Genomics 93 (2009) 449–460

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Contents lists available at ScienceDirect

Genomics

journal homepage: www.elsevier.com/locate/ygeno

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

Michelle L. Brinkmeier a, Shannon W. Davis a, Piero Carninci b, James W. MacDonald c, Jun Kawai b, Debashis Ghosh e,1, Yoshihide Hayashizaki b, Robert H. Lyons d, Sally A. Camper a,⁎

a Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, 48109-5618, USA b Omics Science Center, RIKEN Yokohama Institute, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan c Affymetrix and cDNA Microarray Core Facility, University of Michigan Cancer Center, Ann Arbor, MI 48109-0946, USA d Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, 48109-5638, USA e Department of Biostatistics, School of Public Health, University of Michigan, 48109-2029, USA

article info abstract

Article history: We report a catalog of the mouse embryonic pituitary gland transcriptome consisting of ﬁve cDNA libraries Received 13 December 2007 including wild type tissue from E12.5 and E14.5, Prop1df/df mutant at E14.5, and two cDNA subtractions: E14.5 Accepted 24 November 2008 WT–E14.5 Prop1df/df and E14.5 WT–E12.5 WT. DNA sequence information is assembled into a searchable Available online 11 February 2009 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 Keywords: Cap trapper were detected as part of the pituitary transcriptome, representing most expected ones, which validated Homeobox gene library coverage, and many novel ones, underscoring the utility of this resource as a discovery tool. We took a Prop1 similar approach for signaling-pathway members with novel pituitary expression and found 157 genes Gene expression related to the BMP, FGF, WNT, SHH and NOTCH pathways. These genes are exciting candidates for regulators Ames dwarf of pituitary development and function. © 2008 Elsevier Inc. All rights reserved.

Introduction its growth [1–5]. The overall effect of these signaling molecules is to induce the regional expression of numerous critical transcription The pituitary gland is a pivotal component of the endocrine system factors, which control the differentiation of the hormone secreting due to its role in controlling a wide range of fundamental bodily cells between E13.5 and birth. activities including growth, pubertal development, thyroid gland Failed or incomplete differentiation of anterior pituitary cells function, and the capacity to cope with stress. These critical functions during embryonic development can result in the genetically hetero- are regulated by six different hormones secreted by five specialized geneous disorder, Multiple Pituitary Hormone Deficiency (MPHD). cell types within the anterior pituitary gland, in response to local Molecular genetic analysis of spontaneous and genetically engineered stimulatory and inhibitory peptides released by the hypothalamus. mouse mutants with pituitary defects has revealed a functional role Pituitary development in the mouse begins at embryonic day (E) for many homeobox transcription factors in the etiology of MPHD in 9.5 and continues after birth. All of the hormone-secreting cells in the mouse and man. These include Prop1, Pou1f1, Hesx1, Lhx3, Lhx4, Otx2, anterior lobe derive from a single primordial tissue, Rathke's pouch, Pitx1, and Pitx2 [reviewed by [6–8]. Lesions in these genes only which forms from the oral ectoderm adjacent to the base of the account for approximately half of the known genetic MPHD, sug- developing brain. Formation of Rathke's pouch occurs in contact with gesting that many of the key factors involved in the process of a specialized region of the neuroectoderm, the infundibulum, which pituitary cell proliferation and differentiation remain unknown. will later form the posterior lobe of the pituitary gland. Mutations in PROP1 are the most common known genetic cause of The molecular mechanisms underlying pituitary organ growth MPHD in humans [9]. Patients with PROP1 mutations commonly and cell differentiation are complex. Signaling molecules including exhibit deficiencies in Growth Hormone (GH), Prolactin (PRL), FGF, BMP, NOTCH, WNT and SHH family members have roles in Thyroid Stimulating Hormone (TSH) and gonadotropins, with evol- establishing regional identity within Rathke's pouch and contribute to ving adrenocorticotropin deficiency (ACTH). Similarly, the sponta- neousAmesdwarfmousemodel(Prop1df/df), that harbors a homeodomain mutation in Prop1 displays a phenotype that includes ⁎ Corresponding author. Fax: +1 734 763 5831. hypoplasia of the anterior pituitary accompanied by nearly complete E-mail address: [email protected] (S.A. Camper). 1 Current address: Department of Statistics and Huck Institute of Life Sciences, Penn loss of the hormone-secreting cell types that produce GH (somato- State University, University Park, PA 16802, USA. tropes), PRL (lactotropes), and TSH (thyrotropes) [10–13].

Interestingly, genetic background has a more significant impact on the wane in expression by E14.5 such as Hesx1 [12]. We also expected to viability of Prop1 deficient mice than any functional difference find genes normally activated by Prop1, and expressed at E14.5 in WT between the Prop1df and Prop1null alleles, suggesting that genetic mice, but absent in the E14.5 Prop1df/df mutant, such as Pou1f1 [10] and modifiers may influence the clinical features of some MPHD patients Notch2 [3]. Using the Cap-trapper method to enrich for full-length [13]. clones [39–43], followed by aggressive subtraction and normalization, Prop1 expression is first detectable in Rathke's pouch at E10–10.5. we produced full-length cDNA libraries and generated DNA sequence It reaches a peak at E12.5 after which time expression declines rapidly from 56,716 cDNA clones. The comparison and global analysis of the [14]. A time of intense cell proliferation occurs in the developing transcripts contained within each library reveals expression of 12,009 pituitary from E12.5–E14.5, which precedes the appearance of the unique genes that we have integrated into a comprehensive searchable differentiated pituitary cell types. The effects of mutations in Prop1 database. We demonstrate the integrity, validity, utility and novelty of and other critical transcription factors including Pitx2, Lhx3 and Hesx1 the database by identifying 45 homeobox genes expressed in the are evident at this time [15–17]. This suggests that potential Prop1 developing anterior pituitary gland and 157 genes related to the BMP, targets and other important as yet unknown transcription factors may FGF, WNT, NOTCH or SHH signaling pathways, many of which were not be expressed around E12.5 and/or E14.5. previously known to be part of the developing pituitary transcriptome. At E12.5 the anterior pituitary is composed primarily of proliferating, undifferentiated cells. There is a very small group of non- Results proliferating cells that have begun to differentiate and express Foxl2 and the alpha subunit of the gonadotropins and thyrotropin, Cga [18]. The embryonic pituitary encyclopedia is a rich source for gene discovery At this developmental time Prop1df/df and WT pituitaries are indistinguishable in size and morphology [13,19]. By E14.5 there are We sought to investigate the depth and complexity of the developing more cells in the anterior lobe of normal mice, and they appear loosely pituitary transcriptome, while maximizing novel gene discovery and packed and glandular. In contrast, the pituitary glands of Prop1df/df developing full-length clones that could be exploited for functional mutants are highly dysmorphic at E14.5 because cells apparently fail studies of the developing pituitary gland. ‘Phase I’ involved end to migrate away from the dorsal aspect of the gland and colonize the sequencing of clones from three embryonic pituitary libraries (E12.5 anterior lobe. They exit the cell cycle but fail to differentiate [13,19,20]. WT, E14.5 WT and E14.5 Prop1df/df) until less than 10% of the new We exploited the Prop1df/df model to gain insights into the sequences produced novel information [38]. 20,270 sequences were embryonic pituitary transcriptome. We used cDNA subtractive hybri- obtained from the 5′ end, 51,379 were sequenced from the 3′ end, and dization between Prop1df/df and wild type pituitary gland primordia at 14,933 were sequenced from both ends. Here, we present the E14.5 to identify differentially expressed genes in mutant and normal identification and characterization of the cDNAs from these three embryonic pituitary [21]. We took a differential display approach to libraries. We also present two additional libraries that were generated by define an expression profile for the developing anterior pituitary at a subtractive hybridization analysis between E14.5 WT and E14.5 E12.5 and E14.5 by comparing Rathke's pouch tissue from wild type Prop1df/df (Sub1) and between E14.5 WT and E12.5 WT (Sub2). samples taken at these times [22]. Collectively, these studies repres- We conducted single pass end-sequencing of 56,716 unique cDNA ented the first expression profiles of the developing pituitary gland clones from these five libraries. The sequences were each queried using and also paved the way for demonstrating the role of the Wnt BLAST analysis against the UniGene unique subset of the NCBI Mus signaling pathway in regulating pituitary growth. More specifically, musculus database (Build #159, www.ncbi.nlm.nih.gov/UniGene/). these studies identified Wnt5a, Aes and Tcf7l2 in the developing The same sequences were also subjected to BLAST analysis against the pituitary. We have now demonstrated functional roles for each of mouse-specific Reference Sequence (RefSeq) (Release #20, www.ncbi. these genes in pituitary development through the analysis of corres- nlm.nih.gov/RefSeq/). Clones were identified based on the first BLAST ponding mutant mouse models [2,23–25]. identity recorded with an expect value of 1x10e-5 or lower. This Numerous gene expression profiling studies have been carried out analysis identified 51,352 clones present in the encyclopedia. 5364 using adult pituitary tissue and cell lines, but only a few studies have clones were not identified by BLAST analysis. While some of these addressed developmental changes in pituitary gene expression aside clones have poor sequence quality through the polyA stretch at the 3′ from those with the Prop1df/df model [26,27]. Alterations due to aging end that interferes with BLAST analysis, we anticipate that some of and caloric restriction have been investigated [28] and some studies these clones will be identified when compared to more recent UniGene with cell lines have implicated transcription factors as potential regu- and Refseq builds. Further analysis to determine the number of unique lators of development and cell proliferation [29,30]. The expression genes represented revealed 12,009 different cDNA clones distributed studies with adult pituitary tissue and cell lines identified collections between the five libraries. of potentially pituitary-specific genes [31,32], candidates for tumor biomarkers and mechanistic involvement in pituitary adenoma Table 1 formation [reviewed in [33], and pathways induced by hormonal or Number and distribution of cDNA clones in the embryonic pituitary encyclopedia growth factor treatment [34–37]. Our studies with the Prop1df/df model have been proven valuable for novel gene discovery and dissection of the genetic differences between the wild type and mutant pituitary; although, a major limitation for functional studies has been the lack of full-length cDNAs available. In addition, previous approaches have not addressed the depth and complexity of the developing pituitary transcriptome specifically. To overcome these limitations we generated a full-length cDNA encyclopedia of the mouse embryonic pituitary gland comprised of 5 transcriptome volumes including three samples and genotypes: E12.5 wild type pituitary (E12.5 WT), E14.5 wild type pituitary (E14.5 df/df a E14.5 WT-E14.5 Prop1.df/df WT) and E14.5 Prop1 pituitary [38], and two subtracted libraries: b – df/df – E14.5 WT-E12.5 WT. E14.5 WT E14.5 Prop1 , WT E14.5 WT E12.5. We selected these c Example interpretation: clones sequenced from the E12.5 WT library matched 4192 df/df times and genotypes because we expected E14.5 Prop1 mutants to different UniGene entries,1246 were unique to this library, 3029 were found in all libraries. continue to express genes that are normally expressed at E12.5 and d Library-specific transcripts are on the diagonal, shaded in gray. M.L. Brinkmeier et al. / Genomics 93 (2009) 449–460 451

