Characterization of Kiss1 neurons using transgenic mouse models

Abstract

Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptor (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or β-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Melanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed melanocortin receptors type 4 (MC4R). Specifically in the preoptic area, 18-28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction.

Figure 1

Schematic diagram of the derivation of Kiss1-Cre transgenic mice. In order to generate the transgenes, the entire coding region of Kiss1 and an additional 48-bp downstream of the Kiss1 stop codon were replaced by the coding sequence of Cre recombinase followed by an SV40 polyadenylation signal (pA) and an frt site. The J2-3, J2-4 and J2-6 lines were derived from the RP24-299B2 Kiss1 BAC. Kiss1’s and the transgenes’ transcriptional start sites (+1), Kiss1’s exons (denoted by numbered rectangles), and the start codons (ATG) and stop codons (TGA or TAG) for Kiss1 and Cre, respectively, are all indicated. The amounts of mouse chromosomal sequence present in the BACs and either upstream of Kiss1’s start codon or downstream of Kiss1’s stop codon also are indicated. Small green arrows denote the locations of oligonucleotide primers used for genotyping.

Figure 2

Comparative distribution of Kiss1 mRNA in wild-type female mice (perfused on diestrus) and Cre-activity in Kiss1-Cre reporter female mice (line J2-4). A, C, E, G, darkfield photomicrographs of hypothalamic sections showing the distribution of Kiss1 mRNA in the anteroventral periventricular nucleus (AVPV, A), in the anterior periventricular nucleus (PeN, C) and in two rostro-to-caudal levels of the arcuate nucleus (Arc, E, G). B, D, F, H, fluorescent photomicrographs of hypothalamic sections showing the distribution of βGalactosidase immunoreactivity (βGal-ir) in the AVPV (B), in the PeN (D) and in two levels of the Arc (F, H). 3v, third ventricle. Scale bar: A–H = 400 μm.

Figure 3

Distribution of Cre activity in additional brain sites. A, fluorescent photomicrograph showing βGalactosidase immunoreactivity (βGal-ir) in the cerebral cortex (layers 2–3 and 5–6) of a female mouse from line J2-4. B, darkfield photomicrograph showing the distribution of Kiss1 mRNA in the cerebral cortex (layers 2–3 and 6) of a C57BL/6 female mouse. C, brightfield photomicrograph showing the hybridization signal (silver grains) of Kiss1 riboprobe at cellular level (arrows), in the layers 2–3 of the neocortex. Observe the absence of hybridization signal in the layer 1. D–F, fluorescent photomicrographs showing Cre activity (Kiss1 reporter mice, green fluorescent protein immunoreactivity/GFP-ir) in the lateral septum (LS, D), in the anterodorsal preoptic nucleus (ADP, E), and in the medial nucleus of the amygdala (MeA, F). Abbreviations: 3v, third ventricle; ac, anterior comissure; CPu, caudate putamen; ic, internal capsule; LV, lateral ventricle; MS, medial septum; opt, optic chiasm. Scale bar: A–B = 400 μm, C = 10 μm, D–F = 200 μm.

Figure 4

Validation of the Kiss1-Cre mouse model, line J2-4. A–B, brightfield photomicrographs showing the colocalization of βGalactosidase immunoreactivity (βGal-ir, brown cytoplasm) and Kiss1 mRNA (silver grains, arrows) in cells of the anteroventral preoptic nucleus (AVPV, A) and in the arcuate nucleus (Arc, B). C–H, fluorescent photomicrographs showing the colocalization of green fluorescent protein immunoreactivity (GFP-ir, green cytoplasm) and ERα immunoreactivity (ERα -ir, red nuclei) in neurons of the AVPV (female, line J2-4, C–E) and of the Arc (F–H). Note the high number of dual labeled neurons (yellow nuclei) in E and H (arrows in C and E indicate the same cell). Scale bar: A–C = 200 μm. Abbreviations: 3v, third ventricle; ox, optic chiasm; PeN, anterior subdivision of the periventricular nucleus. Scale bar: A–B, E = 100 μm; C–H = 200 μm.

Figure 5

Kiss1 gene expression in male and female wild type mice. A–B, bar graphs showing relative levels of Kiss1 mRNA in various brain sites. Note the moderate levels of Kiss1 mRNA in the cerebral cortex and the low levels in the amygdala. Data shown is mean ± SEM, relative to hypothalamic mRNA levels (Hypothalamus mean Ct values: A = 27.27, B = 25.83; Cerebral Cortex mean Ct values: A = 27.28, B = 27.60). Abbreviations: amy, amygdala; b.stem, brainstem; c.ctx, cerebral cortex; cereb, cerebelo; hyp, hypothalamus; olf b., olfactory bulb.

