Rescue of an aggressive female sexual courtship in mice by CRISPR/Cas9 secondary mutation in vivo
We had previously reported a mouse line carrying the Atypical female courtship (HoxD Afc ) allele, where an ectopic accumulation of Hoxd10 transcripts was observed in a sparse population of cells in the adult isocortex, as a result of a partial deletion of the HoxD gene cluster. Female mice carrying this allele displayed an exacerbated paracopulatory behavior, culminating in a severe mutilation of the studs’ external genitals. To unequivocally demonstrate that this intriguing phenotype was indeed caused by an illegitimate function of the HOXD10 protein, we use CRISPR/Cas9 technology to induce a microdeletion into the homeobox of the Hoxd10 gene in cis with the HoxD Afc allele.
Females carrying this novel HoxDDel(1–9)d10hd allele no longer mutilate males. We conclude that a brain malfunction leading to a severe pathological behavior can be caused by the mere binding to DNA of a transcription factor expressed ectopically. We also show that in HoxD Afc mice, Hoxd10 was expressed in cells containing glutamate decarboxylase (Gad1) and Cholecystokinin (Cck) transcripts, corroborating our proposal that a small fraction of GABAergic neurons in adult hippocampus may participate to some aspects of female courtship.
KeywordsSocial behavior Hippocampus HOXD10 Genome editing CRISPR/Cas9
Atypical female courtship allele of the HoxD locus
fluorescent in situ hybridization
optimal cutting temperature
Although the heterozygous HoxD Afc genotype proved semi-lethal in both sexes, only sexually mature females displayed an aberrant courtship behavior. When placed with a male for mating, and regardless of the male genotype (i.e. HoxD Afc heterozygous or wildtype), females repeatedly bit and injured the male’s penises, often up to their complete ablation. In such adult HoxD Afc heterozygous mice, ectopic Hoxd10 transcript accumulation was found in numerous scattered cells in the hippocampus , while Hox genes are never expressed rostral to the hindbrain and its derivatives .
The detailed protocol for the derivation of the CRISPR/Cas9 induced HoxDDel(1–9)d10hd allele was described in . Briefly, the Hoxd10 CRISPR/Cas9 allele was produced by pronuclear injection of the pX330:hSpCas9 (Addgene ID 42230) vector with the AGAGCGTTAACCTCACCGAC guide sequence oligo cloned as recommended. A small deletion occurred initiating in the guide sequence that removed 10 nucleotides (CCGACAGGCA) corresponding to mm10 positions chr2: 74695262–74695271. The deletion predicted a HOXD10 protein product with a truncated homeodomain after position 40. Breeding over more than three filial generations indicated that the indel segregated with the HoxDDel(1–9) deficiency. Both HoxDDel(1–9)d10hd, HoxDDel(1–9) and HoxDDel(4–9) stocks were maintained by serial backcrosses to (B6CBA)F1 females. Experimental adult females were 12–24 weeks of age.
For in situ hybridization analyzes, freshly dissected brains were mounted in the Optimal cutting temperature (OCT) compound and stored at − 80 °C. In most experiments, pairs of hemi-brains of HoxD Afc and HoxD(Del4–9) heterozygous or wild type control adult females were mounted in the same block, cut, collected on the same slides and processed together to allow for direct comparison of the Hoxd10 signals under identical conditions. Usually four parallel sub-series of 14 µm thick coronal cryo-sections were collected, air-dried and stored at − 80 °C. One of the sub-series was stained with Cresyl violet and the position of the sections along the Coronal Allen Brain Atlas was determined. On the day of hybridization, slides were thawed, air-dried and fixed in 4% paraformaldehyde in PBS. In situ hybridizations were carried out at 63.5 °C overnight, followed by stringency washes at 61 °C. The binding of the antisense probe was revealed either by the NBT/BCIP alkaline phosphatase substrate (e.g. Allen Brain Institute http://mouse.brain-map.org/gene), or with the FASTRED alkaline phosphatase substrate (Sigma, SIG-31072) to detect DIG labeled probes, and the Tyramide amplification procedure (PerkinElmer SAT700001EA), followed by 1:100 dilution of Streptavidin Alexa Fluor 488 conjugate (Invitrogene S32354 to detect Fluorescein labeled probes.