tribution of 12,894 cDNA clones between the E12.5 WT, E14.5 WT and E14.5 Prop1df/df libraries (Fig. 2a). Overall, there is similar overlap between transcripts detected in these three libraries, where any two of the three libraries considered share at least 11–12% clone representation. Each of the three libraries contains a signiﬁcant pro- portion of apparently library-speciﬁc genes (14%), suggesting that each library contributes an important collection of unique genes to the encyclopedia that may be dependent on developmental stage and genotype.

The two subtracted libraries each add a dimension of genetic novelty to the encyclopedia

We compared the relative distribution of 13,035 cDNA clones between the E14.5 WT, E14.5 Prop1df/df and Sub1 libraries (Fig. 2b). The Sub1 library (E14.5 WT minus E14.5 Prop1df/df) should theoreti- cally represent those genes expressed at E14.5 in normal pituitaries but not in Prop1df/df mutants. This trend is observed, suggesting that the subtraction was effective. Gene representation in the E14.5 WT and Sub1 libraries overlaps by 16% compared to a 13% overlap between E14.5 Prop1df/df and Sub1. The percentage of genes expressed in both E14.5 WT and E14.5 Prop1df/df is also 10%. This implies that the Sub1 and E14.5 Prop1df/df libraries are as different as the E14.5 WT and E14.5 Prop1df/df libraries in terms of the expressed genes being detected or sampled. In addition to those genes that were detected in both E14.5 Fig. 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). WT and Sub1, there are also genes detected in only one library. Over half of these (2644) are represented by a single cDNA clone (shown in the first Uniquely represented genes constitute 10–14% of the total expressed column). A continually decreasing trend is observed, such that as the number of genes, which equates to 1612 genes in the case of the Sub1 library, replicates or cDNA clones increases, the number of genes represented by these clones demonstrating that it adds a dimension of genetic novelty to the decreases. Number of Replicates (X axis) refers to the number of clones representing encyclopedia. each unique UniGene entry. Frequency (Y axis) refers to the number of unique UniGene entries represented. We evaluated the E12.5 WT, E14.5 WT and Sub2 libraries, comparing 14,509 clones (Fig. 2c). The Sub2 and E14.5 WT libraries should share expression of a larger subset of genes compared to Table 1 lists the total number of cDNA clones sequenced from each E14.5 WT and E12.5 WT, and this trend was observed. E14.5 WT and library, the number of library-specific genes represented, and the Sub2 common transcripts overlap by 17%, compared to an overlap of distribution of cDNA clones relative to each of the five libraries in 10% between E14.5 WT and E12.5 WT. This overlap is equivalent to different pair-wise combinations. In terms of library-specific tran- that observed between Sub2 and E12.5 WT (13%) suggesting that scripts, 1246 genes in the encyclopedia were unique to E12.5 WT, 642 this subtraction was also effective. The Sub2 library adds new to E14.5 WT, 1256 to E14.5 Prop1df/df, 629 to the subtraction of E14.5 information to the encyclopedia as 2283 (16%) unique cDNA clones Prop1df/df from E14.5 WT, and 1264 from the subtraction of E12.5 WT present in the Sub2 library were not sampled in the E14.5 WT or from E14.5 WT. 631 genes were expressed universally in all libraries. E12.5 WT libraries.

The majority of genes are represented by a small number of clones Gene Ontology (GO) terminology reveals novel library-specific biological processes The underlying principle of our approach was to develop a tool for novel gene discovery that addressed the depth and complexity of the We wanted to determine the unique biological processes embryonic pituitary transcriptome. Our sequencing effort was represented in each library as a means for identifying the genes designed to maximize gene identification and enrich for those genes that contribute to the morphological and functional differences in that were rare rather than abundant. The extremely high ranking of pituitary development [13,19]. Using Gene Ontology (GO) terms we these libraries with regard to novelty in a pool consisting of 246 probed the five sets of genes uniquely sampled from each library for libraries generated from 71 different tissues is evidence of success enriched processes and also analyzed the set of genes that were [38]. The E14.5 Prop1df/df library ranked 1/246, where 24% of the detected universally. Twelve GO terms were significantly enriched unique clones sequenced in this library were found to be novel, and (unadjusted p-valueb0.01) in the universally expressed transcript the libraries from E14.5 WT and E12.5 WT also ranked highly, 8/246 set (Table 2). Enriched terms include ‘translation’, ‘RNA splicing’, and 16/246, respectively. Another measure of success is to ascertain ‘protein folding’ and ‘protein transport’, fundamental biological the distribution of clone representation per unique gene with the processes that are active in all tissues, irrespective of developmental expectation that the majority will have few replicates. This trend was stage or genotype. observed in all five libraries. Fig. 1 shows the results for the E14.5 WT The E14.5 WT library contains three significantly over-represented library following analysis. The same trend was observed in all of the GO terms; ‘spermidine metabolic process’, ‘hydrogen peroxide other libraries (data not shown). catabolic process’, and ‘defense response to bacterium’ (Table 2). While these were unexpected, it is not difficult to envision how genes All three non-subtracted cDNA libraries contribute unique genetic in these categories could be engaged in other known processes that information occur predominantly at this time. For example, spermidine markedly increases DNA binding activity of certain transcription factors in the To gain greater insight into the unique cDNA distribution between pituitary gland [44]. It may, therefore, play an important role in the the three non-subtracted libraries, we compared the relative dis- changing transcription characteristic of the developing pituitary at 452 M.L. Brinkmeier et al. / Genomics 93 (2009) 449–460

