Visualisation of Kiss1 Neurone Distribution Using a Kiss1-CRE Transgenic Mouse

Abstract

Kisspeptin neuropeptides are encoded by the Kiss1 gene and play a critical role in the regulation of the mammalian reproductive axis. Kiss1 neurones are found in two locations in the rodent hypothalamus: one in the arcuate nucleus (ARC) and another in the RP3V region, which includes the anteroventral periventricular nucleus (AVPV). Detailed mapping of the fibre distribution of Kiss1 neurones will help with our understanding of the action of these neurones in other regions of the brain. We have generated a transgenic mouse in which the Kiss1 coding region is disrupted by a CRE-GFP transgene so that expression of the CRE recombinase protein is driven from the Kiss1 promoter. As expected, mutant mice of both sexes are sterile with hypogonadotrophic hypogonadism and do not show the normal rise in luteinising hormone after gonadectomy. Mutant female mice do not develop mature Graafian follicles or form corpora lutea consistent with ovulatory failure. Mutant male mice have low blood testosterone levels and impaired spermatogenesis beyond the meiosis stage. Breeding Kiss-CRE heterozygous mice with CRE-activated tdTomato reporter mice allows fluorescence visualisation of Kiss1 neurones in brain slices. Approximately 80-90% of tdTomato positive neurones in the ARC were co-labelled with kisspeptin and expression of tdTomato in the AVPV region was sexually dimorphic, with higher expression in females than males. A small number of tdTomato-labelled neurones was also found in other locations, including the lateral septum, the anterodorsal preoptic nucleus, the amygdala, the dorsomedial and ventromedial hypothalamic nuclei, the periaquaductal grey, and the mammillary nucleus. Three dimensional visualisation of Kiss1 neurones and fibres by CLARITY processing of whole brains showed an increase in ARC expression during puberty and higher numbers of Kiss1 neurones in the caudal region of the ARC compared to the rostral region. ARC Kiss1 neurones sent fibre projections to several hypothalamic regions, including rostrally to the periventricular and pre-optic areas and to the lateral hypothalamus.

Keywords: CLARITY; Kiss-CRE; mouse; neuronal distribution; tdTomato; transgenic.

Figure 1

Generation of Kiss1 ^{tm1(Cre‐ GFP )Coll} mutant mice and hypogonadism. (a) Gene targeting strategy. The Kiss1 gene consists of three exons with the coding regions shown in black. The targeting vector contains a CRE‐GFP transgene immediately downstream of the Kiss1 initiation codon so that CRE expression is regulated by the Kiss1 promoter. (b, c) Confirmation of a null mutation by immunocytochemistry for kisspeptin expression (arrowed) in the arcuate nucleus (b) and the anteroventral periventricular nucleus (AVPV) (c) regions of the hypothalamus. Scale bar = 150 μm. (d) Female reproductive organs showing thread‐like uteri in mutants. U, uterus; Od, oviduct; Ov, ovary. Scale bar = 0.5 cm. (e) Male reproductive organs showing reduced growth. T, testes; Ep, epididymis; Vd, vas deferens. Scale bar = 1 cm. (f) Histology of ovaries showing corpora lutea (CL) in wild‐type and heterozygous mice but no CL in the mutant mice. Large numbers of atretic follicles (At, arrows) were found in the mutant mice. Scale bar = 200 μm. (g) Histology of testes showing spermatozoa (Sz) in the seminiferous tubules of wild‐type and heterozygous mice but none in the mutant testes. Sc, spermatocytes; Sp, spermatids. Scale bar = 100 μm.

Figure 2

Hormone profiles of Kiss1 ^{tm1(Cre‐ GFP )Coll} mutant mice. Blood plasma luteinsing hormone (LH) levels in males (a) and females (b). No difference in basal LH was found between wild‐type and mutant mice, although mutant mice failed to show the post‐gonadectomy (GDX) rise in LH. Data were analysed by a nonparametric anova (Kruskal–Wallis test, P < 0.005) with a Dunn's multiple comparison post‐test. (c) Blood plasma testosterone levels were significantly reduced in mutant mice. The data were analysed by a nonparametric Mann–Whitney test. The number of mice in each group is indicated on the histogram.

Figure 3

Fluorescent visualisation of Kiss1 neurones. Kiss1 neurones were visualised by breeding Kiss‐CRE mice with floxed tdTomato mice Gt(ROSA)26Sor ^{tm9( CAG ‐tdTomato)Hze)}. (a) Fluorescent Kiss1 neurones in the arcuate nucleus (ARC) and the anteroventral periventricular nucleus (AVPV) regions of females. (b) Fluorescent Kiss1 neurones in ARC and AVPV regions of males. (c) Co‐localisation of tdTomato neurones with kisspeptin protein expression in the ARC (gonadectomised mouse). Scale bar = 10 μm. (d) Lateral septum. Scale bar = 50 μm. (e) Amygdala. Scale bar = 25 μm (f) Mammillary nucleus. Scale bar = 50 μm. (g) CLARITY image showing Kiss1 neurone distribution in rostral ARC and AVPV regions of female mice. (h) CLARITY images showing the increase in the number of fluorescent Kiss1 neurones in peri‐pubertal (post‐natal day 33; P33) and post‐pubertal (post‐natal day 56; P56) mice. (i). CLARITY image of the AVPV region in a female mouse and a false colour image to show depth of Kiss1 cells. (j) CLARITY image of the ARC region in a female mouse and false colour image to show depth of Kiss1 cells. 3V, third ventricle; ARC, arcuate; AVPV, anteroventral periventricular nucleus. All scale bars in CLARITY images = 200 μm.

Figure 4

Distribution of Kiss‐CRE/tdTomato labelled neurones in the female mouse brain. Coronal brain images were taken from the online Mouse Brain Library resource 30. TdTomato positive cell bodies are represented by red circles and fibres represented by red lines. LSI, lateral septum, intermediate; ADP, anterodorsal preoptic nucleus; AVPV, anteroventral periventricular nucleus; AMG, amygdala; DMH, dorsomedial hypothalamus; ARC, arcuate nucleus; PAG, periaquaductal grey; MM, medial mammillary nucleus.

Figure 5

Fibre distribution in the Kiss‐CRE/tdTomato female mouse brain. (a) Montage of Z‐stack series of the ventral hypothalamus of a P56 age mouse brain. Immunocytochemistry of 2,2′‐thiodiethanol (TDE)‐cleared horizontal slices enabled the visualisation of tdTomato fibre projections. Scale bar = 200 μm. (b) Schematic horizontal view of the ventral hypothalamus (approximately the same scale as in a) illustrating the fibre projections from Kiss1 neurones. Anteroventral periventricular nucleus (AVPV) Kiss1 neuronal projections are indicated by by solid black arrows. Arcuate nucelus (ARC) Kiss1 projections are indicated by white arrows. Amygdala Kiss1 projections are indicated by dotted arrows. 3V, 3rd ventricle; AHA, anterior hypothalamic area; AMG, amygdala; AOB, accessory olfactory bulb; LHA, lateral hypothalamic area; LPO, lateral preoptic area, MEPO, median preoptic nucleus; MPA, medial preoptic area; MPN medial preoptic nucleus; Rch, retrochiasmatic area; SCN, suprachiasmatic area; OVLT, vascular organ of lamina terminalis, PVpo, periventricular preoptic nucleus; VTM, ventral tuberomammillary nucleus.