|Home | About | Journals | Submit | Contact Us | Français|
We identified a novel fertile, autosomal recessive mutation, called peewee and that results in dwarfing, in a region-specific ENU-induced mutagenesis. These mice at litter size were smaller those of other strains. Histological analysis revealed that the major organs appear normal, but abnormalities in cellular proliferation were observed in bone, liver and testis. Haplotype analysis localized the peewee gene to a 3.3-Mb region between D5Mit83 and D5Mit356.3. There are 18 genes in this linkage area, and we also performed in silico mapping using the PosMed program, which searches for connections among keywords and genes in an interval, but no similar phenotype descriptions were found for these genes. In the peewee mutant compared to the normal, C57BL/6J mouse, only Slc10a4 expression was lower. Our preliminary mutation analysis examining the nucleotide sequence of three exons, two introns and an untranslated region of Slc10a4 did not find any sequence difference between the peewee mouse and the C57BL/6J mouse. Detailed analysis of peewee mice might provide novel molecular insights into the complex mechanisms regulating body growth.
Growth-retarded mice provide a valuable model system with which to elucidate the molecular mechanisms that interplay among growth, body size and genetic influences. Some spontaneous genetic mutations in mice cause dwarfism by altering endocrine systems, such as Snell dwarf (dw), Ames dwarf (df), and little (lit) mice (Bartke 1964; Eicher and Beamer 1976; Schaiber and Gowen 1961; Snell 1929). These mutant models were important for elucidating the mechanisms of growth hormone regulation and the role of transcription factors such as Prop1 (Sornson et al. 1996) and Pit1 (Li et al. 1990) as regulators underlying neuroendocrine axis activation and organization. Genetic mutations in these two factors cause defects in anterior pituitary cell types, resulting in poor or no production of GH, prolactin, and TSH, but normal basal levels of FSH and LH (Bartke 1964, 1965; Cheng et al. 1983; Sinha et al. 1975; van Buul-Offers 1983). However, dwarfism is not limited to disorders of the pituitary gland and hypothalamus. Besides central endocrine mechanisms, peripheral defects retard growth, as seen in hypothyroid (hyt), congenital goiter (cog), growth-retarded (grt), thyroid peroixdase (tpo), Pax8, and discoidin domain receptor 2 (slie) homozygous mutant mice (Beamer et al. 1987; Beamer et al. 1981; Flamant et al. 2002; Kano et al. 2008; Takabayashi et al. 2006; Yoshida et al. 1994).
Mice homozygous for dwarf mutations often manifest infertility (Bartke 2000). The infertility in Snell dwarf mice, for example, appears to be due to gonadal dysfunction arising from a lack of neuro-endocrine axis activation in either sex (Bartke 2000; Bartke and Lloyd 1970; Smith and MacDowell 1931).
In mice, forward genetic screens are powerful tools with which to identify biological pathways and the genetic components of complex phenotypes. A program was conducted to mutagenize the mouse genome with ENU and to specifically recover mutations in the region spanned by the rump-white (Rw) inversion on Chr 5 (Wilson et al. 2005). Rw is a radiation-induced mutation causing depigmentation of the posterior and ventral abdomen in heterozygotes and embryonic lethality in homozygotes (Stephenson et al. 1994). We discovered a novel ENU-induced mutant mouse with fertility defects and dwarfing, peewee.
In this study, we characterize and map the peewee mutant. Detailed analysis of peewee mice might provide novel molecular insights into the complex mechanism of body growth.
The peewee mutation was generated through the ENU mouse mutagenesis project by Dr. Simon John (The Jackson Laboratory). The peewee mice were maintained on a mixed genetic background of C57BL/6J (including the Rump white (Rw) inversion) and C3H/HeJ (Schimenti and Bucan 1998; Wilson et al. 2005). Homozygous mice were obtained by intercrossing homozygous peewee mice. The peewee mice will be available at the Bio Resource Center of RIKEN, Japan.
All mice used in this study were bred and maintained in vivariums at either the University of Tokyo or The Jackson Laboratory. Their respective institutional Animal Care and Use Committees approved all procedures in these studies. A minimum of three mice were analyzed at each time point.
Blood chemical anaysis was conducted in Rodents Multi-Analyte Profiles by Rules-Based Medicine, Inc., (Austin, TX).
