The squiggle tail (squig) mutation in mice is associated with a deletion in the mesenchyme homeobox 1 (Meox1) gene
BMC Research Notes volume 15, Article number: 305 (2022)
We have taken a positional approach to assign the spontaneous squiggle tail (squig) mutation in mice to a specific gene defect.
A large panel of backcross mice was produced and characterized to map squig to high genetic resolution on mouse Chromosome (Chr) 11. Two overlapping candidate genes that co-localized with squig (Meox1, for mesenchyme homeobox 1; and Gm11551, which encodes a lncRNA located entirely within the first intron of Meox1) were fully sequenced to discover any squig-specific defects. This analysis revealed a 3195 bp deletion that includes all of Meox1, Exon 1 but does not disrupt Gm11551. We recommend that the squig mutation be renamed Meox1squig, and suggest that this variant may offer an appropriate animal model for Klippel-Feil syndrome 2 (KFS2) in humans.
The recessive squiggle tail mutation (abbreviated squig) arose spontaneously in the BALB/cJ inbred mouse strain at The Jackson Laboratory (Bar Harbor, ME, USA) in 2013, and has been maintained on a segregating, coisogenic background since that time. Mice homozygous for squig display a shortened and very curly tail and are frequently smaller than their non-mutant littermates (see Additional file 1: Figure S1). In 2016, Karst et al.  mapped squig to mouse Chromosome (Chr) 11, based on the analysis of single nucleotide polymorphisms (SNPs) among a small set of F2 homozygotes, but the genetic resolution achieved was not sufficient to suggest any causative gene.
As a basis for assigning the squiggle tail phenotype to a specific genetic cause—which would facilitate the further analysis of this interesting variant and help to identify an orthologous human disorder—here we have fine-mapped squig with respect to various microsatellite and SNP markers on mouse Chr 11. This analysis identified a small set of co-localizing candidate genes, and we now suggest that one of these, Meox1 (for mesenchyme homeobox 1), harbors the squig defect.
Mice from the standard inbred strains C57BL/6 J (JAX stock #000664) and BALB/cJ (JAX stock #000651), and co-isogenic BALB/cJ-squig/GrsrJ mice (JAX stock #026620) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The squig mutation was maintained at CCSU by crossing heterozygotes with mutant homozygotes. Mutants were reliably identified (with at least 99.3% penetrance in our colony) by their short and curly tails that are apparent from birth (see Additional file 1: Figure S1). At the end of the study, mice were killed by cervical dislocation or by use of CO2 gas added to a chamber (typically their home cage) using a compressed gas cylinder fitted with a flow meter adjusted to displace only 30–70% of the chamber volume per minute (consistent with the recommendations of the AVMA Guidelines for the Euthanasia of Animals, 2020 Edition). Only the P.I. (TRK) who was trained at The Jackson Laboratory (Bar Harbor, ME) and has over 30 years of experience, performed euthanasia.
DNA isolation and marker typing
Genomic DNA was isolated from 2 mm tail-tip biopsies taken from two- to three-week-old mice using NucleoSpin® Tissue kits (Macherey–Nagel, Düren, Germany; distributed by Clontech Laboratories, Inc., Mountain View, CA, USA), as directed. DNA samples from standard inbred and mutant strains that we do not routinely maintain in our colony were purchased from The Jackson Laboratory’s Mouse DNA Resource.
The polymerase chain reaction (PCR) was performed in 13 µl reactions using the Titanium® PCR kit from Clontech Laboratories, as directed. Oligonucleotide primers for PCR were designed and synthesized by Integrated DNA Technologies, Inc. (Coralville, IA, USA), based on sequence information available online [2, 3]. To score PCR product sizes for dimorphic microsatellite markers, reactions plus 3 µl loading buffer were electrophoresed through 3.5% NuSieve® agarose (Lonza, Rockland, ME, USA) gels. Gels were stained with ethidium bromide and photographed under ultraviolet light. In addition to eight standard microsatellite markers  on Chr 11, eight DNA markers based on single nucleotide polymorphisms previously reported to differ between strains BALB/cJ and C57BL/6 J [2, 3] were scored. These markers (herein designated SNP#) are described in detail in Additional file 2: Table S1 and Additional file 3: Table S2.