Gad1 and Cck antisense riboprobes were synthesized using cDNA plasmid clones as templates (http://www.imagenes-bio.de). Briefly, mouse Gad1 cDNA clone IRAKp961I2154Q was linearized with Kpn1 and transcribed by T7 polymerase (Promega, #P2075). Mouse Cck cDNA clone IRAVp968E034D was linearized with EcoRI and transcribed with T3 polymerase (Promega, #P2083). Mouse Hoxd10 cDNA clone  was digested with EcoRI and transcribed by T7 polymerase. Labeled nucleotides were incorporated using digoxigenin (DIG) RNA Labeling Mix (Roche 1122707390), or Fluorescein RNA Labeling Mix (Roche 11685619910). We successfully detected Hoxd10 with DIG, yet not when a fluorescein labeled antisense cRNA probe was used. This may reflect a higher sensitivity of the alkaline phosphatase enzymatic reaction, which was also supported by the easier detection of the Gad1 and Cck signals with DIG/FAST RED, as compared to the fluorescein/Tyramide enhancement. In double fluorescent in situ hybridization (FISH) experiments Hoxd10 specific red signal was scored at probe concentrations, when red stained cellular profiles were detected only in the HoxD Afc , and not in either control samples, indicating that conditions were appropriate for specific detection of Hoxd10 transcripts.
Gad1/Cck colocalizations were obtained from 3 pairs of brains, 14 sections from 3 independent hybridization reactions: 431 cells were Gad1 positive in the Cornu Ammonis (CA), of which 89 were Gad1/Cck double positive. Hoxd10/Gad1 colocalizations were obtained from one pair of brains, seven sections from five independent hybridization reactions: 131 cells were Gad1 positive in CA of which 5 were Gad1/Hoxd10 double positive. The difference in incidences of Hoxd10 over total Gad1 (prop 1 = 0.03816794) and Cck over total Gad1 (prop 2 = 0.20649652) were statistically significant (p = 1.149e−05, 2-sample test for equality of proportions with continuity correction). Cck/Hoxd10 colocalizations were obtained from one pair of brains, five sections from two independent hybridization reactions: 39 cells were Cck positive in CA, of which 9 were Cck/Hoxd10 double positive. The difference in incidences of Hoxd10 over total Gad1 (prop 1 = 0.03816794) and Hoxd10 over total Cck (prop 3 = 0.23076923) were statistically significant (p = 0.0004497, 2-sample test for equality of proportions with continuity correction). In all cases, any Hoxd10 positive cells proved positive for either Gad1 or Cck depending on the probe mix under investigation. The product of prop2 and prop3 was in good agreement with prop1 (0.046 vs 0.038), therefore we concluded that further technical repetitions with these same techniques were unlikely to bring additional information.
To confirm the causal role of Hoxd10 ectopic expression in this unusual behavior, we induced a deletion in the homeobox of the Hoxd10 gene in cis with the HoxD Afc allele as a secondary mutation (Fig. 1a). Non-homologous end joining of genomic DNA after exposure to a single guide RNA and the Cas9 endonuclease in fertilized eggs resulted in a 10 base-pair long deficiency in the Hoxd10 homeobox, giving rise to the HoxDDel(1–9)d10hd allele. This mutant allele had lost the third alpha-helix of the HOXD10 homeodomain necessary for the binding of this transcription factor to its DNA target sites (Fig. 1b), due to a protein truncation from the 40th position of the homeodomain onwards, replacing 34 residues by a 10 residues frameshifted sequence (Fig. 1c).