As previously reported, peewee was never observed to recombine in the Rw inversion, which spans the ~27- to 74-Mb interval on chromosome 5. To reduce the critical region, a mating cross of Balb/cCrSlc × peewee was established to generate F1 offspring. The heterozygous F1 mice, which do not exhibit dwarfism, were intercrossed for the initial linkage mapping study. The resultant F2 progeny mice were weighed at 10 weeks of age to distinguish affected animals. Genomic DNA from F2 offspring was analyzed using microsatellite markers to construct a genetic map. Microsatellite marker primer pairs for the genome scan were purchased from Research Genetics; Invitrogen Life Technologies, Inc. (Carlsbad, CA). For genomic PCR amplification, 25 ng of tail DNA was used in a 10 μl volume containing 50 mM KCl, 10 mM Tris-Cl pH 8.3, 2.5 mM MgCl2, 0.2 mM oligonucleotides, 200 μM dNTP, and 0.02 U BIOTAQ DNA polymerase (Bioline Inc., Randolph, MA). The cycling conditions were 95°C for 2 min, followed by 49 cycles at 94°C, 20 sec; 50°C, 20 sec; 72°C, 30 sec; and a 7 min extension at 72°C. PCR products were separated by electrophoresis on a 4% MetaPhor agarose gel (FMC, Rockland, ME) and visualized by ethidium bromide staining.
Mutation analysis was performed by direct sequencing of genomic DNA and cDNA using primers that were designed flanking the entire genomic region of Slc10a4 and the cDNA of Tec. Genomic PCR and RT-PCR were performed using primers spanning the coding regions of each gene within the peewee critical interval (Supplemental Table 1). For genomic PCR and RT-PCR, 25 ng of genomic DNA or cDNA was used in a 20 μl volume containing 0.5 μM oligonucleotides, 200 μM dNTP, and 0.4 U Phusion DNA polymerase (Finnzymes OY, Espoo, Finland). The cycling conditions were 98°C for 30 sec, followed by 35 cycles at 98°C, 10 sec; 58°C, 30 sec; 72°C, 30 sec; and a 7 min extension at 72°C. The appropriate bands were excised. DNA was recovered using the NucleoSpin Extract II (Macherey-Nagel GmbH & Co., KG, Düren, Germany) per the manufacturer's instructions. Purified genomic PCR and RT-PCR products were sequenced using the 3130 Genetic Analyzer (Applied Biosystems Japan, Tokyo, Japan).
Bone samples were collected from wild-type and peewee mice at 2 and 10 weeks of age (n>3 per sample and age). Bone was decalcified and fixed in Bouin's reagent overnight. Samples were dehydrated, embedded in paraffin, cut into 5 μm sections, and processed for hematoxylin and eosin, TUNEL, or immunohistochemical staining.
To localize PCNA in peewee and wild-type homozygotes, polyclonal antibodies to PCNA were used (sc-56; Santa Cruz Biotechnology Inc., Santa Cruz, CA). Following deparaffinization, the sections were boiled in sodium citrate buffer (pH 6.0) for 5 min in a microwave oven for antigen retrieval. The sections were pre-incubated with 5% normal goat serum for 30 min, followed by 2 hr incubation with the primary antibody (1:500), then with biotinylated secondary antibody (1:400; Vector Laboratories, Burlingame, CA) for 1 hr, and next with streptavidin-biotin-HRP complex (Vectastain Elite ABC, Vector Laboratories) for 30 min. As a negative control, sections without the primary antibody were processed (data not shown). All the reactions were carried out at room temperature. Terminal deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining was performed according to the manufacturer's protocol (DeadEnd Colorimetric TUNEL System; Promega, Madison, WI). PCNA- and TUNEL-positive cells were counted across each tissue on sections in three wild-type and three peewee mice. PCNA staining was quantitated by counting 500 cells each in random sections.
For semi-quantitative RT-PCR, total RNAs were isolated from brain, thymus, lung, heart, spleen, intestine, ovary and eye of wild-type and peewee mutant mice using TRIzol reagent (Invitrogen). cDNA was synthesized from total RNA with the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen). Table 1 lists the primers used to detect Gabrg1, Gabra2, Cox7b2, Gabra4, Gabrb1, Commd8, Atp10d, Corin, Nfxl1, Zar1, EG545758, Cnga1, Npal1, Txk, Tec, Slain2, Slc10a4, Fryl and housekeeping gene Gapdh (also called G3PDH). PCR was performed using AccuPrime Pfx DNA Polymerase (Invitrogen). The amplification conditions used were 98°C, 30 s followed by 15-25 cycles of 98°C, 10 s, 58°C, 30 s; 72°C, 30 s. The number of cycles used ensured that the reaction could be quantified within the log phase of the amplification reaction.
