Recql4-deficient mice as a model for RTS by inactivating the gene product through the deletion of a specific region in the helicase domain.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Generation of Recql4-deficient mice
We disrupted exon 13 of the Recql4 gene by replacing six base pairs (2045–2050) with a targeting vector (Fig. 1B). Exon 13 (180 bp) is the last RecQ helicase domain-coding exon whose length is a multiple of 3, and therefore can be deleted without shifting the reading frame. This exon encodes a domain that plays an essential role in helicase activity, and its deletion of 90 amino acids results in the removal of one-sixth of the helicase domain. Recql4 belongs to the helicase super family II (SFII) that includes seven conserved motifs, of which exon 13 encodes motif III (12,13). On the basis of an X-ray structural analysis of the MjDEAD putative RNA helicase, Story et al. (14) have suggested that the TAT (SAT) sequence in motif III plays a critical role in helicase activity through the hydrolysis of ATP, and its binding to templates (14). Others have shown that mutation of the SAT motif in mouse eIF4a (also part of SFII) inhibits its helicase activity (15), as does mutation of motif III of mouse Blm (16), and that mutation of TAT in the E. coli RecG helicase inhibits branch migration (17). All this suggests a crucial role for motif III in helicase activity.

The targeting vector was transfected into embryonic stem cell line R1 and the targeted cells were selected in G418 and ganciclovir followed with screening by Southern hybridization (18,19). We isolated 350 G418-resistant clones and obtained eight ES cell lines in which homologous recombination had occurred between the targeting vector and the mouse Recql4 locus. We checked for random integration of the targeting vector in these cell lines and found that one out of the eight contained a random integration in addition to the homologous-recombined integration. We also performed karyotype analysis and discarded two cell lines for which the chromosome abnormality was greater than 20%. After the injection of the remaining five ES cell lines into C57BL/6 blastocysts, we successfully obtained chimeric mice from three of them. Two of these lines, PC26 and PC107, were used for further analyses. Heterozygous F₁ +/- mice were intercrossed to obtain -/- mice. Southern hybridization and sequencing revealed that homologous recombination occurred precisely at both left and right arms (Fig. 1C). PCR, using primers targeted to the cDNA region encoding the RecQ-helicase domain (Fig. 1B), revealed the presence of an exon 13-deleted transcript in the primary mouse embryonic fibroblasts (MEFs) and testis of the mutant mice. We isolated the mRNA transcripts expressed in the -/- MEFs and the testis, and determined their cDNA sequences. The results indicated that an aberrant splicing occurred precisely between exons 12 and 14 at their original boundaries (Fig. 1D). Furthermore, a quantitative PCR estimated the expression level of mutant transcript in the testis of Recql4-deficient mice to be between 1 and 2% (Fig. 1E). We found no trace of the exon 13-deleted transcript in other tissues we investigated (brain, thymus, spleen or liver). In addition, we also detected a substantial number of short transcripts covering exon 1 to exon 12 that can code truncated products (data not shown). It is interesting that we succeeded in obtaining a Recql4 mutant mouse, while the knockout mice reported previously were lethal. This would suggest the existence of a critical region for development just before the helicase domain which is absent in the mice reported previously. Note also that all mutations identified in RTS patients have been within or after the helicase domain. (Fig. 1A)

Growth retardation of Recql4-deficient mice
Approximately 40% of the -/- newborn mice died just after birth and 80% of the remaining mice died within 2 days. Altogether, 95% of the mice died within 2 weeks. The body weight of -/- newborns was 60% of +/- and +/+ mice (Fig. 2A). The average body weight of newborns was 0.86±0.12, 1.51±0.21 and 1.48±0.17 g for -/-, +/- and +/+ mice, respectively (Fig. 2B). Growth retardation was also observed in the -/- adult mice. The average body weight of -/- adult mice at 4 and 10 weeks was approximately half and one-third that of wild mice, respectively (Fig. 2C and D). We followed the growth of the mutant mice over 2 months and found a significant difference in the growth rate among -/- mice in both sexes (data not shown). Growth retardation was also found in embryonic day 14.5 embryos (data not shown). To determine whether some exon 13-deleted Recql4-deficient mice are embryonic lethal, we performed genotyping of mice obtained by Caesarian section just before delivery and found the ratio +/+ : +/- : -/-=19 : 43 : 14, indicating no lethality of the -/- mice during their embryogenesis.

View larger version (31K): [in this window] [in a new window]   Figure 2. Growth retardation in Recql4-deficient mice. (A) Photograph of newborn Recql4-deficient mice. (+/+), (+/-) and (-/-) are littermates. (B) Average whole-body weight of newborn Recql4-deficient mice. (C) Photograph of 10 week old Recql4-deficient mice. (+/+) and (-/-) are littermates. (D) Average whole body weight of 4 week old Recql4-deficient mice. (E) Growth curves of MEFs derived from wild-type, heterozygous and Recql4-deficient mice. Two different MEF lines established from different Recql4-deficient mouse strains derived from the ES cell clones PC26 and PC107, were used for the analysis. Standard error is shown.

