Data assist 3

For gene expression analyses, mRNAs obtained from FACS-sorted tanycytes were reverse transcribed using SuperScript III Reverse Transcriptase (Life Technologies) and a linear preamplification step was performed using the TaqMan PreAmp Master Mix Kit protocol (P/N 4366128, Applied Biosystems). Real-time PCR was carried out on an Applied Biosystems 7900HT Fast Real-Time PCR System, using exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystems) as follows: DARPP32 (Ppp1r1b: Mm00454892_m1), ERα (Esr1: Mm00433149_m1), ERβ (Esr2: Mm00599821_m1), PR-A/B (Pgr: Mm00435628_m1), TGF-β1 (Tgfb1: Mm01178820_m1) and Sema7a (Sema7a: Mm01171202_m1). Control housekeeping genes were r18S (18S: Hs99999901_s1) and ACTB (Actb: Mm00607939_s1). Gene expression data were analysed using SDS 2.4.1 and Data Assist 3.0.1 software (Applied Biosystems).

For gene expression analyses, cDNA obtained from RT-PCR were reverse transcribed using SuperScript III Reverse Transcriptase (ThermoFisher, Invitrogen). Real-time PCR was carried out on Applied Biosystems 7900HT Fast Real-Time PCR System using exon-span-specific TaqMan Gene Expression Assays (Applied Biosystems, Carlsbad CA). The list of primers used for these experiments is the following: Amh (Mm00431795_g1), Gnrh1 (Mm01315605), Amhr2 (Mm00513847_m1); Acvr1 (Mm01331069_m1); Bmpr1a (Mm00477650_m1); Bmpr1b (Mm03023971_m1). Control housekeeping genes: Rn18s (Hs99999901-s1) and Actb (Mm00607939). Amperase activation was achieved by heating to 50°C for 2 min, before denaturing at 95°C for 20 s, followed by 40 cycles of 1 s 95°C with a 20 s extension time at 60°C. Gene expression data were analyzed using SDS 2.4.1 and Data Assist 3.0.1 software (Applied Biosystems, Carlsbad, CA), with ActB and Rn18s as control house-keeping mRNA following a standardized procedure (Schmittgen and Livak, 2008 (link)). Values are normalized relative to control values and expressed, as appropriate, to 1.

Sample sizes for physiological and neuroanatomical studies and gene expression analyses were estimated based on prior experience and those represented in the extant literature. Typically, mice taken from at least two different litters for each group were used. No stringent randomization method was used to assign subjects in the experimental groups or to process data.
Quantitative RT-PCR gene expression data were analyzed using SDS 2.4.1 and Data Assist 3.0.1 software (Applied Biosystems, Carlsbad, CA). All other analyses were performed using Prism 5 (GraphPad Software). Data sets were assessed for normality (Shapiro-Wilk test) and variance. Where appropriate a one-way or two-way ANOVA followed by post hoc testing (specified in the figure legends) was performed and for non-Gaussian distributions, a Kruskal-Wallis test followed by Dunn’s multiple comparison test was used – indicated in figure legends. Exact P/adjusted p values are given in figure legends where possible. α was set at 0.05 for all experiments excluding WES data.

RNA and cDNA from lung tissue for real-time PCR arrays were prepared according to the method described in our previous study [22 (link)]. Data were analyzed using DataAssist 3.01 software (freeware by Applied Biosystems, Foster, CA, USA) with reference to ribosomal protein L13A (Rpl13a). The list of genes is shown in Table S9.

For gene expression analyses, messenger RNAs obtained from FACS-sorted GnRH neurons were reverse transcribed using SuperScript III Reverse Transcriptase (Life Technologies) and a linear preamplification step was performed using the TaqMan PreAmp Master Mix Kit protocol (Applied Biosystems). Real-time PCR was carried out on Applied Biosystems 7900HT Fast Real-Time PCR System using exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystems): Gnrh1 (Gnrh1-Mm01315605_m1), Amhr2 (AMH2r-Mm00513847_m1), Alk2 (Acvr1-Mm01331069_m1), Alk3 (Bmpr1a-Mm00477650_m1), Alk6 (Bmpr1b-Mm03023971_m1), Smad 1 (Smad1-Mm00484723_m1), Smad 4 (Smad4-Mm03023996_m1), Smad 5 (Smad5- Mm03024001_g1) and Smad 8 (Smad8/9-Mm00649885_m1). Control housekeeping genes: r18S (18S-Hs99999901_s1); Actb (Actb-Mm00607939_s1). Quantitative real-time PCR was performed using TaqMan Low-Density Arrays (Applied BioSystems) on an Applied BioSystems 7900HT thermocycler using the manufacturer's recommended cycling conditions. Gene expression data were analysed using SDS 2.4.1 and Data Assist 3.0.1 software (Applied BioSystems).