Fig. 2. Venn diagrams illustrate the relationships between the five libraries in the encyclopedia. (A) Comparison of a total of 12,894 genes from the E12.5 WT, E14.5 WT and E14.5 Prop1df/df. (B) Comparison of a total of 13,035 genes from the E14.5 WT, E14.5 Prop1df/df and Sub1 libraries. (C) Comparison of a total of 14,509 genes from the E12.5 WT, E14.5 WT and Sub2 libraries. this time. The ‘defense response to bacterium’ GO term is also signi- ‘knowns’, consisting of some homeobox genes with well-character- ficantly enriched in the Prop1df/df library (Table 2) and is represented ized roles in the developing pituitary gland and related gene family by more genes, compared to the E14.5 library (data not shown). members. Searches for gene name, UniGene ID, and DNA or peptide The E14.5 Prop1df/df library sampling was enriched for six GO terms sequences identified nine homeobox genes in the database (Table 3). (Table 2), five of which are not over-represented in any of the other Pou1f1, a target of Prop1 [14] was detected in the E14.5 WT library, and library samples, providing clues regarding the types of processes not in the E14.5 Prop1df/df library, as expected. Lhx3, Pitx1 and Pitx2, occurring in the Prop1df/df pituitary that could differ from the normal and two members of the Six gene family, were identified in several state. For example, ‘Response to oxygen radical’ was enriched, a libraries [6,48–51]. Other expected genes detected included Otx2, process that is involved in apoptosis, which is elevated in Prop1df/df Prop1, and Pax6 [6,7,52]. Three of the expected homeobox genes, Lhx4, [19,45]. Two significantly over-represented GO terms emerged from Hesx1, and Pou3f4, were not detected in the database following in the E12.5 WT library sample (Table 2). ‘Positive regulation of calcium silico searching. Absence from the database can be an artifact of the signaling’ precedes the catabolism of spermidine in the pituitary, sampling strategy for sequencing. For example, Hesx1 was detected by which was represented at E14.5, and the role for calcium signaling is PCR at the expected developmental stages, suggesting that deeper well documented [46,47]. The Sub1 and Sub2 library samples sequencing would have revealed the presence of these genes (data not collectively contain four over-represented GO terms, which do not shown). appear in any of the other samples (Table 2). ‘Exocytosis’ and To confirm the identity and integrity of the clones identified ‘response to pH’ may provide a platform on which to launch future computationally from end sequence, at least one clone representing functional studies for some of the genes represented. each homeobox gene identified in Table 3 was sequenced from both ends and examined to determine whether the libraries were enriched Validation of the embryonic pituitary encyclopedia for full-length clones. In most cases the clones were full-length compared to the NCBI Mus musculus UniGene database (Build #159) To validate the five libraries that comprise the embryonic pituitary validating the Cap-trapper technique for producing full-length clones. encyclopedia we queried the database for the presence of several This validation strategy revealed that the high throughput first pass M.L. Brinkmeier et al. / Genomics 93 (2009) 449–460 453

Table 2 database is searchable using a combination of text searching with Enriched gene ontology (GO) terms the terms ‘homeo’, ‘homeobox’ and ‘homeodomain’ or querying the GO terma Description p valueb database using the 60 amino acid homeodomain consensus se- Universally expressed gene set quence [53]. The most comprehensive method was to search using GO:0006412 Translation 0.0000012 gene names that are included in a list of homeodomain containing GO:0006457 Protein folding 0.0000027 proteins at Prosite (http://ca.expasy.org/prosite/). We recovered GO:0015671 Oxygen transport 0.000043 numerous additional homeobox genes expressed in the encyclopedia GO:0008380 RNA splicing 0.00013 GO:0006397 mRNA processing 0.00029 (Table 4). GO:0009408 Response to heat 0.00143 GO:0006986 Response to unfolded protein 0.00235 Differential expression analysis confirms transcription of novel GO:0015031 Protein transport 0.00242 homeobox genes GO:0046907 Intracellular transport 0.00419 GO:0006334 Nucleosome assembly 0.00629 GO:0000398 Nuclear mRNA splicing, via spliceosome 0.00739 We analyzed 23 of these genes with respect to expression in the GO:0030198 Extracellular matrix organization and biogenesis 0.00907 developing pituitary gland at E12.5, E14.5, and E18.5 using a combination of in situ hybridization analysis, RT-PCR, and quantitative PCR. Fig. 3 E12.5 WT shows a subset of the in situ hybridization results. Dlx3 and Rax GO:0019068 Virus assembly 0.0007 fi GO:0050850 Positive regulation of calcium-mediated signaling 0.0077 expression was con rmed at E14.5 (Fig. 3a and c) and Dlx1 and Zhx1 at E14.5 (Fig. 3b and d). For PCR analyses cDNA was generated from E12.5, E14.5 WT E14.5, and E18.5 WT, and E14.5 Prop1df/df pituitary primordia. The GO:0008216 Spermidine metabolic process 0.002 quality of these cDNAs was confirmed using a series of primers specific GO:0042744 Hydrogen peroxide catabolic process 0.002 for Prop1, Pou1f1, Hesx1,andHprt1 (Fig. 4, data not shown). Using GO:0042742 Defense response to bacterium 0.005 standard RT-PCR expression of 12 genes was confirmed during pituitary E14.5 Prop1df/df gland development. Meis1, Adnp, Prrx2, Lhx2, Zfhx3, Pknox2, Pbx2,and GO:0042742 Defense response to bacterium 0.0003 Meis2 were expressed at similar levels at all time points analyzed in GO:0006952 Defense response 0.0012 wild type and in Prop1df/df Rathke's pouch cDNA. Meox2, Emx2, Otx2, GO:0048015 Phosphoinositide-mediated signaling 0.0062 GO:0051046 Regulation of secretion 0.0062 and Zeb2 appeared to exhibit developmentally regulated expression, GO:0000305 Response to oxygen radical 0.0083 with the peak expression of Meox2 at E12.5, Emx2 at E14.5, and Zeb2 at GO:0006111 Regulation of gluconeogenesis 0.0083 E18.5 (Fig. 4a). In addition, expression of Meox2 and Otx2 appeared elevated in Prop1df/df mutant Rathke's pouch compared to wild type – df/df Sub1 (E14.5 WT E14.5 Prop1 ) controls. Quantitative Real Time PCR was used to confirm the results GO:0030174 Regulation of DNA replication initiation 0.002 GO:0007264 Small GTPase mediated signal transduction 0.005 suggested by standard RT-PCR (Fig. 4b). cDNA was generated from additional E12.5, E14.5, E18.5 WT, and E14.5 Prop1df/df pituitary Sub2 (E14.5 WT–E12.5 WT) primordia and the quality confirmed using primer sets for Prop1 and GO:0009268 Response to pH 0.002 Pou1f1. All samples were done in triplicate and normalized to Hprt1. GO:0006887 Exocytosis 0.004 The fold activation was determined by comparison to E14.5 WT, which a www.geneontology.org. was set to 1. Both Meox2 and Otx2 peak in expression at E12.5 and are b GO terms with an unadjusted p-valueb0.01 are listed. elevated 7.9 and 6 fold, respectively, relative to E14.5 WT. The peak expression of Emx2 is between E12.5 and E14.5, while Zeb2 peaks later in sequencing correctly identifies greater than 95% of the clones development at E18.5. Meox2 and Otx2 are slightly elevated in the E14.5 accurately. The misidentified clones tended to be ones with poor Prop1df/df mutants relative to E14.5 WT by 1.59 fold and 2.06 fold, expect values from the initial BLAST analysis. respectively. Zeb2, Adnp,andMeis1 are decreased in the Prop1df/df mutants relative to E14.5 WT by 2.09, 2.15, and 2.39 fold, respectively. Identification of additional homeobox genes in the embryonic pituitary Prrx2 does not exhibit developmental regulation or differences between encyclopedia wild type and Prop1df/df. Taken together these results suggest that Meox2, Otx2, Zeb2, Adnp,andMeis1 could merit further evaluation as Given the critical roles of numerous homeobox genes in the dev- downstream targets of Prop1. eloping pituitary gland, we used the database as a tool for discovering The identification of individual homeobox genes in the specific additional homeobox genes expressed in the pituitary gland. The libraries does not necessarily correlate with the actual expression