In silico positional cloning was carried out using PosMed, developed by RIKEN (http://omicspace.riken.jp/PosMed/). It provides a bioinformatics approach to find the most promising candidate genes in a candidate interval restricted by genetic mapping analysis.
Descriptive statistics (mean and SEM) were calculated for the body weight of peewee and C57BL/6J mice, the number of PCNA- and TUNEL- positive cells, and the expression of Slc10a4 in the tissues of wild-type and peewee mutant mice. Mann-Whitney tests were used to detect significant statistical differences among the experimental groups. Differences were considered significant at P<0.05 (StatView, SAS Institute, Cary, NC).
Peewee is an autosomal recessive mutation, and is induced by ENU mutagenesis directed at the Rw inversion region of proximal mouse chromosome 5. At birth, peewee mutants are indistinguishable from wild-type mice, and peewee mutant mice have no abnormalities other than their stature. Their weight gain is slower and they lack the juvenile growth spurt observed in wild-type littermates after weaning (Fig. 1). The litter size was significantly smaller compared with other strains (Supplementary Fig. 1).
To investigate the physiological and morphological consequences of the peewee mutant, the major organs of homozygous wild-type and peewee mutant mice were compared at 10 weeks of age. No gross morphological abnormalities were observed in the organs and tissues examined (Supplemental Fig. 2). Plasma analysis showed that EGF, INF-δ, IL-α, Apo-A1, leptin, IL-6 and OSM levels were significantly decreased, and fibrinogen, myoglobin, TNF-α, VEGF, MIP-1α, MDC and IL-10 levels were significantly increased in peewee mutant mice (Supplementary Table 2).
As indicated by the plasma analysis, some cell growth factors were altered in the peewee mutant, which could promote cellular proliferation or cell death. In order to determine whether a greater incidence of cellular proliferation or apoptosis occurs in peewee mutants, we performed immunohistochemical analysis labeling with PCNA and TUNEL assays in juvenile (2-week-old) wild-type and peewee mutant mice in proliferative tissue. Similar levels of PCNA positive staining were present in thymus (Fig. 2A and B), lung (Fig. 2 C and D) and small intestine (Fig.2 G and H) of wild-type and peewee mutants. However, more significant numbers of positive cells were observed in chondrocytes of bone (Fig. 2 K and L) and fewer positive cells in hepatocyte of liver (Fig. 2 E and F) and spermatogonia of testis (Fig. 2 I and J) of peewee mutant compared to wild-type. The altered ratio of PCNA- positive cells between wild-type and peewee mutant mice at 2 weeks of age indicates a tissue-specific imbalance of proliferation (Fig. 2M). At 2 weeks of age, similar levels of TUNEL-positive cell staining were present in liver, testis, and bone of wild-type and peewee mutants (Fig. 3).
We initially scanned the Rw inversion region using four microsatellite markers at approximately 50 cM intervals (Fig. 4A). Initial genome scanning indicated significant linkage between D5Mit232 and D5Mit356.3 (Fig. 4A). A high-resolution genetic map of the region was constructed using the flanking markers D5Mit83 and D5Mit356.3 on 492 F2 progeny from a peewee homozygote × Balb/cCrSlc) F1 intercross and their associated phenotype (Fig. 4B). The fine structure genetic map encompassed an estimated 3.3 Mb physical region on Chromosome 5 containing the peewee mutation. According to annotations in the M. musculus genome assembly version 37 (http://www.ensembl.org), the peewee critical region contained 18 gene loci.
Eighteen candidate genes were identified within the peewee critical region (Table 2). We performed in silico positional cloning using PosMed program, which searches for connections among keywords and genes in an interval. We entered a keyword, dwarf, in the PosMed and identified five candidate genes--Gabra2, Gabra4, Corin, Txk and Tec—that are related to the peewee phenotype in the critical region (Table 2).
