glis3 restricts pit1-precursors during zebrafish adenohypophysis development

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.附属数据集_Rurale等人（Excel文件）：本数据集用于支撑一项关于GLIS3在斑马鱼腺垂体（adenohypophysis, AH）发育与功能中的作用的研究。该研究分析了glis3敲低（Knockdown, KD）对垂体细胞分化、下丘脑-垂体功能以及渗透压调节的影响。 本数据集包含对应本研究正文图及补充材料中所有图表的原始数值数据。根据实验类型的不同，统计学分析采用GraphPad Prism 10软件进行学生t检验或曼-惠特尼检验。具有统计学显著性的数值以红色标注。 每个子文件夹均以对应的图表命名，具体如下： 图1_K图：针对受精后24小时（hours post fertilization, hpf）的对照组（CTRL）和glis3敲低胚胎，对nkx2.2a、pax7、lim3、prop1及pit1的整体原位杂交染色面积（以像素为单位）进行测量。采用固定感兴趣区域（region of interest, ROI）包裹垂体原基，使用Fiji软件对染色面积进行定量分析。结果以每组7枚胚胎的均值±标准差表示。 图1_L图：针对受精后24小时的对照组、glis3敲低组及拯救组胚胎混合样本，采用实时荧光定量逆转录PCR（quantitative real-time reverse transcription PCR, qRT-PCR）定量检测nkx2.2a、pax7、lim3、prop1及pit1的mRNA表达水平。采用ΔΔCt法进行数据分析，结果以3次独立实验的均值±标准差表示，以对照组转录本水平为1进行归一化处理。 图2_A图：针对受精后26、30及33小时的对照组、glis3敲低组及拯救组胚胎混合样本，采用qRT-PCR定量检测pit1的mRNA表达水平。采用ΔΔCt法进行数据分析，结果以3次独立实验的均值±标准差表示，以对照组转录本水平为1进行归一化处理。 图2_E图：针对受精后26、30及33小时的对照组和glis3敲低胚胎，对pit1的荧光原位杂交信号体积（以立方微米为单位）进行定量分析。为每个z堆栈设置固定ROI，使用Volocity软件（尼康）进行体积量化。结果以每组15枚胚胎的均值±标准差表示。 图2_F图：针对受精后36小时的对照组、glis3敲低组及拯救组胚胎混合样本，采用qRT-PCR定量检测prl、smtla、smtlb、gsua及pomca的mRNA表达水平。采用ΔΔCt法进行数据分析，结果以3次独立实验的均值±标准差表示，以对照组转录本水平为1进行归一化处理。 图2_L图：针对受精后60小时的对照组、glis3敲低组及拯救组胚胎混合样本，采用qRT-PCR定量检测prl、smtla、smtlb、gsua、tshba、pomca及gh的mRNA表达水平。采用ΔΔCt法进行数据分析，结果以3次独立实验的均值±标准差表示，以对照组转录本水平为1进行归一化处理。 图3_A图：针对受精后120小时的对照组、glis3敲低组及拯救组幼鱼混合样本，采用qRT-PCR定量检测prl、tshba、prhrh、trh及th的mRNA表达水平。采用ΔΔCt法进行数据分析，结果以3次独立实验的均值±标准差表示，以对照组转录本水平为1进行归一化处理。 图3_G图：针对受精后120小时的对照组、glis3敲低组及拯救组幼鱼混合样本，在基础条件或施加20 nM三碘甲状腺原氨酸（T3）后，采用qRT-PCR定量检测prl、tshba、trh及th的mRNA表达水平。采用ΔΔCt法进行数据分析，结果以3次独立实验的均值±标准差表示，以对照组转录本水平为1进行归一化处理。 图4_A图：针对受精后120小时的对照组和glis3敲低幼鱼混合样本，采用酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）定量检测催乳素（ng/ml）含量。结果以3次独立实验的均值±标准差表示。 