Table 3 Libraries contain homeobox genes implicated in pituitary development

Gene Gene name Unigene IDa Number of clones identiﬁedb pituitary cDNA source symbol E12.5 E14.5 E14.5 Sub1c Sub2d WT WT Prop1df/df Lhx3 LIM homeobox protein 3 Mm.386765 5 3 2 5 Otx2 Orthodenticle homolog 2 Mm.134516 1 Pax6 Paired box gene 6 Mm.3608 1 Pitx1 Paired-like homeodomain transcription factor 1 Mm.135195 1 1 2 3 Pitx2 Paired-like homeodomain transcription factor 2 Mm.246804 2 1 2 5 Pou1f1 POU domain, class 1, transcription factor 1 Mm.457971 3 Prop1 Paired like homeodomain factor 1 Mm.1386 2 2 4 Six3 Sine oculis-related homeobox 3 homolog Mm.370208 1 1 1 3 Six6 Sine oculis-related homeobox 6 homolog Mm.57138 4 8 5 1 2

a NCBI UniGene accession number. b Number of cDNA clones identiﬁed in each library corresponding to indicated gene. Blank indicates none. c E14.5 WT–E14.5 Prop1df/df. d E14.5 WT–E12.5 WT. 454 M.L. Brinkmeier et al. / Genomics 93 (2009) 449–460

Table 4 Identiﬁcation of additional homeobox genes in the embryonic pituitary encyclopedia

Gene Gene name Number of clones identified a pituitary cDNA source Homeobox b symbol E12.5 WT E14.5 WT E14.5 Prop1df/df Sub1 Sub2 class Adnp Activity-dependent neuroprotective protein 1 3 3 POU-like Dlx1 Distal-less homeobox 1 1 3 Dll Dlx3 Distal-less homeobox 3 1 Dll Emx2 Empty spiracles homolog 2 3 1 EMS Hlx H2.0-like homeo box 1 HLX Homez Homeodomain leucine zipper-encoding 2 1 ZF Isl1 Islet 1 transcription factor 2 1 LIM Lass2 LAG1 homolog, ceramide synthase 2 3 2 Lass3 LAG1 homolog, ceramide synthase 3 1 Lass4 LAG1 homolog, ceramide synthase 4 3 3 Lass5 LAG1 homolog, ceramide synthase 5 1 1 2 Lass6 LAG1 homolog, ceramide synthase 6 3 2 Lhx2 LIM homeobox protein 2 [69] 1 LIM Meis1 Myeloid ecotropic viral integration site 1 1 TALE Meis2 Myeloid ecotropic viral integration site-related gene 1 [66,71] 2 1 TALE Meox2 Mesenchyme homeobox 2 1 TALE Msx1 Homeobox, msh-like 1 [67,86] 1 1 1 MSH Nkx2-4 NK2 transcription factor related, locus 4 2NK Nkx3-1 NK-3 transcription factor, locus 1 3NK Pax1 Paired box gene 1 2 2 PRD Pbx2 Pre B-cell leukemia transcription factor 2 1 3 TALE Pknox1 Pbx/knotted 1 homeobox 1 [64] 1 TALE Pknox2 Pbx/knotted 1 homeobox 2 1 TALE Pou4f1 POU domain class 4, transcription factor 1 [37] 1 1 POU Pou6f1 POU domain class 4 transcription factor 1 [70] 1 POU Prrx2 Paired related homeobox 2 1 PRD Prrxl1 Paired related homeobox protein-like 1 2 PRD Rax Retina and anterior neural fold homeobox [63] 2 2 1 PRD Satb1 Special AT-rich sequence binding protein 1 2 1 3 CUT Six1 Sine oculis-related homeobox 1 homolog 5 2 4 10 6 SIX Six4 Sine oculis-related homeobox 4 homolog 1 3 2 SIX Tgif1 TGFB-induced factor homeobox 1 1 1 TALE Tgif2 TGFB-induced factor homeobox 2 2 TALE Zeb2 Zinc finger E-box binding homeobox 2 [60] 1ZF Zfhx3 Zinc finger homeobox 3 1 2 ZF Zhx1 Zinc fingers and homeoboxes protein 1 2 ZF

a Number of cDNA clones identiﬁed in each library corresponding to indicated gene. b Homeobox class is indicated where known. None known for Lass family.

profile of that gene. For instance, Meis1 was only identified in the revealing a total of 61 genes expressed in the developing pituitary E12.5 library; however, our RT-PCR data show that it is expressed at gland related to the BMP (N=20), FGF (N=13), NOTCH (N=2), SHH E12.5, E14.5, and E14.5 Prop1df/df embryos. Therefore, it is mislead- (N=1) or WNT (N=25) signaling pathways. A subset corresponding ing to assume that the identification of a gene in a specific library or to the FGF pathway is shown in Table 5, and the complete list is even a subtracted library provides accurate differential expression available in Supplementary Table 1.17additionalgeneswere information. identified that are involved in functional crosstalk between two or more signaling pathways (Table 5 and Supplementary Table 1). Database analysis implicates additional signaling molecules in pituitary As an alternative strategy we searched the library for GO terms development related to signaling and for specific gene names for genes known to be involved in the BMP, FGF, NOTCH, SHH, and WNT pathways. This Fundamental to the current understanding of the genetic mechan- search identified an additional 72 genes (Supplement Table 1). This isms driving pituitary development has been the identification of list has not been verified by end sequencing yet. numerous signaling pathways that exert their effects on the pituitary primordia. Previous studies have demonstrated the importance of Spatial expression of three Bmp-related genes in the developing pituitary individual members of the BMP, FGF, NOTCH, SHH and WNT signaling pathways in directing normal pituitary development [1–3,5,23– We searched the set of 61 signaling genes described above for 25,54,55]. A continuing challenge in the field is to identify additional those with previously demonstrated roles in the developing pituitary. members of these pathways and to determine how these various Twelve “known” genes are in the set including Aes and Tle4 [23], signaling pathways interact with each other during pituitary organo- Bmpr1a and Bmpr1b [56], Chrd [55], Fgf10 [1] Nkx2-1 [4], Hey1 and genesis to promote development of the organ. Notch2 [3], Wnt5a [2,25,55], Axin2 [2,57] and Fzd5 [21] (Supplemen- We used Genomatix software (www.genomatix.de/) to build tary Table 1). Previously we presented the expression pattern for the functional genetic networks for signaling pathways known to affect BMP signaling related genes Nbl1 and Fstl1, found in our develop- pituitary development and queried the database for the expression of mental library, at E12.5 and E14.5, and for Id3 at E10.5 [54].We these genes. For example, in the case of the BMP pathway, a group of expanded the analysis of Id3 and examined the expression of Id2 and 78 Bmp-related genes emerged, and we identified 20 of them in the Tgfbi (Fig. 5). Id2 expression was detected in the dorsal aspect of encyclopedia. This analysis was conducted for additional pathways, Rathke's pouch at E12.5 (Fig. 5a). Expression persists at E14.5 in the M.L. Brinkmeier et al. / Genomics 93 (2009) 449–460 455

additional 16 genes in our encyclopedia that are involved in functional cross talk during organ development, including pituitary (Supple- mentary Table 1). A subset of these (4/16) is shown in Table 5. 4/16 genes of the pathway interaction genes identified belong to the helix–loop–helix transcription factor family (Table 5). These include Ascl1 and Id1. The zebrafish Ascl1 orthologue (Ascl1a)isa critical gene for pituitary cell differentiation and cell survival [58]. Zebrafish Fgf3 mutants fail to express Ascl1a, suggesting that Ascl1a acts downstream of Fgf. Zebrafish Fgf3 is orthologous to mammalian Fgf10, known for its function in mammalian pituitary development, implying a conserved mechanism for FGF signaling and Ascl1 expression [1]. Additionally, Id1 mediated expression of Bmp2 leads to reduced Ascl1 expression [59]. Ascl1, Id1, Id2, Id3, and Id4 are all implicated as differentially expressed in pituitary tumors [28,60–62]. Together these studies suggest a functional link between FGF and BMP signaling and identify the Id gene family and Ascl1 as interesting candidates in mediating cell differentiation in the developing pituitary. This example demonstrates the utility of the embryonic pituitary encyclopedia in providing a foundation for furthering our understanding of signaling in the developing pituitary.

