DOI: 10.5281/zenodo.15043737
title: glis3 restricts pit1-precursors during zebrafish adenohypophysis development

description: Supporting Data_Rurale et al (Excel file): this dataset supports the study investigating the role of glis3 in zebrafish adenohypophysis (AH) development and function. The study analyzes the effects of glis3 knockdown (KD) on pituitary cell differentiation, hypothalamic-pituitary function, and osmoregulation.

This dataset provides all raw numerical data corresponding to the graphs presented in the main text figures and supplementary materials of this study. Depending on the experiment, statistics were calculated using Student-t-test or Mann-Whitney Test using GraphPad Prism 10 Software. Statistically significant values are colored in red.

Each subfolder is named according to the corresponding figure, as follows:



Fig. 1_Graph K: Measurement of the area (in pixels) of whole-mount in situ hybridization staining for nkx2.2a, pax7, lim3, prop1, and pit1 in CTRL and glis3KD embryos at 24 hpf. A fixed ROI enclosing the pituitary anlage was used, and the stained area was quantified with Fiji Software. Results are expressed as mean ± standard deviation of 7 embryos per group.

Fig. 1_Graph L: quantification of the expression of nkx2.2a, pax7, lim3, prop1, and pit1 mRNAs by qRT-PCR in pools of CTRL, glis3KD, and rescue embryos at 24 hpf. ddCT method was used to analyse the results, expressed as mean ± standard deviation from three independent experiments. Results were normalized considering transcript levels of CTRL as 1.  

Fig. 2_Graph A: quantification of the expression of pit1 by qRT-PCR in pools of CTRL, glis3KD, and rescue embryos at 26, 30, and 33 hpf. ddCT method was used to analyse the results, expressed as mean ± standard deviation from three independent experiments. Results were normalized considering transcript levels of CTRL as 1.

Fig. 2_Graph E: Quantification of volume (in um3) of fluorescent in situ hybridization of pit1 in CTRL and  glis3KD embryos at 26, 30, and 33 hpf. A fixed ROI for each z-stack was used and quantified with Volocity Software (Nikon). Results are expressed as mean ± standard deviation of 15 embryos per group.

Fig. 2_Graph F: quantification of the expression of prl, smtla, smtlb, gsua, and pomca mRNAs by qRT-PCR in pools of CTRL, glis3KD, and rescue embryos at 36 hpf. ddCT method was used to analyse the results, expressed as mean ± standard deviation from three independent experiments. Results were normalized considering transcript levels of CTRL as 1.  

Fig. 2_Graph L: quantification of the expression of prl, smtla, smtlb, gsua, tshba, pomca, and gh  mRNAs by qRT-PCR in pools of CTRL, glis3KD, and rescue embryos at 60 hpf. ddCT method was used to analyse the results, expressed as mean ± standard deviation from three independent experiments. Results were normalized considering transcript levels of CTRL as 1.  

Fig. 3_Graph A: quantification of the expression of prl, tshba, prhrh, trh, and th mRNAs by qRT-PCR in pools of CTRL, glis3KD, and rescue larvae at 120 hpf. ddCT method was used to analyse the results, expressed as mean ± standard deviation from three independent experiments. Results were normalized considering transcript levels of CTRL as 1.  

Fig. 3_Graph G: quantification of the expression of prl, tshba, trh, and th mRNAs by qRT-PCR in pools of CTRL, glis3KD, and rescue larvae at 120 hpf in basal condition or after the administration of 20 nM T3. ddCT method was used to analyse the results, expressed as mean ± standard deviation from three independent experiments. Results were normalized considering transcript levels of CTRL as 1.  

Fig. 4_Graph A: quantification of prolactin (ng/ml) by ELISA assay in pools of CTRL and glis3KD larvae at 120 hpf. Results are expressed as mean ± standard deviation from three independent experiments.

Fig. 4_Graph D: quantification of prolactin immunofluorescence of CTRL and glis3KD larvae at 120 hpf. A fixed ROI enclosing gills, pectoral fins, pronephric ducts, or forebrain (FB) nuclei was used. The mean fluorescence intensity (MFI) was quantified with Fiji Software. Results are expressed as mean ± standard deviation of 15 embryos per group, derived from at least 15 independent injections. 

