Rescue of an aggressive female sexual courtship in mice by CRISPR/Cas9 secondary mutation in vivo

Objective We had previously reported a mouse line carrying the Atypical female courtship (HoxDAfc) 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 HoxDAfc allele. Results 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 HoxDAfc 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.


Introduction
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 [1], while Hox genes are never expressed rostral to the hindbrain and its derivatives [2].
For in situ hybridization analyzes, freshly dissected brains were mounted in the Optimal cutting temperature Open Access BMC Research Notes *Correspondence: Denis.Duboule@epfl.ch 1 Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland Full list of author information is available at the end of the article (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. image nes-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 [1] 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.
The double FISH procedure was carried out as in [4]. Pictures were taken with HBO 100 illumination using the appropriate filter sets to visualize red, green and blue fluorescence signals (set 43, 10 and 49 respectively), on a Zeiss Axioplan 2 microscope (Fig. 1f-h). Hoxd10 red hybridization signals were accepted as positive if the signal could be seen with a 5×/0.25 n.a. 0.17 Zeiss FLUAR objective using filter set 43. Upon higher magnification, a clear cytoplasm signal zone included a negative zone corresponding to the position of a cell nucleus (perikaryon). Images were taken with a Leica DFC300 FX digital color camera. Brightness and contrast were adjusted in Photoshop CS3. Red and blue or green and blue double color images were generated using the HDR2 plug-in.  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.

Results
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). Nonhomologous 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 HoxD Del(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 HoxD-Del(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 [1]. 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 [3]. 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 [6].

Discussion
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 [7]. CCK signaling was previously associated with a sex-dependent control of behavior and its level seems to be modulated during the estrus cycle [8]. 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 [9]. Altogether, this is consistent with a gender-specific role of CCK positive GABAergic cells in the modulation of behavior [10]. 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 [11].

Limitations
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.