Effect of CCKBR(A) loss-of-function mutation on gonadal development.

(A) Expression of CCKBR(A) and two identified GnRH receptors in LH and FSH cells. Expression data were taken from a transcriptome of sorted pituitary cells of transgenic Nile tilapia (Oreochromis niloticus, previously obtained by Hollander-Cohen et al (26). Each dot represents a FACS fraction from a bulk of 20 pituitaries (n=8, 4 groups of males and 4 groups of females). The expression of each gene in each cell type is normalised to its expression in non-gonadotroph pituitary cells. (two-way ANOVA, * p<0.05,). (B) RNA expression of CCKBR(A) (white) identified by hybridization chain reaction (HCR) in transgenic zebrafish pituitaries expressing RFP in LH cells (magenta) and GFP in FSH cells (green) (scale bar = 10µm), the right panel is a magnification of the white square in the left panel. (C) Immunohistochemical staining of CCK (white) in transgenic zebrafish expressing GFP in FSH cells (left panel, scale bar = 50 µm and 8 µm) or GFP in GnRH neurons (right panel, scale bar = 100 µm and 10 µm). In B and C, staining from adult fish was performed on whole head sections 15 µm thick. (D) H&E staining of body cross-sections (dorsoventral axis) of adult WT, heterozygous (CCKBR(A)wt/+12, CCKBR(A)wt/+7/, CCKBR(A)wt/-1), and KO zebrafish (CCKBR(A)+12/+12, CCKBR(A) +7/+7,CCKBR(A)-1/-1). An inset of the red square in each image on the right displays a magnified view of the gonad. On the top right of each panel is the sex distribution for each genotype. (E) Gonad areas of mutant zebrafish. (n((+/+), (+/-), (-/-))=10/6/17,one way ANOVA, ****p<0.0001). (F) The distribution of cell types in the gonads of WT, heterozygous and KO zebrafish. (n((+/+),(+/-), (-/-))=10/6/17, two-way ANOVA, * p<0.05, ** p<0.001, *** p<0.0001, ****p<0.00001). (G) Gonadotroph mRNA expression in the pituitaries of the three genotypes (n((wt/wt),(wt/-), (-/-))=10/8/9, one-way ANOVA, * p<0.05, ** p<0.01, *** p<0.001) .

LH and FSH cells exhibit distinct spontaneous activity patterns in vivo.

(A) A confocal image of the pituitary shows RCaMP2 expression in both cell types and GFP expression in FSH cells (scale bar = 100 µm). (B) A diagram describing the setup of the in vivo experiments. The dissected zebrafish were placed in a chamber with a constant flow of water to the gills and imaged in an upright two-photon microscopy. (C) A representative image of in vivo calcium activity (see Figure2-supplementary movie 2). On the top left is a merged image depicting FSH cells in green and LH cells in magenta. The other top panels show sequential calcium imaging calcium rise in FSH cells is marked by white arrows. The bottom panels show the calcium of LH and FSH cells in two different imaged pituitaries, one where only FSH cells were active (Fish 1) and another where both cell types were active (Fish 2, traces ΔF/F, see Figure 2- Figure supplement 1A . for heatmap of the calcium traces, scale bar = 20 µm). (D) The properties of spontaneous calcium transients (ΔF/F) in LH cells and FSH cells in three males and one female and their means. (unpaired t-test, ** p<0.001). Analysis was performed using pCLAMP 11. (E) Left: cross correlation analysis between active ROI to the rest of the cell. The color-coded data points are superimposed on the imaged cells and represent the maximum cross-correlation coefficient between a calcium trace of a region of interest (ROI) and that of the rest of the cells in the same population. Right: is a matrix of maximum cross-correlation coefficient values between all the cells. (scale bar = 20 µm). (F) Summary of the mean max cross-correlation coefficient values of calcium traces in each cell population of repeated in vivo calcium imaging assays (n (calcium sessions) =16, see Figure 2-Figure supplement 1B for all measurements,. unpaired t-test, *** p<0.0001).

GnRH induces a synchronized increase of calcium in all LH cells and partial increase in FSH cells.