For DNA sequence analysis, about 1.5 µg of individual PCR amplimers were purified and concentrated into a 30 µl volume using NucleoSpin® PCR Clean-up kits, and then shipped to the Keck Foundation Resource Laboratory at Yale University (New Haven, CT, USA) for primer-extension analysis.
A “3-primer” test for detecting Meox1squig alleles
To rapidly determine genotypes at the squig locus (especially among phenotypically wild type mice) we used a standard PCR assay that employed three primers: a single forward primer (F1, 5’-GTTACCAGGAGGTGCTCAAA-3’) that annealed 5’ to the Meox1 deletion and two reverse primers—one that annealed within the Meox1 deletion (R1, 5’-GTGAAATGTGAGAGAGGAGAGG-3') and one that annealed 3’ to the deletion, within Gm11551, Exon 2 (R2, 5’-CCAGATCCCAGCAATCAAGATA-3'). Primers F1 and R1 direct the amplification of a 268 bp product specific to wild type BALB/cJ templates; the F1, R2 primer pair direct the amplification of a 456 bp product specific to squig templates.
To genetically map the squig mutation, F1 heterozygotes (made by crossing BALB/c-squig/squig mice with standard C57BL/6 J mice) were crossed with squig/squig mutants, producing 1008 backcross (N2) offspring that segregated for alternative alleles of squig and numerous molecular markers. Guided by Karst’s previous mapping efforts , DNAs isolated from this N2 panel were typed for eight, PCR-scorable microsatellite (dinucleotide repeat) markers  known to map to distal Chr 11. Additional file 4: Figure S2 shows the string of markers transmitted by the F1 parent to each of these 1008 N2 progeny. This haplotype analysis suggested that the squig gene must be located within the 3.2 cM region between D11Mit59 and D11Mit360 (Fig. 1A). This genomic interval includes Rpl27 (for ribosomal protein L27), and, because defects in the related genes Rpl24 and Rpl38 have been shown to cause tail abnormalities in mice [5, 6] respectively, we investigated Rpl27 as the potential basis of squig. However, DNA sequence analysis of all exons of Rpl27 in squig mutants (data not shown) revealed no defects compared to wild type.
DNA samples from the 32 mice identified as having a crossover between D11Mit59 and D11Mit360 were typed next for eight SNPs known to lie in that 3.24 Mb interval. These eight SNP markers are described in detail in Additional file 2: Table S1 and Additional file 3: Table S2 and are designated herein as SNP#. This analysis located six crossovers that fell centromeric to squig (between SNP10 and SNP6), and one crossover that fell distal to squig (between SNP13 and SNP14) (see Fig. 1B), thus restricting the location of squig between SNP10 and SNP14 (and very near SNP6 and SNP13, which were not meiotically separated from each other or from squig).
The 0.6 Mb span from SNP10 to SNP14 includes 18 expressed genes (Fig. 1C) (which, incidentally, do not include Rpl27). While four of these genes (Arl4d, Sost, Dusp3 and Cd300lg) have been associated with abnormal bone morphology or mineralization  the homeodomain-containing transcription factor gene, Meox1 (for mesenchyme homeobox 1), became our primary gene candidate due to its well-established role in axial skeleton formation [7,8,9,10]. DNA sequence analysis of all Meox1 exons in wild type BALB/cJ and BALB/c-squig/squig genomic DNA revealed a 3195 bp deletion that extends from the Meox1 promoter region to include all of Exon 1 and part of Intron 1–2 (Fig. 2). The deletion does not extend as far as predicted gene Gm11551  which encodes a lncRNA that is entirely contained within Intron1-2 of Meox1 and is transcribed from the complementary DNA strand (see Fig. 1D).