 
Next, using MEFs established from two mouse strains developed from the two different ES cell lines, PC26 and PC107, cell proliferation was determined. A significant decrease in cell proliferation was observed in the MEFs from the -/- mice, but not in the MEFs from +/- or +/+ mice (Fig. 2E). All the MEFs from the various genotypes exhibited reduction of growth after day 6, probably as a result of high cell density as the wild-type MEFs became confluent at day 9.

Skin abnormalities of Recql4-deficient mice
In order to know whether the Recql4-deficient mice represent the abnormal phenotypes observed in the RTS patients, we investigated skin of the mutant mice. The majority of the mutant mice exhibited several skin abnormalities. Most newborn mutant mice initially developed a normal coat, but spontaneous hair loss was subsequently noted on the neck, back and behind the forelegs around 6 weeks after birth. During this period, some of the mutant mice also showed colorless hair on the caudal and abdominal regions in addition to the neck and back (Fig. 3A and C). Although the extent of the hair loss varied, the lesions progressively developed and reached 20% of the whole body surface. Furthermore, the hairless skin became partially eroded in the course of the next few months, sometimes accompanied by slight bleeding (Fig. 3B). These lesions healed through crust formation, but without regrowth of hair. The lesions were found in areas of physical contact, suggesting that the skin of the mutant mice is brittle. Dry skin was most remarkable in the tail and was found in 60% of the mutant mice at 3–4 months of age (Fig. 3D). All skin samples were prepared from the same region of each mouse, the middle of the back. Histological analyses of the skin showed hypoplastic epidermis, dermis and subcutaneous tissue (Fig. 3F and H) when compared with control mice (Fig. 3E and G). The mutant mice also exhibited smaller dermal papilla, lower hair follicle density, and thinner layers of inner and outer root sheaths (Fig. 3H) as compared to control mice (Fig. 3G). No sign of inflammation (capillary dilatation, or perivascular infiltration in the dermis) was observed, however, which is inconsistent with the definition of poikiloderma, one of the most common symptoms of RTS (Fig. 3E–H).

View larger version (61K): [in this window] [in a new window]   Figure 3. Skin abnormalities observed in the Recql4-deficient mice. Most mutant mice showed (A) hair loss (17 weeks), (B) erosive lesions (22 weeks), (C) colorless hair (22 weeks), and (D) sclerotic appearance of the tail (33 weeks). (E, F, G and H) HE staining of skin sections of Recql4+/- controls (E, G) and Recql4-deficient mice (F, H) at 13 weeks of age. Hypoplastic alterations were marked at the epidermis (H), dermis and subcutaneous tissue (F). Hair follicles and dermal papillae were also small (H). E, epidermis. D, dermis. Sc, subcutaneous tissue. Hf, hair follicle. Dp, dermal papilla. Original magnification: x100 (E, F), x400 (G, H). Scale bars indicate 20 µm (E, F) and 5 µm (G, H).

 
Hypoplasia of several tissues
Histological examination of the bone revealed significantly fewer trabeculas in the metaphyseal regions (Fig. 4B) compared with control mice (Fig. 4A). The number and size of villi in the small intestine were smaller in the mutant mice (Fig. 4D) than in control mice (Fig. 4C). Fewer dividing cells in the crypts and connective tissue were observed in the mutant mice (data not shown). These findings suggest that the defect in Recql4 activity particularly affects actively proliferating intestinal cells.

View larger version (82K): [in this window] [in a new window]   Figure 4. Abnormalities in other tissues in the Recql4-deficient mice. HE staining of the bone at 15 weeks (A,B), small intestines (C,D), thymi (E,F) and spleen (G,H) at 4 weeks using control +/- mice (A, C, E, G) and Recql4-deficient mice (B, D, F, H). The mutant mice showed significantly fewer bone trabecula (B), smaller villi (D), poor cortical-medullary boundary in the thymus (F) and disappearance of the white pulp in the spleen (H). C, cortex. M, medulla. Wp, white pulp. Original magnification: x100 (A, B), x400 (C, D), x200 (E–H). Scale bar, 20 µm (A, B), 5 µm (C, D), 10 µm (E–H).