Discussion

In previous studies we described a partial expression profile of the developing pituitary gland and paved the way for understanding the role of WNT signaling in pituitary development using a combination of differential display PCR [22] and subtractive hybridization [21]. The datasets generated in these studies were fairly small (b400 genes in each), but the gene discovery was significant, providing proof of principle that generation of a more comprehensive pituitary transcriptome profile would likely enable significant gene discovery and Fig. 3. In situ hybridization of four homeobox genes with novel expression in the developing pituitary gland. In situ hybridization analysis of Dlx3 at E14.5 (A) Dlx1 at provide insight into the transcription factors and signaling molecules E12.5 (B), Rax at E14.5 (C) and Zhx1 at E12.5 (D) in the developing pituitary indicates expressed in the developing pituitary. robust expression. (A) At E14.5 Dlx3 is expressed strongly in the intermediate lobe (as In this study we generated a full-length cDNA encyclopedia of well as in the posterior pituitary (PP) and throughout the expanding Rathke's pouch the developing pituitary at wild type E12.5 and E14.5 and in the (RP). (B) At E12.5 Dlx1 is expressed throughout Rathke's pouch and the intermediate df/df lobe. Expression appears concentrated in the dorsal aspect of the pouch, as shown by Prop1 pituitary at E14.5 using an approach designed to enrich for the white arrow. Dlx1 expression is also present in the ventral diencephalon (VD). Rax full-length clones, maximize gene discovery, and permit high through- (C) and Zhx1 (D) are expressed throughout the developing Rathke's pouch (RP). Sense put gene recovery [38,40–43]. Using sequence data from these libraries controls corresponding to each gene are shown in the top left hand corner of each we built a comprehensive database through which to access transcript panel. information. It is searchable by Gene Ontology (GO) terminology; direct sequence query, including nucleic and amino acid sequence; expanding anterior pituitary (Fig. 5b) but appears restricted to those gene name or text-string options; motifs for transcription factor cells closest to the lumen of the pouch. Id2 does not appear to be binding; and UniGene ID and database-specific clone number. This expressed in the proliferating cells of the expanding Rathke's pouch. encyclopedia contains 12,009 unique transcripts, represented by 56,716 By E16.5 Id2 expression is barely detectable in the pituitary (data not cDNA clones, and accounts for over half of the known genes in the shown). Id3 expression was detected in Rathke's pouch at E12.5, mouse genome (22,723) (http://www.ensembl.org/Mus_musculus/ enriched in the dorsal part of the pouch and in the intermediate lobe index.html). Thus, the developing pituitary transcriptome has depth (Fig. 5c). Similar to Id2, Id3 expression does not appear in the proli- and complexity, and our study provides clones ideal for use in func- ferating cells of the expanding Rathke's pouch. Expression of Id3 tional studies such as cell culture transfection and the generation of continues at E14.5 (Fig. 5d) in a similar pattern, restricted to the dorsal transgenic mice. pouch and intermediate lobe and is barely detectable by E16.5 (data Each of the libraries contained within the encyclopedia includes a not shown). Tgfbi showed a striking expression pattern. While there set of library-specific cDNA clones representing between 629 (Sub1) are no transcripts in Rathke's pouch itself, the gene is expressed in the and 1264 (Sub2) unique transcripts. Exclusivity in any one library is mesenchymal tissue immediately surrounding the pouch at E12.5 (Fig. not a true readout of the pituitary transcriptome, but rather, a 5e). At E14.5, this expression continues more strongly and is also reflection of the sequencing approach taken. Novelty and enrichment detectable surrounding the posterior pituitary (Fig. 5f). Like Id2 and for rare transcripts were favored in place of the complete sequencing Id3, Tgfbi expression wanes by E16.5. These studies confirm expression of any one library. Despite this limitation, the GO terminology analysis of these genes in the pituitary and support the encyclopedia as reflec- suggests that certain biological processes are significantly enriched in tion of pituitary gene expression. a library-specific manner. The genes represented by these processes could provide the basis for future novel mechanistic studies of Interplay of signaling pathways in the developing pituitary embryonic pituitary development. We identified a total of 45 homeobox genes that are expressed in We surveyed the signaling genes represented in the encyclopedia the developing pituitary gland, expanding significantly on the subset of using Genomatix software to identify those genes that displayed a homeobox genes with well-characterized roles. Transcripts for seven functional overlap between two or more of the five pathways defined of these newer genes have been detected in pituitary cell lines (Lhx2, in Supplementary Table 1. This analysis revealed the presence of an Meis2, Pknox1, Pou6f1), pituitary adenomas (Pou4f1, Zeb2), or other 456 M.L. Brinkmeier et al. / Genomics 93 (2009) 449–460

Fig. 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. Hprt1 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 Hprt1. 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 Hprt1. developing vertebrates (Rax) [37,60,63–71]. These genes correspond to E14.5 pituitary (Fig. 4), and Zhx1 has been detected at e12.5 by in situ over fifteen different homeodomain classes including the LIM, Paired hybridization (data not shown). Mutations in one of these genes, ZEB2 (Prd), Paired-like (Prd-like) and POU homeobox families [reviewed by or ZFHX1B, cause Mowat Wilson Syndrome, a human disorder char- [72]]. Given the importance of known genes from these gene families acterized by a distinctive facial phenotype in association with mental in pituitary organogenesis such as Prop1, Pou1f1, Pitx1, and Pitx2, the retardation, microcephaly, and short stature [73]. Because this gene is identification of Adnp (POU-like), Prrx2 (Prd), and Prrxl1 (Prd), expressed in human pituitary, it is intriguing to hypothesize that suggests that these genes may also play important roles in driving affected individuals with this syndrome have a pituitary defect [60]. cell proliferation and/or differentiation during the formation of the Several genes belonging to the TALE family of homeobox genes pituitary gland. Mutations in some of these genes may underlie some were identified in the embryonic pituitary (Pbx2, Meox2, Meis1, Meis2, cases of MPHD, where the current genetic cause is unknown. Pknox2, Tgif1, and Tgif2). Genes from this family are required to specify Interestingly, Meox2, Otx2, Emx2, and Zeb2 are differentially cell fate in numerous organs including the spleen, and they may play a expressed in the developing Rathke's pouch between E12.5 and similar role in the developing pituitary [74]. The TALE gene Pbx1 E18.5. No obvious differences in pituitary cell specification are noted in regulates expression of the Fshb gene in pituitary gonadotrope-like Emx2 mutant mice [87]. Meox2 and Otx2 are elevated in the Prop1df/df cells and Gnrh1 in the hypothalamus [64,75]. MEIS2 interacts with E14.5 pituitary relative to E14.5 wild type, and Prop1df/df newborns LHX2 to regulate Cga expression in pituitary gonadotrope-like cells have ectopic, elevated expression of Otx2 compared to wild type [66]. Our expression analysis of Meox2 suggests that Prop1 may littermates (Mortensen, unpublished observation). Meis1 and Adnp repress its expression during pituitary development. are reduced in the Prop1df/df E14.5 pituitary relative to E14.5 WT. The One of the current challenges in the field is to determine how the dynamic expression patterns of these homeobox genes in the various signaling pathways interact with each other during pituitary developing pituitary raise the question of whether they are regulated organogenesis. BMP and FGF signals influence anterior pituitary cell by Prop1 and whether they have functional roles in pituitary differentiation [1,55], and SHH signaling may participate in boundary development. formation with FGF and BMP signaling in the ventral diencephalon Some of the remaining homeobox genes identified represent novel [54,55]. Cross talk between the WNT, BMP, FGF and SHH pathways is classes of homeobox genes, not previously found in the developing evident in Tcf7l2, Wnt5a, and noggin mutants, which exhibit pituitary pituitary. For example, three zinc finger (ZF) homeobox genes were developmental abnormalities [2,24,54]. Using the embryonic pituitary identified in the encyclopedia (Table 4). Zeb2 expression was detected encyclopedia we identified 79 additional BMP, FGF, NOTCH, SHH or at low levels at E12.5, expression levels increased at both E14.5 and WNT pathway members with novel expression in the developing E18.5 in normal mice and expression was reduced in the Prop1df/df pituitary. One such example is Fgf13, which is critical for neural M.L. Brinkmeier et al. / Genomics 93 (2009) 449–460 457

Table 5 genotyping was performed as described [21], and pituitaries were a Identification of signaling-related genes in the embryonic pituitary encyclopedia pooled for RNA extraction and cDNA generation following genotyping. Gene symbol Gene name CD1 mice were purchased from Charles River Laboratories (Wilming- Fgf-relatedb ton, MA) as timed pregnancies and pituitary glands were dissected Fgf10 Fibroblast growth factor 10 from E12.5, E14.5, and E18.5 embryos. Pituitaries were pooled for RNA Fgf13 Fibroblast growth factor 13 extraction and cDNA generation. The University of Michigan Commit- Fgf14 Fibroblast growth factor 14 tee on Use and Care of Animals approved all experiments. Fgf17 Fibroblast growth factor 17 Fgfr1 Fibroblast growth factor receptor 1 Fgfr2 Fibroblast growth factor receptor 2 Library generation Fibp Fibroblast growth factor intracellular binding protein Frs3 Fibroblast growth factor receptor substrate 3 Pituitary tissue for library generation was prepared as described Spry1 Sprouty homolog 1 (Drosophila) [38]. Briefly, starting from 50 µg of total RNA, first strand cDNA was Spry2 Sprouty homolog 2 (Drosophila) Spred2 Sprouty-related, EVH1 domain containing 2 prepared with an anchored oligo-dT primer adapter [80] and the Sp3 Trans-acting transcription factor 3 cDNA was cap-selected [81] and subsequently cloned in a Lambda FLC Tbx3 T-box 3 I vector [41]. Libraries were excised into plasmids and sequenced. E14.5 WT was given the library ID K7, E14.5 Prop1df/df was K8, and BMP-relatedc Id2 Inhibitor of DNA binding 2 E12.5 WT was K9 [38]. Additionally, subtracted libraries were pre- df/df Id3 Inhibitor of DNA binding 3 pared from E14.5 WT minus E14.5 Prop1 and E14.5 WT minus Tgfβi Transforming growth factor, beta induced E12.5 WT, using established procedures [82].

