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. 2009 May;93(5):449-60.
doi: 10.1016/j.ygeno.2008.11.010. Epub 2009 Feb 11.

Discovery of transcriptional regulators and signaling pathways in the developing pituitary gland by bioinformatic and genomic approaches

Michelle L Brinkmeier  1 , Shannon W Davis, Piero Carninci, James W MacDonald, Jun Kawai, Debashis Ghosh, Yoshihide Hayashizaki, Robert H Lyons, Sally A Camper
Affiliations

Discovery of transcriptional regulators and signaling pathways in the developing pituitary gland by bioinformatic and genomic approaches

Michelle L Brinkmeier et al. Genomics. 2009 May.

Abstract

We report a catalog of the mouse embryonic pituitary gland transcriptome consisting of five cDNA libraries including wild type tissue from E12.5 and E14.5, Prop1(df/df) mutant at E14.5, and two cDNA subtractions: E14.5 WT-E14.5 Prop1(df/df) and E14.5 WT-E12.5 WT. DNA sequence information is assembled into a searchable database with gene ontology terms representing 12,009 expressed genes. We validated coverage of the libraries by detecting most known homeobox gene transcription factor cDNAs. A total of 45 homeobox genes were detected as part of the pituitary transcriptome, representing most expected ones, which validated library coverage, and many novel ones, underscoring the utility of this resource as a discovery tool. We took a similar approach for signaling-pathway members with novel pituitary expression and found 157 genes related to the BMP, FGF, WNT, SHH and NOTCH pathways. These genes are exciting candidates for regulators of pituitary development and function.

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Figures

Figure 1
Figure 1. Distribution of clone representation per unique transcript for the E14.5 WT library
4339 unique UniGene entries are contained in the E14.5 WT library (Table 1). Over half of these (2644) are represented by a single cDNA clone (shown in the first column). A continually decreasing trend is observed, such that as the number of replicates or cDNA clones increases, the number of genes represented by these clones decreases. Number of Replicates (X axis) refers to the number of clones representing each unique UniGene entry. Frequency (Y axis) refers to the number of unique UniGene entries represented.
Figure 2
Figure 2. Venn diagrams illustrate the relationships between the five libraries in the encyclopedia
(A) Comparison of a total of 12,799 genes from the E12.5 WT, E14.5 WT and E14.5 Prop1df/df. (B) Comparison of a total of 5,765 genes from the E14.5 WT, E14.5 Prop1df/df and Sub1 libraries. (C) Comparison of a total of 7,590 genes from the E12.5 WT, E14.5 WT and Sub2 libraries.
Figure 3
Figure 3. In situ hybridization of four homeobox genes with novel expression in the developing pituitary gland
In situ hybridization analysis of Dlx3 at E12.5 (A) and Dlx1 (B), Rax (C) and Zhx1 (D) at E14.5 in the developing pituitary indicates robust expression. (A) At E12.5 Dlx3 is expressed strongly in the intermediate lobe (IL), shown by the white arrow, as well as in the posterior pituitary (PP) and throughout the expanding Rathke's pouch (RP), though predominantly in the dorsal aspect. The dorsal aspect is indicated by the black diagonal line. (B) At E14.5 Dlx1 is expressed throughout Rathke's pouch and the intermediate lobe. Expression appears concentrated in the ventral aspect of the pouch, as shown by the white arrow. Dlx1 expression is also present in the ventral diencephalon (VD). Rax (C) and Zhx1 (D) are expressed throughout the developing Rathke's pouch (RP). Sense controls corresponding to each gene are shown in the top left hand corner of each panel.
Figure 4
Figure 4. Expression of homeobox genes in the developing wild type and Prop1df/df pituitary
(A) Pituitary expression of a set of homeobox genes identified in the embryonic pituitary encyclopedia was validated by RT-PCR using cDNA generated from pituitary primordia at E12.5, E14.5, E18.5, and Prop1df/df E14.5. The homeobox genes Meox2, Emx2, Otx2, and Zeb2 show differential expression between E12.5-E18.5 and in the Prop1df/df E14.5 pituitary, while Meis1, Adnp, and Prrx2 showed similar expression in all samples. Hprt is a control for RNA and cDNA quality. Differential expression was quantitated using Real Time PCR (B). The quality of the cDNA samples was confirmed by demonstrating the expected pattern of Prop1 and Pit1 expression. Meox2, Emx2, Otx2, Prrx2, Meis1 and Adnp expression levels were validated using pituitary cDNA from E12.5 WT (white bars), E14.5 WT (dark gray bars), E18.5 WT (light gray bars). Different E12.5 cDNA preparations were used in the top and bottom panels of B. All samples were done in triplicate and standardized to Hprt. Fold activation and standard deviations were calculated relative to E14.5 WT based on the ΔCt values of each sample compared to the ΔCt of Hprt.
Figure 5
Figure 5. Developmental expression of 3 novel Bmp-related genes in the embryonic pituitary gland
(A) Expression of Id2 is predominantly in the intermediate lobe (IL) and around the lumen of the pituitary. Expression appears restricted to the cells that are not in the expanding proliferating Rathke's pouch (delineated by the black dashed line). Expression is also detected in the ventral diencephalon (VD). This expression pattern persists at E14.5 (B). (C) At E12.5 Id3 is expressed in the IL and dorsal part of the cells surrounding the pituitary lumen (indicated by the horizontal black line) and this expression pattern continues at E14.5 (D). (E) At E12.5 Tgfbi was detected in the mesenchymal tissue immediately surrounding the developing pituitary (shown by the black arrows). By E14.5 (F), Tgfbi expression becomes stronger in the mesenchyme and is also detected surrounding the posterior pituitary (PP) (shown by the black arrows).
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