 
We also examined the histology of several lymphoid organs. The thymi from the six mutant mice were all disproportionately smaller. The cortical–medullary boundary in the thymi of the mutant mice was unclear (Fig. 4E and F). The spleens of the mutant mice were not disproportionately smaller, but the white pulp areas in the spleen were much smaller and drastically decreased in number (Fig. 4G and H). To determine whether lymphocyte development in the Recql4-deficient mice was abnormal, lymphocytes from the peripheral blood, thymus and spleen were analyzed by flow cytometry using monoclonal antibodies specific for cell surface markers. The ratio of CD4^-CD8^- : CD4⁺CD8⁺ : CD4⁺CD8^- : CD4^-CD8⁺ T cells was not statistically different between mutant and wild-type mice, as was the case for the ratio of IgM⁺ : IgM^- B cells (data not shown). This suggests that the shrunken thymi of the Recql4-deficient mice are not due to the aberrant development of lymphocytes during the stages that we investigated here, as in severe combined immunodeficiency (SCID) mice (20). However, several cases of RTS patients with frequent infections, impaired lymphocyte function, and decreased T lymphocyte and leukocyte counts have been reported (4), while distinct immunological dysfunction has not been observed in these patients. These symptoms resemble the phenotypes in the mutant mice. It is possible that an aberration in development occurs at some earlier stage. Further analyses including fetal liver adoptive transfer into normal mice may shed light on this issue. On the other hand, we cannot exclude the possibility that the shrunken thymi are not caused by the disruption of the Recql4 gene, because hypoplastic lymphoid is sometimes observed in runt mice.

We also evaluated the tooth phenotype of Recql4-deficient mice. Microscopic analysis revealed that both incisor and molar teeth in mutant mice were significantly smaller than those in wild-type littermates. Moreover the morphology of cusps in molar teeth was not well developed in mutant mice. Under X-ray examination we observed poor calcification of the molar crown in some mutant mice. Thus Recql4-deficient mice showed tooth dysgenesis (data not shown).

Normal sensitivity to IR and UV irradiation
Most RecQ helicase genes play a role in preserving the integrity of the genome, and mutation of these genes may induce high sensitivity to ionizing radiation (IR). Interestingly, cells from some RTS patients are sensitive to IR and cells from others are not (4,5). We assessed the sensitivity to ionizing and ultraviolet radiation of MEFs derived from the Recql4-deficient mice. No statistically significant difference in sensitivity to either form of radiation was found between three MEF lines (Table 1).

View this table: [in this window] [in a new window]   Table 1. Sensitivity to UV and IR in the MEFs from Recql4-deficient mice

 
Mouse model of RTS
We have summarized the phenotypes observed in RTS patients and our Recql4-deficient mice in Table 2. While no poikiloderma or visible cataracts were found in the Recql4-deficient mice, several other abnormalities observed in RTS patients were found as mentioned above. Knockout mice have previously been developed for two other RecQ helicase-related human syndromes, WRN (21,22) and BLM (23,24), but the Recql4-deficient mouse is the first to exhibit the marked appearance of premature aging commonly observed in humans with RecQ helicase deficiency.

View this table: [in this window] [in a new window]   Table 2. Comparison of reported symptoms of Rothmund–Thomson syndrome and Recql4-deficient mice

 
Some of the premature aging features of RTS exhibited by our mice include growth retardation, hypoplasia in various tissues, alopecia, and premature graying of fur. Yet these mice (observed for 8 months) do not exhibit poikiloderma, osteosarcoma or other malignancies, or bilateral cataract development. There are two possible explanations for the difference in phenotypes between RTS and the mutant mice: (1) the mutant mice express exon 13-deleted mRNA—the exons downstream of exon 13 in Recql4 are in-frame and expressed; these downstream exons are not in general expressed in human mutation of RECQL4; and (2) sometimes orthologous genes produce different phenotypes in mice and humans. Preparation and analysis of a knockout mouse instead of the exon 13-deleted mouse might resolve this question.

Our study provides significant new evidence that the Recql4 gene is linked to RTS, and we believe that these mice will contribute to the understanding of the role of Recql4 and as an animal model for RTS.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Gene targeting of Recql4
Our targeting vector consists of the left arm of the 4.3 kb genomic region containing exons 1–13 and the right arm of the 1.9 kb region containing exons 13–20 of the mouse Recql4 gene (Fig. 1B). The genomic regions were amplified by high-fidelity and long-range PCR using KOD Plus DNA polymerase (Toyobo, Osaka, Japan). The targeting vector was transfected into R1 cells (18) by means of electroporation, and clones resistant to G418 and ganciclovir were selected and forwarded for further screening by Southern hybridization (19). Genomic DNA digested with restriction enzyme XbaI was transferred to a nylon membrane and hybridized with a fluorescence-labeled probe corresponding to the 0.6 kb region containing exons 21 and 22 (labeled ‘3' probe’ in Fig. 1B). Recql4 +/- ES clones were injected into C57BL/6 blastocysts using a microinjector (Narishige International Inc., Tokyo, Japan) equipped with a piezo-micromanipulator system (Prime Tech Ltd, Ibaraki, Japan). We bred chimeric (over 80%) agouti males with C57BL/6 females and established germline transmission. Genotyping using tails was performed by means of PCR using the following primers, mQ4-5 (+) 30 (5'-CTCGTGGTCTCGCCTCTCCTGTCACTCATG-3'), mQ4-6(-)30 (5'-GCCCACCATGGACAGGCAGGTGCGGAGGAG-3') and pgkNeo5'-1(-)30 (5'-CTTGGGAAAAGCGCCTCCCCTACCCGGTAG-3'). The Recql4 +/- F₁ mice were intercrossed to produce homozygous -/- mice, or crossed with C57BL/6 or littermate +/+ mice to produce a large number of +/- mice (F₂). The experimental design was approved by the animal use committee of the National Institute of Radiological Sciences. PCR primers are available upon request.