Functional cross talkd Database generation Ascl1⁎ Achaete-scute complex homolog-like 1 (Drosophila) Id1⁎ Inhibitor of DNA binding 1 Jun⁎ Jun oncogene Mouse UniGene Build #159 (Mm.seq and Mm.seq.uniq) and Neurod1⁎ Neurogenic differentiation 1 Mouse RefSeq Release 20 were obtained from NCBI FTP servers, a A subset of the 63 genes identified is shown. The complete list is available in along with the ‘blastall’ program used to perform the BLAST Supplementary Figure 1. identifications. Each cDNA sequence was first BLASTed against the b Of the Fgf genes detected, only Fgf10 was previously known to be expressed in the Mm.seq.uniq dataset, and then against the full mouse UniGene set pituitary gland. (Mm.seq). All cDNA sequences were also BLASTed against RefSeq to c The expression patterns of these were studied by in situ hybridization. fi d The asterisk⁎ indicates genes in the basic helix–loop–helix family. provide an alternative identi cation. A database was constructed to contain the sequences themselves, along with Riken identification and a ‘library’ identification, indicative differentiation in Xenopus [76]. Additionally, three Sprouty genes were of the tissue source along with the UniGene and RefSeq identifications, identified in the encyclopedia (Spry1, Spry2 and Spred2). Spry1, Spry2, where obtainable. Web-based search functions were developed to and Spry4 expression was previously reported in E14.5 Rathke's pouch allow exploration of the contents of the various libraries by sequence, [77]. Sprouty genes have highly conserved roles as antagonists of FGF gene name, etc. From the GO Consortium (www.geneontology.org) signaling, which can affect differentiation [78,79]. Thus, several we downloaded a database of GO identifiers in hierarchical format members of the Sprouty family may regulate FGF signaling during (10/31/06 release). This data, along with files from the NCBI ‘Gene’ pituitary organogenesis. database (‘gene2go’, ‘gene2unigene’) allowed us to query our UniGene An important application of our studies lies in the ability to utilize identifications using GO terminology. the full-length cDNA clones. Some examples are the full-length cDNA clones for the Fstl1, Id3, and Six6 genes that were used for expres- Gene Ontology (GO) terminology analysis sion analysis by in situ hybridization in pituitary development in demonstrating the important role of noggin [54]. Full-length cDNA Over-represented Gene Ontology (GO) terms in the set of unique clones for the forkhead transcription factor Foxl2 and numerous sequences from each library as well as the intersecting sequences for members of both the Wnt and Frizzled gene families, (Fzd6, Axin2 and all libraries were detected using a modification of the standard Fisher's Dvl2, see Supplementary Table 1) have been invaluable [2,18]. exact test [83]. Briefly, we selected those sequences that were unique In conclusion, the embryonic pituitary encyclopedia represents a to each library based on the UniGene ID and mapped these IDs to valuable gene discovery tool that can be used to identify novel genes Entrez Gene IDs using the mouseLLMappings package of Bioconductor and pathways in the developing wild type and mutant pituitary (http://www.bioconductor.org). We mapped the union of all (Prop1df/df) and to initiate functional studies. We have just scratched sequences in each library to Entrez Gene to use as the ‘universe’ from the surface and anticipate that this full-length cDNA encyclopedia will which the unique sequences were selected. In other words, the usual act as an ongoing resource for studying pituitary development and heuristic explanation of a Fisher's exact test is the idea of selecting disease. colored balls from an urn. In our case, the unique sequences from each library are the balls that were selected from the urn, and the union of all Materials and methods sequences in each library is the ‘universe’ of balls in the urn. We computed over-representation of GO terms using the topGO package of Mice Bioconductor, which uses the directed acyclic graph (DAG) structure of the GO ontologies to decorrelate significant subordinate terms from Prop1df/+ mouse stocks were obtained from Dr. A. Bartke (South- consideration when estimating the significance of higher-order terms. ern Illinois University, Carbondale, Ill.) in 1988 and maintained at the University of Michigan according to NIH guidelines. Heterozygote In situ hybridization carriers were intercrossed to generate timed pregnancies and the morning after mating was designated as E0.5. Developing pituitary RNA in situ hybridization was performed as described previously glands were dissected from E14.5 embryos with the aid of a dissecting [84]. Dlx1, Dlx3, Rax, Zhx1, Id2, Id3 and Tgfbi cDNAs were isolated from microscope and were individually stored in RNA Later (Ambion, the embryonic pituitary encyclopedia [38]. Id2, Id3 and Tgfbi were Austin, TX) at −20 °C. Genomic DNA was extracted from the yolk sac, linearized with NotI, and Dlx1, Dlx3, Rax and Zhx1 were linearized 458 M.L. Brinkmeier et al. / Genomics 93 (2009) 449–460

Fig. 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 and is excluded from the forming anterior lobe (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).