Figure 6

Distribution of leptin-induced phosphorylation of STAT3 immunoreactivity (pSTAT3-ir) in the female mouse brain (wild type on diestrus). A–L, brightfield photomicrographs showing the distribution of pSTAT3-ir in various brain sites following intraperitoneal injection of saline or leptin in C57BL/6 mice perfused following 45 min or 2h after the treatment. Note the small number of pSTAT3 immunoreactive neurons in the brains of mice treated with saline (A, D, G, J, M). Also note the increased number of pSTAT3 immunoreactive neurons following 2h of leptin treatment in the lateral septum (LS, B–C), in the lateral hypothalamic area (LHA, E–F, H–I), in the dorsal subdivision of dorsomedial nucleus of the hypothalamus (dDMH, H–I) and in the ventral tegmental area (VTA, K–L). M–O, brightfield photomicrograph showing that very few neurons in the anteroventral periventricular nucleus (AVPV) of female mice display leptin-induced-pSTAT3-ir. Abbreviations: 3v, third ventricle; ac, anterior comissure; CPU, caudate/putamen; EW, Edinger-Westphal nucleus; f, fornix; IP, interpeduncular nucleus; LV, lateral ventricle; ox, optic chiasm; vDMH, ventral subdivision of the dorsomedial nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus. Scale bar: A–L = 500 μm, M–O = 200 μm.

Figure 7

Distribution of Kiss1 neurons responsive to leptin. A, bar graph showing number of leptin-induced phosphorylation of STAT3 immunoreactivity (pSTAT3-ir) in hypothalamic nuclei which express Kiss1 reporter gene (anteroventral periventricular nucleus/AVPV, anterior periventricular nucleus/PeN, 3 rostro-to-caudal levels of the arcuate nucleus Arc1-3); B, bar graph showing the number of neurons expressing Kiss1 reporter gene (green fluorescent protein immunoreactivity/GFP-ir); C, percentage of Kiss1 neurons expressing pSTAT3-ir; D, percentage of pSTAT3-ir neurons expressing Kiss1 reporter gene. Females on diestrus from line J2-4 were used in this experiment. E, fluorescent and brightfield photomicrographs showing distribution of leptin-induced pSTAT3-ir and Kiss1 neurons (reporter gene), in a caudal level of the Arc. F–G, fluorescent and brightfield photomicrographs showing Kiss1 neurons (reporter gene) expressing leptin-induced pSTAT3-ir (black nucleus, arrows). H–M, fluorescent photomicrographs showing the distribution of leptin receptor reporter gene (LepR, βGalactosidase immunoreactivity/βGal-ir) in the preoptic area. Note the reduced number of neurons expressing LepR reporter gene in the AVPV (H–J) and PeN (K–M), and the absence of colocalization between LepR and kisspeptin immunoreactivity. Abbreviations: 3v, third ventricle; MPO, medial preoptic nucleus; ox, optic chiasm; PMV, ventral premammillary nucleus. Scale bar: E–F = 200 μm; G = 50 μm; H–J, 400 μm; K–M, 100 μm.

Figure 8

Distribution of Kiss1 neurons expressing leptin-induced phosphorylation of STAT3 immunoreactivity (pSTAT3-ir). A–F, line drawings of two rostro-to-caudal levels of the preoptic area (A–B), of the retrochiasmatic area (RCA, C) and three rostro-to-caudal levels of the arcuate nucleus (Arc, D–F). We found no colocalization of Kiss1-reporter gene (green fluorescent protein immunoreactivity/GFP-ir, green squares) and leptin-induced pSTAT3-ir (black dots) in the anteroventral periventricular nucleus (AVPV) and in the anterior periventricular nucleus (Pe). Colocalization of Kiss1-reporter gene and leptin-induced pSTAT3-ir was only found in the Arc. We also noticed an increase in number of dual labeled neurons (red crosses) toward the caudal levels of the Arc. Females on diestrus from line J2-4 were used in this experiment. Abbreviations: 3v, third ventricle; ac, anterior comissure; DMH, dorsomedial nucleus of the hypothalamus; f, fornix, MPO, medial preoptic nucleus; ox, optic chiasm; PMV, ventral premammillary nucleus; VMH, ventomedial nucleus of the hypothalamus.