We crossed this allele out through three consecutive generations and observed twelve adult females caged with males. Studs were followed for the appearance of injuries at their external genitals. Heterozygous HoxDDel(1–9)d10hd females bred successfully, without any indications of atypical female courtship (0 out of 12). This was in marked contrast with the observation of 12 out of 18 HoxD Afc females carrying the intact Hoxd10 homeobox sequence and showing genital biting . This difference was statistically significant (p = 0.001071) by the 2-sample test for equality of proportions with continuity correction Other abnormal phenotypic traits associated with the HoxD Afc allele, like malocclusion and slow postnatal weight gain were also rescued . These results provide strong genetic evidence of the direct role of the HOXD10 transcription factor in bringing about the courtship aberration observed in HoxD Afc mice.
This atypical female courtship anomaly occurred in animals with a low abundance of Hoxd10 positive cells in adult forebrain, in both sides and at any observed rostra-caudal sections of the hippocampal formation, which display molecular and neuroanatomical characteristics reminiscent of a small subpopulation of GABAergic interneurons [1, 5], as characterized by the detection of both the Gad1 and Cck markers (Fig. 1d, e). Double labeling simultaneous FISH analyses with Hoxd10-dig and Gad1-fluo pair of probes indeed showed Hoxd10 positive cells localized selectively in the hippocampus, distributed in any of the layers of the Cornu Ammonis (CA) fields where it co-localized with Gad1 (Fig. 1f–h). Furthermore, by using Cck-flou and Hoxd10-dig probes simultaneously, we scored the Hoxd10 specific red signal in cells accumulating Cck transcripts (Fig. 1h). As all Cck positive non-principal cells seemed included in the Gad1 labeled pool, and since all Hoxd10 positive cells were part of the Cck positive non-principal pool, we concluded that ectopic Hoxd10 transcripts accumulated in a very sparse subpopulation of Cck positive GABAergic cells. Of note, Hoxd10 like other Hox genes is not expressed in any cells of a normal adult forebrain .
The HoxD Afc phenotype followed a gender-specific pattern of expressivity, limited to sexually receptive females, despite the fact that ectopic expression of Hoxd10 was similar in both sexes. The ectopic presence of this HOX product in CCK positive GABAergic neurons in adult hippocampus may thus interfere with the implementation of a particular genetic program in a sexually dimorphic manner, perhaps through the property of such proteins to exert a dominant negative effect in various contexts . CCK signaling was previously associated with a sex-dependent control of behavior and its level seems to be modulated during the estrus cycle . Also, the inactivation of the Cck2 receptor, which presumably mediates some effects of CCK neuropeptides in postsynaptic neurons, elicits behavioral alterations distinct in females as compared to males . Altogether, this is consistent with a gender-specific role of CCK positive GABAergic cells in the modulation of behavior . A persistent ectopic expression of HOXD10 in CCK positive hippocampal GABAergic cells may thus interfere with the function of these cells in controlling the dynamic physiological status of females during the estrous cycle .
The Identification of GABAergic cells and the co-localization of ectopic Hoxd10 gene product accumulation was carried out relying on in situ hybridization detection of mRNA. This methodology provided a way to circumvent protein-based localization assays due to the absence of the required high-quality antibody. However, this approach does not allow for a rigorous evaluation of the HOXD10 protein distribution.
Study concept and design: all authors. Acquisition of data: JZ. Analysis and interpretation of data: all authors. Drafting of the manuscript: all authors. Critical revision of the manuscript for important intellectual content: all authors. Statistical analysis: JZ. Administrative, technical, and material support: all authors. Study supervision: DD. Both authors read and approved the final manuscript.
We thank N. Flores-Ramirez for help with in situ hybridizations and I. Rodriguez for discussions.
The authors declare that they have no competing interests.
Availability of data and materials
Mouse strains, and probes will be made available upon request.
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Ethics approval and consent to participate
Experiments were conducted according to the Swiss law on animal protection (LPA) under licenses #GE/81/14 and #GE/29/26. Licenses were obtained after approval by the legal and ethical authority, i.e. the ‘commission cantonale de l’expérimentation animale’ in the Canton of Geneva, Switzerland.
This work was supported by funds from the University of Geneva, the Ecole Polytechnique Fédérale (Lausanne), the Swiss National Research Fund (No. 310030B_138662) and the European Research Council grants SystemHox (No. 232790) and RegulHox (No. 588029).
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