Quantitative PCR for the Recql4 transcripts
Total RNA was prepared from adult tissues (brain, heart, thymus, kidney and testis), and MEFs of wild-type and Recql4^-/- mice. RT–PCR was performed using the following primers: mQ4-5(+)30 and mQ4-8(-)30 (5'-CAGCTGGGCACTGCCGCCAAGGCAATGCAG-3'). In the Recql4^-/- mice, no wild type Recql4 transcript was detected in any of these tissues. MEFs and testis of Recql4^-/- mice, however, expressed mutated transcripts. PCR products were cloned into pGEM-T Easy (Promega, Madison, WI, USA) and their sequences were determined. To examine mutant transcripts, sequencing was performed with the synthetic primers 5'-CTGCCTCTCTCAGTGGTCAC-3' and 5'-GACAGGCAGGTGCGGAGGAG-3'. To assess the expression level of exon 13-deleted transcripts in the mutant mice, we performed competitive PCR in which various amounts of total cDNAs from wild type mice testis were combined with 1 µg of total RNA from the mutant mice in each reaction. The fraction of both cDNA samples was normalized by the amount of Gapdh transcript in each sample. Real-time PCR was carried out according to the procedure recommended by the manufacturer.

Histology
Mice were sacrificed to perform histological studies. Briefly, tissues were fixed in 10% buffered formalin, embedded in paraffin blocks and sectioned. Staining was performed with hematoxylin and eosin (HE). The sections were examined and photographed using a microscope, ECLIPSE TE300 (Nikon, Tokyo, Japan) with 40x, 100x and 400x magnification.

Cytological assays
MEFs were propagated from embryonic day 14.5 embryos. In brief, MEFs were isolated and cultured in Dulbecco's modified Eagle's medium (Sigma, St Louis, MO, USA) supplemented with 10% fetal bovine serum, 50 µU/ml of penicillin, 50 µg/ml of streptomycin and 58 µM 2-mercaptoethanol on a 100 mm dish at 37°C and 5% CO₂. For the cell growth assay, 1.0x10⁵ cells were plated onto a 60 mm dish in duplicate. The culture medium was changed every 2 days and cells were counted every 24 h using a Coulter counter (Beckman Coulter, Fullerton, CA, USA). Sensitivity to IR or UV light was determined by a colony formation assay according to a previous report (25). Briefly, cells (1x10³–2x10⁴ cells per dish) were inoculated in various densities onto 100 mm gelatin-coated dishes containing growth medium and incubated 16 h at 37°C in 5% CO₂. After exposure to X-rays (PANTAK HF320, Shimadzu, Kyoto, Japan) or UV (254 nm, GL10 lamp, Toshiba, Tokyo, Japan), cells were cultured for 2 weeks in a complete medium containing 10 mM HEPES in addition to the ingredients described above. Colonies were fixed and stained for counting with 0.5% crystal violet. To assess plating efficiency, 500 cells were plated on a dish and cells were counted 2 weeks later.

	ACKNOWLEDGEMENTS

 
We would like to thank A. Nagy for providing us with the R1 ES cell line, T. Hirobe, K. Yamamoto, A. Hatamochi, Y. Ohyama, A. Nifuji, N. Shibata and Y. Matsuda for helpful discussions and suggestions on histological studies, Y. Nagasaka and H. Suzuki for technical assistance and helpful discussions on the FACS analysis, N. Miyamoto, A. Kurimasa, Y. Takada, T. Saito, T. Hayao and T. Isobe for helpful suggestions on preparing knockout mice and J. Rodrigue for editing assistance during the preparation of this manuscript.

	FOOTNOTES

 
^* To whom correspondence should be addressed at: National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan. Tel: +81 432063219; Fax: +81 432514593; Email: abemasum{at}nirs.go.jp

The authors wish it to be known that, in their opinion, the first four authors should be regarded as joint First Authors.

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