Figure 9

Topographic distribution of Kiss1 neurons and their innervation by melanocortins. A–D, schematic drawings showing the topographic distribution of Kiss1 neurons (green dots, line J2-4), proopiomelanocortin neurons (red dots) and neuropeptide Y neurons (black dots) in the retrochiasmatic area (RCA) and 3 rostro-to-caudal levels of the arcuate nucleus (Arc). E, fluorescent photomicrograph showing the segregated distribution of neurons expressing Kiss1 reporter gene (green fluorescent protein immunoreactivity/GFP-ir, green cytoplasm) and βEndorphin immunoreactivity (βEnd-ir, a POMC product, red cytoplasm). F–G, fluorescent photomicrographs showing close appositions between βEnd immunoreactive fibers and GFP immunoreactive neurons, in the anteroventral periventricular nucleus (AVPV, F) and in the Arc (G). H, darkfield photomicrograph showing expression of melanocortin 4 receptor (MC4R) mRNA (hybridization signal, silver grains) in the AVPV of a female mouse. I–K, fluorescent photomicrographs showing the colocalization of MC4R reporter gene (GFP-ir) and kisspeptin immunoreactivity (kisspeptin-ir) in the AVPV (arrows). Abbreviations: 3v, third ventricle; MPO medial preoptic nucleus; ox, optical chiasm. Scale bar: A–D = 600 μm; E, H = 400 μm; I–K = 100 μm, F–G, 50 μm.

Figure 10

Kiss1 neurons differentially express glutamate and GABA. A, C, darkfield photomicrographs showing the distribution of glutamic acid decarboxylase (GAD-67) mRNA (hybridization signal, silver grains) in the anteroventral preoptic nucleus (AVPV, A) and in the arcuate nucleus (Arc, C). C, brightfield photomicrograph showing colocalization of GAD-67 mRNA (silver grains) and Kiss1 reporter gene (βGalactosidase immunoreactivity/βGal-ir, brown cytoplasm, arrows) in the AVPV (line J2-4). B, D, darkfield photomicrographs showing the distribution of vesicular glutamate transporter type 2 (vGluT2) mRNA (hybridization signal, silver grains) in the AVPV (B) and in the Arc (D). F, brightfield photomicrograph showing colocalization of vGluT2 mRNA (silver grains) and Kiss1 reporter gene (βGal-ir, brown cytoplasm, arrows) in the Arc (line J2-4). Abbreviations: 3v, third ventricle; DMH, dorsomedial nucleus of the hypothalamus; f, fornix; MPO medial preoptic nucleus; ox, optic chiasm; PMV, ventral premammillary nucleus; VMH, ventromedial nucleus of the hypothalamus.. Scale bar: A–B, D–E = 400 μm; C, F = 100 μm.

Figure 11

Expression of genes of interest in GFP neurons collected by fluorescence activated cells sorting (FACS). A–B, graphical representation of FACS of cells from the arcuate nucleus indicating cells collected as part of the GFP-enriched pools (Kiss1-Cre/GFP line J2-4). Observe in B that no fluorescent cell was detected in wild-types. C–F, bar graphs showing relative expression levels of Kiss1 mRNA (A), NPY mRNA (B), POMC mRNA (C) and LepR mRNA (D) in Kiss1-GFP neurons from the AVPV/PeN and Arc, from NPY-GFP neurons and from POMC-GFP neurons. G–I, bar graphs showing relative expression levels of ERα mRNA (E), MC4R mRNA (F), vGluT2 and GAD67 mRNA (F) in Kiss1-GFP neurons from the AVPV/PeN and Arc. Data shown is mean ± SEM, relative mRNA levels compared to Kiss1 mRNA in Kiss1 positive neurons of the Arc (A, mean Ct value = 21.67), to NPY mRNA in NPY positive neurons (B, mean Ct value = 20.54), to POMC mRNA in POMC positive neurons (C, mean Ct value = 17.95), to Kiss1 mRNA in Kiss1 positive neurons of the Arc (D, mean Ct value = 25.31), to ERα mRNA in Kiss1 positive neurons of the Arc (E, mean Ct value = 22.21), to MC4R mRNA in Kiss1 positive neurons of the Arc (F, mean Ct value = 23.27), to vGluT2 mRNA in Kiss1 positive neurons of the Arc.