Fig. 4_Graph E: quantification of the expression of prolactin receptors (prlra, prlrb) and ionocytes expressed in gills and pronephric ducts (atp1a1a.5, atp1b1b, ca2, slc9a3.2, slc12a3, slc12a10.2, trpv6, aqp1a, and aqp3a)   by qRT-PCR in pools of CTRL and glis3KD larvae at 120 hpf. ddCT method was used to analyse the results, expressed as mean ± standard deviation from three independent experiments. Results were normalized considering transcript levels of CTRL as 1.  

Fig. 4_Graph G: number and frequency of abdominal edema observed in CTRL and glis3KD larvae at 120 hpf. Results are expressed as frequency > 300 larvae per group derived from three independent experiments. 

Fig. 4_Graph L: number and frequency of CTRL and glis3KD embryos that excrete (E) or retain (R) the Rhodamine B-dextran (RBD) solution after 15 mins (T0), 24 hours (T1), or 48 hours (T2) post-injection. Results are expressed as frequency of 45 embryos per group derived from three independent experiments. 


 



Supplementary Fig. 1_Graph A: quantification of the expression of pitx3, eya1, six1a, and dlx3b mRNAs by qRT-PCR in pools of CTRL, glis3KD, and rescue embryos at 16 hpf. ddCT method was used to analyse the results, expressed as mean ± standard deviation from three independent experiments. Results were normalized considering transcript levels of CTRL as 1.  

Supplementary Fig. 1_Panels B-D: number of tshba-positive cells in 11 CTRL, glis3KD, and PTU-induced hypothyroid larvae at 120 hpf. Cofocal z-stacks of tshba were used to manually count single cells. 

Supplementary Fig. 1_Graph F: quantification of tshba by qRT-PCR in CTRL, glis3KD, and PTU-induced hypothyroid larvae at 120 hpf, in basal condition (1.2% DMSO), or treated with increasing doses of levohyroxine (L-T4, 25, 50, and 100 nM). ddCT method was used to analyse the results, expressed as mean ± standard deviation from three independent experiments. Results were normalized considering transcript levels of CTRL (1.2% DMSO) as 1.  

Supplementary Fig. 1_Graph G: quantification of tshba by qRT-PCR in CTRL, glis3KD, and PTU-induced hypothyroid larvae at 120 hpf, in basal condition (1.2% DMSO), or treated with increasing doses of triiodothyronine (T3, 5, 10, and 20 nM). ddCT method was used to analyse the results, expressed as mean ± standard deviation from three independent experiments. Results were normalized considering transcript levels of CTRL (1.2% DMSO) as 1.  


 



Supplementary Table 1: number of cells positive for prl, smtla, smtlb, gsua, and pomca of CTRL and glis3KD embryos at 36 hpf. Cofocal z-stacks of FISH were used to manually count single cells. Results are expressed as mean ± standard deviation of 20 embryos per group derived from at least three independent experiments. 

Supplementary Table 2: number of cells positive for prl, smtla, smtlb, gsua, tshba, gh, and pomca of CTRL and glis3KD embryos at 60 hpf. Cofocal z-stacks of FISH were used to manually count single cells. Results are expressed as mean ± standard deviation of 20 embryos per group derived from at least three independent experiments. 

Supplementary Table 3: number of cells positive for prl and tshba of CTRL and glis3KD larvae at 120 hpf. Cofocal z-stacks of FISH were used to manually count single cells. Results are expressed as mean ± standard deviation of 20 embryos per group derived from at least three independent experiments. 

Supplementary Table 4: quantification of volume (in um3) of fluorescent in situ hybridization of prl, tshba, trh, and th in CTRL and  glis3KD larvae at 120 hpf. A fixed ROI for each z-stack was used and quantified with Volocity Software (Nikon). Results are expressed as mean ± standard deviation of 15 larvae per group.

Supplementary Table 5: list of primers used for qRT-PCR.

Supplementary Table 5: list of primers used for WISH/FISH probes.