Journal: Disease Models & Mechanisms

Article Title: Characterization of the human GnRH neuron developmental transcriptome using a GNRH1 -TdTomato reporter line in human pluripotent stem cells

doi: 10.1242/dmm.040105

Figure Lengend Snippet: RNA -seq at two time points of differentiation toward GnRH neurons. (A) The GnRH neuron differentiation protocol begins with 10 days Dual Smad inhibition (DM+SB), followed by 10 days FGF8 treatment to differentiate anteriorly patterned neuronal progenitor cells. To investigate the effect of FGF8 on the RNA profile of the cells, we collected FGF8-treated and non-treated cells at day (D)20. The final seven days of the protocol included Notch inhibition by DAPT. Based on TdTomato reporter expression, we collected the D27 TdTomato-positive samples by FACS sorting on D27. (B) RNA-seq and differential expression analyses were performed between these samples ( n =4) in three paired comparisons. (C-G) Results from D27TdT + versus D20FGF8 differential gene expression analysis. (C) Transcriptome of GnRH neurons versus FGF8-treated progenitor pool, showing the top 50 upregulated (red, top) and downregulated (green, bottom) genes differentially expressed in D27TdT + versus D20FGF8. Genes previously implicated with an association to puberty timing or expression in, or in close association with, newly formed GnRH neurons (animal models) are indicated (+). (D) Top 20 GO biological processes, and the number of genes per category, enriched in all the significantly upregulated (red, left) and downregulated (green, right) genes D27TdT + versus D20FGF8. IPA was used to draw a pathway containing connections within top 50 upregulated (E) and downregulated (F) genes, and their cytoplasmic localization. (G) IPA pathway of ISL1 upstream and downstream interactions represented in differential expression analysis 1. Lfc, log fold change. See Tables S3,S4 for references associated with panels C, E, F and G.

Article Snippet: Primary antibodies used were: rabbit polyclonal anti-ISL1 antibody (Abcam, ab20670, 1:500), guinea pig anti-GnRH antibody (EH#1018, 1:8000; a generous gift from Dr Erik Hrabovszky), previously characterized in embryonic and post-mortem human hypothalami ( ; ), and anti-TAG1 (made in goat, R&D Systems, AF2215, 1:500); 3×5 min washes in 0.01 M PBS were followed by incubation in appropriately conjugated secondary antibodies (dilution 1:400): anti-rabbit 568 (made in donkey, Invitrogen, A10042); anti-guinea pig 488 (made in donkey, Jackson ImmunoResearch, 706-545-148); anti-goat 647 (made in donkey, Invitrogen, A21082), for 1 h before incubation with Hoechst 1:1000.

Techniques: RNA Sequencing, Inhibition, Expressing, Quantitative Proteomics, Gene Expression

Journal: Disease Models & Mechanisms

Article Title: Characterization of the human GnRH neuron developmental transcriptome using a GNRH1 -TdTomato reporter line in human pluripotent stem cells

doi: 10.1242/dmm.040105

Figure Lengend Snippet: ISL1 expression in hPSC-derived and human fetal GnRH neurons. (A) Immunocytochemical staining with ISL1 and GnRH antibodies at day 27 of differentiation showed nuclear staining of ISL1 in GnRH-positive neurons. Result has been replicated in 3 experiments. (B) Schematic of a GW10.5 human fetus head (coronal view). Box indicates the area represented in immunohistochemical staining in C and D. (C,D) GnRH (green), ISL1 (red) and TAG-1 (white) expression in a coronal section of a GW10.5 fetus. ISL1 is expressed in GnRH neurons located in the nose and entering the forebrain (olfactory bulbs, OB). D shows a magnified view of the boxed area in C. (E) Schematic of a GW10.5 human fetus brain (coronal view). Box indicates the area represented in immunohistochemical staining in F and G. (F,G) ISL1 (red) is expressed in GnRH neurons that have migrated into the forebrain (septum). G shows a magnified view of the boxed area in F. White arrows show GnRH/ISL1 double-labeled cells. Arc, arcuate nucleus; OE, olfactory epithelium; LV, lateral ventricle; NEP: neuro-epithelium; 3V: third ventricle. Scale bars: 50 µm in A; 250 µm in C,F; 20 µm in D; 40 µm in G.

Article Snippet: Primary antibodies used were: rabbit polyclonal anti-ISL1 antibody (Abcam, ab20670, 1:500), guinea pig anti-GnRH antibody (EH#1018, 1:8000; a generous gift from Dr Erik Hrabovszky), previously characterized in embryonic and post-mortem human hypothalami ( ; ), and anti-TAG1 (made in goat, R&D Systems, AF2215, 1:500); 3×5 min washes in 0.01 M PBS were followed by incubation in appropriately conjugated secondary antibodies (dilution 1:400): anti-rabbit 568 (made in donkey, Invitrogen, A10042); anti-guinea pig 488 (made in donkey, Jackson ImmunoResearch, 706-545-148); anti-goat 647 (made in donkey, Invitrogen, A21082), for 1 h before incubation with Hoechst 1:1000.

Techniques: Expressing, Derivative Assay, Staining, Immunohistochemical staining, Labeling