with SacII. T3 polymerase was used in all cases to generate antisense concentrations were 1×PCR Reaction buffer (Roche, Indianapolis, IN), probes. 1.5 mM MgCl2 (Perkin Elmer, Waltham, MA), 200 mM dNTP mix (Roche), and 0.4 U GoTaq polymerase (Perkin Elmer) per 25 μl RT-PCR and PCR reaction. Standard reactions contained 25–50 ng template cDNA. All PCR reactions were performed using the following conditions: RNA was extracted from pituitary tissue dissected from CD1 mouse 94 °C; 4 min, followed by 30 cycles of 94 °C; 30 s, PCR-specific embryos at E12.5, E14.5, and E18.5 and stored in RNAlater (Ambion, annealing temperature; 30 s and 72 °C; 30 s, followed by 1 cycle of Foster City, CA) following the recommended commercial protocol. 72 °C; 10 min. PCR-specific annealing temperatures and primers are RNA was further purified via RNAqueous 4PCR (Ambion, Foster City, listed in Supplementary Table 2. CA). cDNA was prepared from total RNA using the Superscript First Quantitative Real Time PCR was performed using TaqMan Gene Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA). Briefly, Expression Assays On Demand (Applied Biosystems, Foster City, CA). 5 μg total RNA was incubated at 42 °C for 50 min in a 10 μl reaction Briefly25–50 ng of template cDNA was added to 10 μl Taqman containing 2 μl cDNA buffer, 4 μl MgCl2,2μl0.1μl DTT, and 1 μlof Universal PCR Master Mix (Applied Biosystems), 8 μl double distilled Superscript II RT enzyme. Standard PCR reaction components at final H2O, and 1 μl TaqMan gene specific primer set. All samples were done M.L. Brinkmeier et al. / Genomics 93 (2009) 449–460 459 in triplicate and standardized to Hprt1. Real Time PCR was performed [18] B.S. Ellsworth, N. Egashira, J.L. Haller, D.L. Butts, J. Cocquet, C.M. Clay, R.Y. Osamura, S.A. Camper, FOXL2 in the pituitary: molecular, genetic, and developmental using the Prism 7000 Sequence Detection System (Applied Biosys- analysis, Mol. Endocrinol. 20 (2006) 2796–2805. tems). Fold activation and standard deviations were determined as [19] R.D. Ward, L.T. Raetzman, H. Suh, B.M. Stone, I.O. Nasonkin, S.A. Camper, Role of previously described [85]. PROP1 in pituitary gland growth, Mol. Endocrinol. 19 (2005) 698–710. [20] R.D. Ward, B.M. Stone, L.T. Raetzman, S.A. Camper, Cell proliferation and vascu- larization in mouse models of pituitary hormone deficiency, Mol. Endocrinol. 20 Acknowledgments (2006) 1378–1390. [21]K.R.Douglas,M.L.Brinkmeier,J.A.Kennell,P.Eswara,T.A.Harrison,A.I. This work was supported by the following grants: National Patrianakos, B.S. Sprecher, M.A. Potok, R.H. Lyons Jr., O.A. MacDougald, S.A. Camper, Identification of members of the Wnt signaling pathway in the Institutes of Health R37HD30428 and HD34283 (SAC), NIH Grant embryonic pituitary gland, Mamm. Genome 12 (2001) 843–851. R01GM72007 (JM, DG), University of Michigan Center for Computa- [22] K.R. Douglas, S.A. Camper, Partial transcriptome of the developing pituitary gland, – tional Medicine and Biology (SAC, RHL, DG), University of Michigan Genomics 70 (2000) 335 346. [23] M.L. Brinkmeier, M.A. Potok, K.B. Cha, T. Gridley, S. Stifani, J. Meeldijk, H. Clevers, DNA Sequencing Core Facility (RHL) and Research Grant for the RIKEN S.A. Camper, TCF and Groucho-related genes influence pituitary growth and Genome Exploration Research Project from the Ministry of Education, development, Mol. Endocrinol. 17 (2003) 2152–2161. Culture, Sports, Science and Technology of the Japanese Government [24] M.L. Brinkmeier, M.A. Potok, S.W. Davis, S.A. Camper, TCF4 deficiency expands ventral diencephalon signaling and increases induction of pituitary progenitors, (YH) and a grant for the Genome Network Project from the Ministry of Dev. Biol. 311 (2007) 396–407. Education, Culture, Sports, Science and Technology, Japan (YH). We [25] K.B. Cha, K.R. Douglas, M.A. Potok, H. Liang, S.N. Jones, S.A. Camper, WNT5A thank A.H. Mortensen, M.A. Potok, and N.M. Solomon for their signaling affects pituitary gland shape, Mech. Dev. 121 (2004) 183–194. [26] L.E. Ellestad, W. Carre, M. Muchow, S.A. Jenkins, X. Wang, L.A. Cogburn, T.E. Porter, contributions to this work. Gene expression profiling during cellular differentiation in the embryonic pituitary gland using cDNA microarrays, Physiol Genomics 25 (2006) 414–425. Appendix A. Supplementary data [27] A.H. Mortensen, J.W. MacDonald, R.D. Ward, S.A. Camper, Comparative Gene Expression PROP1 and PIT1 Newborn Pituitaries, Endo 2007, Toronto, ON, 2007. [28] H. Chen, Gene expression by the anterior pituitary gland: effects of age and caloric Supplementary data associated with this article can be found, in restriction, Mol. Cell. Endocrinol. 222 (2004) 21–31. the online version, at doi:10.1016/j.ygeno.2008.11.010. [29] H.P. Mohammad, D.D. Seachrist, C.C. Quirk, J.H. Nilson, Reexpression of p8 contributes to tumorigenic properties of pituitary cells and appears in a subset of prolactinomas in transgenic mice that hypersecrete luteinizing hormone, Mol. References Endocrinol. 18 (2004) 2583–2593. [30] C.C. Quirk, D.D. Seachrist, J.H. Nilson, Embryonic expression of the luteinizing [1] J. Ericson, S. Norlin, T.M. Jessell, T. Edlund, Integrated FGF and BMP signaling hormone beta gene appears to be coupled to the transient appearance of p8, a controls the progression of progenitor cell differentiation and the emergence of high mobility group-related transcription factor, J. Biol. Chem. 278 (2003) pattern in the embryonic anterior pituitary, Development 125 (1998) 1005–1015. 1680–1685. [2] M.A. Potok, K.B. Cha, A. Hunt, M.L. Brinkmeier, M. Leitges, A. Kispert, S.A. Camper, [31] A.I. Su, M.P. Cooke, K.A. Ching, Y. Hakak, J.R. Walker, T. Wiltshire, A.P. Orth, R.G. WNT signaling affects gene expression in the ventral diencephalon and pituitary Vega, L.M. Sapinoso, A. Moqrich, A. Patapoutian, G.M. Hampton, P.G. Schultz, J.B. gland growth, Dev. Dyn. 237 (2008) 1006–1020. Hogenesch, Large-scale analysis of the human and mouse transcriptomes, Proc. [3] L.T. Raetzman, S.A. Ross, S. Cook, S.L. Dunwoodie, S.A. Camper, P.Q. Thomas, Natl. Acad. Sci. U. S. A. 99 (2002) 4465–4470. Developmental regulation of Notch signaling genes in the embryonic pituitary: [32] S. Tanaka, K. Tatsumi, K. Okubo, K. Itoh, S. Kawamoto, K. Matsubara, N. Amino, Prop1 deficiency affects Notch2 expression, Dev. Biol. 265 (2004) 329–340. Expression profile of active genes in the human pituitary gland, J. Mol. Endocrinol. [4] N. Takuma, H.Z. Sheng, Y. Furuta, J.M. Ward, K. Sharma, B.L. Hogan, S.L. Pfaff, H. 28 (2002) 33–44. Westphal, S. Kimura, K.A. Mahon, Formation of Rathke's pouch requires dual [33] X. Qian, B.W. Scheithauer, K. Kovacs, R.V. Lloyd, DNA microarrays: recent induction from the diencephalon, Development 125 (1998) 4835–4840. developments and applications to the study of pituitary tissues, Endocrine 28 [5] M. Treier, S. O'Connell, A. Gleiberman, J. Price, D.P. Szeto, R. Burgess, P.T. Chuang, A. (2005) 49–56. P. McMahon, M.G. Rosenfeld, Hedgehog signaling is required for pituitary gland [34] R.A. Abbud, R. Kelleher, S. Melmed, Cell-specific pituitary gene expression profiles development, Development 128 (2001) 377–386. after treatment with leukemia inhibitory factor reveal novel modulators for [6] D. Kelberman, M.T. Dattani, The role of transcription factors implicated in anterior proopiomelanocortin expression, Endocrinology 145 (2004) 867–880. pituitary development in the aetiology of congenital hypopituitarism, Ann. Med. [35] J.M. Kerr, D.F. Gordon, W.W. Woodmansee, V.D. Sarapura, E.C. Ridgway, W.M. 38 (2006) 560–577. Wood, Growth arrest of thyrotropic tumors by thyroid hormone is correlated with [7] I. Matsuo, S. Kuratani, C. Kimura, N. Takeda, S. Aizawa, Mouse Otx2 functions in the novel changes in Wnt-10A, Mol. Cell. Endocrinol. 238 (2005) 57–67. formation and patterning of rostral head, Genes Dev. 9 (1995) 2646–2658. [36] E. Wurmbach, T. Yuen, B.J. Ebersole, S.C. Sealfon, Gonadotropin-releasing hormone [8] D.P. Szeto, C. Rodriguez-Esteban, A.K. Ryan, S.M. O'Connell, F. Liu, C. Kioussi, A.S. receptor-coupled gene network organization, J. Biol. Chem. 276 (2001) Gleiberman, J.C. Izpisua-Belmonte, M.G. Rosenfeld, Role of the Bicoid-related 47195–47201. homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary [37] H. Zhang, J.S. Bailey, D. Coss, B. Lin, R. Tsutsumi, M.A. Lawson, P.L. Mellon, N.J. development, Genes Dev. 13 (1999) 484–494. Webster, Activin modulates the transcriptional response of LbetaT2 cells to [9] S. Mody, M.R. Brown, J.S. Parks, The spectrum of hypopituitarism caused by PROP1 gonadotropin-releasing hormone and alters cellular proliferation, Mol. Endocri- mutations, Best Pract. Res. Clin. Endocrinol. Metab. 16 (2002) 421–431. nol. 20 (2006) 2909–2930. [10] B. Andersen, R.V. Pearse II, K. Jenne, M. Sornson, S.C. Lin, A. Bartke, M.G. Rosenfeld, [38] P. Carninci, K. Waki, T. Shiraki, H. Konno, K. Shibata, M. Itoh, K. Aizawa, T. Arakawa, The Ames dwarf gene is required for Pit-1 gene activation, Dev. Biol. 172 (1995) Y. Ishii, D. Sasaki, H. Bono, S. Kondo, Y. Sugahara, R. Saito, N. Osato, S. Fukuda, K. 495–503. Sato, A. Watahiki, T. Hirozane-Kishikawa, M. Nakamura, Y. Shibata, A. Yasunishi, N. [11] P.J. Gage, M.L. Brinkmeier, L.M. Scarlett, L.T. Knapp, S.A. Camper, K.A. Mahon, The Kikuchi, A. Yoshiki, M. Kusakabe, S. Gustincich, K. Beisel, W. Pavan, V. Aidinis, A. Ames dwarf gene, df, is required early in pituitary ontogeny for the extinction of Nakagawara, W.A. Held, H. Iwata, T. Kono, H. Nakauchi, P. Lyons, C. Wells, D.A. Rpx transcription and initiation of lineage-specific cell proliferation, Mol. Hume, M. Fagiolini, T.K. Hensch, M. Brinkmeier, S. Camper, J. Hirota, P. Mombaerts, Endocrinol. 10 (1996) 1570–1581. M. Muramatsu, Y. Okazaki, J. Kawai, Y. Hayashizaki, Targeting a complex [12] P.J. Gage, M.L. Roller, T.L. Saunders, L.M. Scarlett, S.A. Camper, Anterior pituitary transcriptome: the construction of the mouse full-length cDNA encyclopedia, cells defective in the cell-autonomous factor, df, undergo cell lineage specification Genome. Res. 13 (2003) 1273–1289. but not expansion, Development 122 (1996) 151–160. [39] P. Carninci, C. Kvam, A. Kitamura, T. Ohsumi, Y. Okazaki, M. Itoh, M. Kamiya, K. [13] I.O. Nasonkin, R.D. Ward, L.T. Raetzman, A.F. Seasholtz, T.L. Saunders, P.J. Gillespie, Shibata, N. Sasaki, M. Izawa, M. Muramatsu, Y. Hayashizaki, C. Schneider, High- S.A. Camper, Pituitary hypoplasia and respiratory distress syndrome in Prop1 efficiency full-length cDNA cloning by biotinylated CAP trapper, Genomics 37 knockout mice, Hum. Mol. Genet. 13 (2004) 2727–2735. (1996) 327–336. [14] M.W. Sornson, W. Wu, J.S. Dasen, S.E. Flynn, D.J. Norman, S.M. O'Connell, I. Gukovsky, [40] P. Carninci, Y. Nishiyama, A. Westover, M. Itoh, S. Nagaoka, N. Sasaki, Y. Okazaki, M. C. Carriere, A.K. Ryan, A.P. Miller, L. Zuo, A.S. Gleiberman, B. Andersen, W.G. Beamer, Muramatsu, Y. Hayashizaki, Thermostabilization and thermoactivation of thermo- M.G. Rosenfeld, Pituitary lineage determination by the Prophet of Pit-1 home- labile enzymes by trehalose and its application for the synthesis of full length odomain factor defective in Ames dwarfism, Nature 384 (1996) 327–333. cDNA, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 520–524. [15] M.A. Charles, H. Suh, T.A. Hjalt, J. Drouin, S.A. Camper, P.J. Gage, PITX genes are [41] P. Carninci, Y. Shibata, N. Hayatsu, M. Itoh, T. Shiraki, T. Hirozane, A. Watahiki, K. required for cell survival and Lhx3 activation, Mol. Endocrinol. 19 (2005) Shibata, H. Konno, M. Muramatsu, Y. Hayashizaki, Balanced-size and long-size 1893–1903. cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC [16] B.S. Ellsworth, D.L. Butts, S.A. Camper, Mechanisms underlying pituitary hypo- family allows enhanced gene discovery rate and functional analysis, Genomics 77 plasia and failed cell specification in Lhx3-deficient mice, Dev. Biol. 313 (2008) (2001) 79–90. 118–129. [42] P. Carninci, Y. Shibata, N. Hayatsu, Y. Sugahara, K. Shibata, M. Itoh, H. Konno, Y. [17] H. Suh, P.J. Gage, J. Drouin, S.A. Camper, Pitx2 is required at multiple stages of Okazaki, M. Muramatsu, Y. Hayashizaki, Normalization and subtraction of cap- pituitary organogenesis: pituitary primordium formation and cell specification, trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of Development 129 (2002) 329–337. new genes, Genome. Res. 10 (2000) 1617–1630. 460 M.L. Brinkmeier et al. / Genomics 93 (2009) 449–460