图4_D图：针对受精后120小时的对照组和glis3敲低幼鱼，对其催乳素免疫荧光信号进行定量分析。采用固定ROI包裹鳃区、胸鳍、原肾管或前脑（forebrain, FB）核团，使用Fiji软件计算平均荧光强度（mean fluorescence intensity, MFI）。结果以每组15枚幼鱼的均值±标准差表示，数据来自至少15次独立注射实验。 图4_E图：针对受精后120小时的对照组和glis3敲低幼鱼混合样本，采用qRT-PCR定量检测催乳素受体（prlra、prlrb）及在鳃和原肾管中表达的离子细胞相关基因（atp1a1a.5、atp1b1b、ca2、slc9a3.2、slc12a3、slc12a10.2、trpv6、aqp1a及aqp3a）的mRNA表达水平。采用ΔΔCt法进行数据分析，结果以3次独立实验的均值±标准差表示，以对照组转录本水平为1进行归一化处理。 图4_G图：统计受精后120小时的对照组和glis3敲低幼鱼的腹部水肿发生数量与频率。结果以每组超过300尾幼鱼的频率表示，数据来自3次独立实验。 图4_L图：统计对照组和glis3敲低胚胎在注射罗丹明B-葡聚糖（Rhodamine B-dextran, RBD）溶液后，于15分钟（T0）、24小时（T1）及48小时（T2）时的排出（E）或滞留（R）情况。结果以每组45枚胚胎的频率表示，数据来自3次独立实验。 补充图1_A图：针对受精后16小时的对照组、glis3敲低组及拯救组胚胎混合样本，采用qRT-PCR定量检测pitx3、eya1、six1a及dlx3b的mRNA表达水平。采用ΔΔCt法进行数据分析，结果以3次独立实验的均值±标准差表示，以对照组转录本水平为1进行归一化处理。 补充图1_B-D组图：统计受精后120小时的对照组、glis3敲低组及丙硫氧嘧啶诱导的甲状腺功能减退幼鱼中tshba阳性细胞数量。通过共聚焦z堆栈图像手动计数单个阳性细胞。 补充图1_F图：针对受精后120小时的对照组、glis3敲低组及丙硫氧嘧啶诱导的甲状腺功能减退幼鱼，在基础条件（1.2%二甲基亚砜，DMSO）或施加递增剂量左甲状腺素（L-T4，25、50及100 nM）后，采用qRT-PCR定量检测tshba的mRNA表达水平。采用ΔΔCt法进行数据分析，结果以3次独立实验的均值±标准差表示，以对照组（1.2% DMSO）转录本水平为1进行归一化处理。 补充图1_G图：针对受精后120小时的对照组、glis3敲低组及丙硫氧嘧啶诱导的甲状腺功能减退幼鱼，在基础条件（1.2% DMSO）或施加递增剂量三碘甲状腺原氨酸（T3，5、10及20 nM）后，采用qRT-PCR定量检测tshba的mRNA表达水平。采用ΔΔCt法进行数据分析，结果以3次独立实验的均值±标准差表示，以对照组（1.2% DMSO）转录本水平为1进行归一化处理。 补充表1：统计受精后36小时的对照组和glis3敲低胚胎中prl、smtla、smtlb、gsua及pomca阳性细胞数量。通过荧光原位杂交的共聚焦z堆栈图像手动计数单个阳性细胞。结果以每组20枚胚胎的均值±标准差表示，数据来自至少3次独立实验。 补充表2：统计受精后60小时的对照组和glis3敲低胚胎中prl、smtla、smtlb、gsua、tshba、gh及pomca阳性细胞数量。通过荧光原位杂交的共聚焦z堆栈图像手动计数单个阳性细胞。结果以每组20枚胚胎的均值±标准差表示，数据来自至少3次独立实验。 补充表3：统计受精后120小时的对照组和glis3敲低幼鱼中prl及tshba阳性细胞数量。通过荧光原位杂交的共聚焦z堆栈图像手动计数单个阳性细胞。结果以每组20枚幼鱼的均值±标准差表示，数据来自至少3次独立实验。 补充表4：针对受精后120小时的对照组和glis3敲低幼鱼，对prl、tshba、trh及th的荧光原位杂交信号体积（以立方微米为单位）进行定量分析。为每个z堆栈设置固定ROI，使用Volocity软件（尼康）进行体积量化。结果以每组15尾幼鱼的均值±标准差表示。 补充表5：qRT-PCR所用引物列表。 补充表5：整体原位杂交/荧光原位杂交探针列表。