Six of the candidate genes (Gabra2, Gabra4, Corin, Zar1, Txk and Tec) in the peewee critical region have already been knocked out in mice, and have not exhibited the dwarfism phenotype observed in peeweee mice. To analyze the expression levels of these candidate genes, we performed semi-quantitative RT-PCR on cDNA of tissue highly expressed for individual genes from both wild-type and peewee homozygotes (Fig. 5A). Among the 15 candidate genes, the levels of Slc10a4 gene expression were relatively low in peewee mutants, and no significant expression differences were observed in any of the other genes, including the genes picked out by PosMed (Fig. 5A). The expression level of Slc10a4 mRNA significantly differed between peewee and wild-type mice, with G3PDH used as an internal control (Fig. 5B).
We examined the genomic sequence of Slc10a4 in peewee and wild-type mice. No nucleotide sequence differences were observed in the genomic DNA of the peewee mice compared to C57BL/6J in 7 kb of Slc10a4 genomic DNA, which included 162 nucleotides in the 5’ and 1096 nucleotides in the 3’- untranslated region.
In this study, we determined the morphogenic and genetic defects associated with a new fertile dwarf mutant mouse, peewee. Mutant mice harboring the peewee mutation showed normal appearance and histology, but we observed altered cellular proliferation in bone, liver and testis. We identified a possible candidate gene for peewee mutation in the Rw region, but did not observe a nucleotide alteration in the gene.
Most mice homozygous for dwarf mutations are a result of reproductive disfunction (Bartke 2000). For example, infertility in the Snell dwarf mice appears to be due to gonadal dysfunction arising from a lack of neuro-endocrine axis activation (Bartke 2000; Bartke and Lloyd 1970; Smith and MacDowell 1931). It has been proposed that decreased GH levels lead directly to diminished circulating insulin and IGF-1, both of which are necessary for normal body size and for aging in Snell, as well as in Ames dwarf mice (Bartke and Brown-Borg 2004; Bartke et al. 1998; Brown-Borg et al. 1996; Flurkey et al. 2002; Flurkey et al. 2001; Kemp 1938; Sornson et al. 1996). Deficiency of pituitary gland function is a direct consequence of a deficiency of growth hormone in many dwarf mice. In Smallie mice, the absence of discoidin domain receptor 2 leads to growth retardation and gonadal dysfunction due to peripheral defects in hormonal-responsive pathways (Kano et al. 2008).
The peewee mutant was generated by a region-specific ENU-induced mutagenesis, which is a powerful and efficient phenotype-driven approach to define gene function relevant to phenotype and disease. Unlike other dwarf mice, the peewee mutants are fertile, although they have smaller litters compared to most inbred and F1 mice. Histological examination of various tissues from peewee mice did not reveal any gross abnormalities. These results confirm that the peripheral reproductive organs and central endocrine mechanisms were normal. The normality of these tissues reveals that the smallness of the litters might not be due to deficiencies in the reproductive system of peewee mice. The small litters could, however, be attributed to their small body size; it is well known that relatively small inbred mice have fewer pups (Mouse Phenome Database (MPD); http://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home).
Plasma analysis to detect alteration in blood chemistry identified apoptotic and cell proliferative factors that could affect growth. To further examine these findings, we performed immunohistochemical analyses using PCNA (to examine proliferation), and TUNEL assay (to examine apoptosis) in the proliferative tissue of juvenile wild-type and peewee mutant mice. In chondrocytes, more proliferative cells were observed in peewee mutant mice than in wild-type mice. Endochondral bone formation has some important processes, including proliferation of chondrocytes (Kronenberg 2003). Further analysis is necessary, but the increased proliferation of chondrocytes could be caused by abnormalities of other processes, such as hypertrophicity of endochondral bone formation, in peewee juvenile mutant mice. Alternatively, repressive factors might be activated in the peewee condrocytes and suppress the proliferation of bone growth. Additionally, low proliferlative activity in the cells of liver and testis and equal levels of apoptosis in various tissues of peewee mutant mice might reflect that the growth retardation of the peewee mutant results from the complete eliminationtotal decreasing of cellular proliferative activity.