Next, we used a “3-primer” PCR test (see Additional file 5: Figure S3A) to rapidly screen for the presence of the squig-associated deletion of Meox1, Exon 1 among 28 standard mouse strains but found no Meox1, Exon 1 defect in any of them (see Additional file 5: Figures S3B and Additional file 6: S4), suggesting that the 3195 bp deletion is specific to the squig mutation.
Because Meox1 and squig map to the same small region on Chr 11, because squig mutants display a severe, specific defect in Meox1, and because engineered mutations in Meox1 produce similar recessive vertebral anomalies [including hemivertebrae, tail kinks and craniovertebral fusions, see [8, 9]] we suggest that squig is a spontaneous mutant allele of Meox1 and recommend that its official designation be changed to Meox1squig.
At least four independent defects in the human MEOX1 orthologue [13,14,15] have been associated with Klippel-Feil syndrome-2 (KFS2), an autosomal recessive condition characterized by a short neck, low occipital hairline and reduced bilateral neck movements resulting from the fusion of cervical vertebrae . Especially because the previously-described mouse variants [8, 9] are no longer extant, we suggest that the Meox1squig variant described herein (available from The Jackson Laboratory as JAX stock #026620) could provide a highly relevant animal model for this inherited human disorder.
Rigorous proof for this gene assignment would require rescue of the mutant phenotype by the single addition of a wild type Meox1 allele, or complementation testing with an extant, engineered Meox1 mutation, for example. These formal tests were not performed here. The location of the antisense lncRNA Gm11551 within the first intron of mouse Meox1 may suggest a cis regulatory relationship  that warrants further investigation, although it is notable that the human MEOX1 orthologue does not harbor a similar lncRNA gene. While the Meox1squig deletion does not disrupt the Gm11551 coding sequence, we did not verify the normal expression or processing of Gm11551 RNA.
Availability of data and materials
All data supporting the results of this article are included in this article and its additional files. Any materials or databases generated in this study are available from the corresponding author upon reasonable request.
Long, non-coding RNA
Single nucleotide polymorphism
Klippel-Feil syndrome 2
- Meox1 :
Mesenchyme homeobox 1
- Rpl27 :
Ribosomal protein L27
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The authors thank undergraduates Edith Anger, Connor Neblock, Nia-Zaire Egsdaille and Joyce Bonila-Moreno for assistance with microsatellite marker typing, and Ms. Mary Mantzaris for excellent animal care.
This study was supported by a University Research grant from the Connecticut State Colleges and University System (grant ARKINP), and two Faculty/Student grants from Central CT State University (grants AFKIZR & AFKINR).
Ethics approval and consent to participate
Mice were housed, fed, and handled in accordance with Federal guidelines, and the Institutional Animal Care and Use Committee at CCSU (Central Connecticut State University) approved of all procedures involving mice (Animal Protocol Application #170). See our euthanasia statement in the Methods section. Animal welfare at CCSU is the responsibility of the Institutional Officer for animal welfare, Dr. Christina Robinson.
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The authors declare that they have no competing interests.
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A wild type heterozygote (left) and a mutant squig/squig mouse (right) at 10 days of age.
Description of SNP markers referred to in the Girard et al. (2022) text.
Location of SNP markers referred to in the Girard et al. (2022) text.
Segregation of alleles of squig and eight microsatellite markers among 1008 intraspecific backcross progeny.
A deletion including Meox1, Exon 1 appears to be specific to the squig mutation.
Original, uncropped photographs that were used to produce Figure S3B (as required by the editor).
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Cite this article
Girard, J.P., Tomasiello, J.F., Samuel-Constanzo, J.I. et al. The squiggle tail (squig) mutation in mice is associated with a deletion in the mesenchyme homeobox 1 (Meox1) gene. BMC Res Notes 15, 305 (2022). https://0-doi-org.brum.beds.ac.uk/10.1186/s13104-022-06192-z