(A)Top: Image of a dissected head with pituitary exposed from the ventral side of the fish used for the ex vivo assays (OB, olfactory bulb; OC, optic chiasm; PIT, pituitary; TH, thalamus; MO, medulla oblongata). Bottom: A diagram describing the ex vivo setup with a constant flow of artificial cerebrospinal fluid (ACSF), a side tube to inject stimuli, and a collecting tube. (B) A representative analysis of basal calcium activity of LH and FSH cells. The left panel is a heatmap of calcium traces (ΔF/F), where each line represents a cell, with the mean calcium trace on top, the separated line at the bottom of each heatmap is the calcium trace of the chosen ROI. The color-coded data points on the right are superimposed on the imaged cells and represent the maximum cross-correlation coefficient between a calcium trace of an active chosen ROI to those of the rest of the cells in the same population, matrix on the right represent the maximum cross-correlation coefficient values between all the cells (see Figure 3-Figure supplement 2 for additional cell activity parameters, scale bar = 20 µm). (C) An analysis of calcium response to GnRH stimulation in two representative imaging sessions, fish 1 where both LH and FSH cells respond and fish 2 where only LH respond. (see Figure 4-Figure supplement 1 for detailed coefficient values distribution in each fish, scale bar = 20 µm). (D) The mean of max cross-correlation coefficient values in each cell type under each treatment (n=10, 3 males, 7 females, one-way ANOVA, **** p<0.0001, see Figure 4-Figure supplement 1A for cell specific vlaues). (E) The percentage of cells responsive to GnRH stimulus (i.e., coefficient values higher than the 80 percentiles of basal values). Each dot represents one fish (n=10, 3 males, 7 females, unpaired t-test, *** p<0.001).

FSH cells are directly stimulated by CCK.

(A) Example of calcium analysis of FSH and LH cells during CCK stimulation: fish1 with only FSH cells responding, and fish2 with FSH and LH cells responding. For each fish the left panels are a heatmaps of calcium traces (ΔF/F), where each line represents a cell. On top of each heatmap is a graph showing the mean calcium trace, the separated line at the bottom of the heat map is the calcium trace of the chosen ROI. On the right are color-coded data points that are superimposed on the imaged cells, showing the maximum cross-correlation coefficient between a calcium trace of a chosen active ROI and those of the rest of the cells in the same population, next to it is a matrix of max cross correlation coefficients between all the cells. (scale bar = 20 µm) (B) The mean of max cross-correlation coefficient values in each cell type (n=7, 4 males, 3 females, one-way ANOVA, **** p<0.0001, see Figure 4-Figure supplement 1B for detailed coefficient values distribution in each fish). (C) The percentage of active cells (i.e., a coefficient value higher than the 80 percentiles of basal levels) during CCK stimulation. Each dot represents one fish (n=7, 4 males, 3 females, unpaired t-test, * p<0.05).

The stimulated calcium activity of LH and FSH cells is associated with hormone secretion. (A and B)

Top: Graphs showing mean calcium trace of 10 LH cells (left panel) or FSH cells (right panel) from consecutive imaging sessions before, during, and after the application of the stimulus (GnRH or CCK). Bottom: Secretion of LH or FSH before or after GnRH (A) or CCK (B) stimulation (dots from the same imaged pituitaries are connected with a line; n=5, paired t-test, *p<0.05). (C) FSH mRNA transcription in the pituitary two hours after injection of CCK or GnRH into live fish (n=10, 5 females and 5 males, see Figure 5-Figure supplement 1 for LH expression; one-way ANOVA, * p<0.05, ** p<0.01). (D) FSH plasma levels after CCK or GnRH injection into live fish (n=10, 5 females and 5 males), (one-way ANOVA, * p<0.05, *** p<0.001).

A model summarizing the two suggested regulatory axes controlling fish reproduction.

The satiety-regulated CCK neurons activate FSH cells . LH cells are directly regulated by GnRH neurons that are gated by CCK, photoperiod, temperature, and behaviour, eventually leading to final maturation and ovulation. Bottom image schematically represents the relative timescale of the two processes and the associated gonadotropin levels. (Created with BioRender.com)