Due to the region-specific targeted ENU mutagenesis, we easily refined the critical region of peewee to distal to the Rw region on chromosome 5. We mapped the peewee locus to a region between D5Mit232 and D5Mit356.3 on the Rw region of chromosome 5 by standard linkage analysis and refined the peewee mutation to a 3.3 Mb interval between markers D5Mit83 and D5Mit356.3. Bioinformatic methodologies were employed by entering the keyword “dwarf” to PosMed™ and by performing a mutant search of the MGI (http://www.informatics.jax.org/) database. We finally idenfied five candidate genes--Gabra2, Gabra4, Corin, Txk and Tec--by PosMed™. Gabra2 and Gabra4 are members of the GABA-A receptor family, and both of these knockout mice mainly show phenotypes of behavior (Knabl et al. 2008; Chandra et al. 2006). Corin is highly expressed in the heart and is the pro-ANP-converting enzyme. Corin-mediated pro-ANP activation may play a role in regulating blood pressure (Yan et al. 2000). Mouse Tec is a nonreceptor-type protein-tyrosine kinase that is highly expressed in many hematopoietic cell lines (Sato et al. 1994). Tec is involved in immune system and bone homeostasis in signaling molecules shared by B cells and osteoclasts (Shinohara et al. 2008). Txk is a member of the Tec subfamily of SRC-type (nonreceptor) tyrosine kinases. Txk expression is mainly detected in T cells and some myeloid cell lines. In this study, none of the candidate genes identified by PosMed™ could be verified, because no similar phenotypes and descriptions were found for these candidate genes. However, if functions and descriptions of candidate genes are well analyzed in the future, PosMed™ should be useful for analyzing relationships between phenotypes and genes, enabling the easy identification of potent candidate genes.
Gene expression of the candidate genes in the peewee critical region was analyzed. The expression of one gene, Slc10a4, a solute carrier, was lower in peewee mice than in wild-type mice both in both brain and pituitary glands. Solute carrier (SLC) families comprise passive transporters, ion-coupled symporters, and antiporters in the plasma membrane and other cellular membrane compartments, and each SLC family is thought to have relationships to physiological, pathological, pathophysiological and pharmacological functions (Hediger et al. 2004). SLC10A4, is an orphan transporter with ubiquitous expression, with high expression in the nervous tissue and pituitary gland (GNF SymAtlas v1.2.4). While SLC10A4 is a solute carrier or transporter of other physiological molecules, it does not appear to transport bile acids (Geyer et al. 2006). Thus far, there is no evidence or reports that associates SLC10A4 function with cell proliferation or other defects in mice, but mutations in another solute carrier, SLC7A7, a member of the SLC family, causes fetal growth retardation by downregulating Igf-1 in the mouse (Sperandeo et al. 2007). In our preliminary experiment, Northern blot analysis indicated that liver Igf-1 mRNA levels did not differ between peewee and wild-type mice (data not shown). This indicates that dwarfism in peewee mice might not be due to hormonal control such as growth hormone or Igf-1 regulation through an SLC10A4 transporter like SLC7A7.
Using recombination mapping, we narrowed the peewee genetic interval to less than 3.3 Mb, but did not find any mutations in a candidate gene, Slc10a4. It is possible that the mutation is in a regulatory element that affects the expression of Slc10a4 outside the genetically defined critical region. The mutation may be in nontranslated transcript elements such as a microRNA. However, there are no such known elements in this region. Thus, DNA sequencing of the entire critical region may be required to identify the peewee mutation. Although Slc10a4 appears to be a good candidate gene with different expression between wild-type and peewee mice, we should consider another possibility that a point mutation in peewee mice causes functional loss of the protein. Transgenic rescue to each candidate gene might provide new evidence about the relationships between the phenotype and the responsible gene in peewee mice. Detailed analysis of the peewee phenotypes and identification of the mutation may provide molecular insights into the complex mechanism of body size and growth regulation.
The litter sizes of peewee mice and inbred line.
Cross sections of various tissues from peewee mutant, stained with hematoxylin and eosin, at 10 weeks of age. Brain (A), pituitary gland (B), eye (C), heart (D), lung (E), spleen (F), skin (G), skeletal muscle (H), pancreas (I), liver (J), stomach (K), intestine (L), kidney (M), uterus (N), ovary (O) and testis (P). Scale bar, 25 μm (C, D, E), 100 μm (F, G, H, J, K, L), 200 μm (A, B, C, M, N, O, P, Q).
Multple analyte profiles in blood of C57BL/6L and peewee
We would like to thank Dr. Neena Haider and Arne Nystuen for their critiques of the manuscript. This research was supported in part by Grants-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Morinaga Foundation, the Foundation for Growth Science and the US National Institute of Health (DK46977 and DK73267).