[43] Y. Shibata, P. Carninci, A. Watahiki, T. Shiraki, H. Konno, M. Muramatsu, Y. [66] D.J. Glenn, R.A. Maurer, MRG1 binds to the LIM domain of Lhx2 and may function Hayashizaki, Cloning full-length, cap-trapper-selected cDNAs by using the single- as a coactivator to stimulate glycoprotein hormone alpha-subunit gene expres- strand linker ligation method, Biotechniques 30 (2001) 1250–1254. sion, J. Biol. Chem. 274 (1999) 36159–36167. [44] N. Kuramoto, K. Inoue, K. Gion, K. Takano, K. Sakata, K. Ogita, Y. Yoneda, [67] Y. Mizokami, N. Egashira, S. Takekoshi, J. Itoh, Y. Itoh, R.Y. Osamura, M. Matsumae, Modulation of DNA binding of nuclear transcription factors with leucine-zipper Expression of MSX1 in human normal pituitaries and pituitary adenomas, Endocr. motifs by particular endogenous polyamines in murine central and peripheral Pathol. (2008). excitable tissues, Brain. Res. 967 (2003) 170–180. [68] L.D. Nolen, D. Amor, A. Haywood, L. St Heaps, C. Willcock, M. Mihelec, P. Tam, F. [45] L.I. Machiavelli, A.H. Poliandri, F.A. Quinteros, J.P. Cabilla, B.H. Duvilanski, Reactive Billson, J. Grigg, G. Peters, R.V. Jamieson, Deletion at 14q22–23 indicates a oxygen species are key mediators of the nitric oxide apoptotic pathway in anterior contiguous gene syndrome comprising anophthalmia, pituitary hypoplasia, and pituitary cells, Nitric Oxide 16 (2007) 237–246. ear anomalies, Am. J. Med. Genet. A. 140 (2006) 1711–1718. [46] S.S. Stojilkovic, Pituitary cell type-specific electrical activity, calcium signaling and [69] M.S. Roberson, W.E. Schoderbek, G. Tremml, R.A. Maurer, Activation of the secretion, Biol. Res. 39 (2006) 403–423. glycoprotein hormone alpha-subunit promoter by a LIM-homeodomain tran- [47] M.J.vandenHurk,D.T.Ouwens,W.J.Scheenen,V.Limburg,H.Gellekink,M.Bai, scription factor, Mol. Cell. Biol. 14 (1994) 2985–2993. E.W. Roubos, B.G. Jenks, Expression and characterization of the extracellular Ca [70] K. Toda, D. Yamamoto, M. Fumoto, N. Ikeshita, E.H. Herningtyas, K. Iida, Y. (2+)-sensing receptor in melanotrope cells of Xenopus laevis,Endocrinology Takahashi, H. Kaji, K. Chihara, Y. Okimura, Involvement of mPOU (Brn-5), a class VI 144 (2003) 2524–2533. POU protein, in the gene expression of Pit-1 as well as PRL, Mol. Cell. Endocrinol. [48] J. Drouin, B. Lamolet, T. Lamonerie, C. Lanctot, J.J. Tremblay, The PTX family of 280 (2008) 20–29. homeodomain transcription factors during pituitary developments, Mol. Cell. [71] A. Visel, C. Thaller, G. Eichele, GenePaint.org: an atlas of gene expression patterns Endocrinol. 140 (1998) 31–36. in the mouse embryo, Nucleic. Acids. Res. 32 (2004) D552–556. [49] C. Gaston-Massuet, C.L. Andoniadou, M. Signore, E. Sajedi, S. Bird, J.M. Turner, J.P. [72] D.E. Watkins-Chow, S.A. Camper, How many homeobox genes does it take to make Martinez-Barbera, Genetic interaction between the homeobox transcription a pituitary gland? Trends. Genet. 14 (1998) 284–290. factors HESX1 and SIX3 is required for normal pituitary development, Dev. Biol. [73] D.R. Mowat, G.D. Croaker, D.T. Cass, B.A. Kerr, J. Chaitow, L.C. Ades, N.L. Chia, M.J. 324 (2008) 322–333. Wilson, Hirschsprung disease, microcephaly, mental retardation, and character- [50] X. Li, K.A. Oghi, J. Zhang, A. Krones, K.T. Bush, C.K. Glass, S.K. Nigam, A.K. Aggarwal, istic facial features: delineation of a new syndrome and identification of a locus at R. Maas, D.W. Rose, M.G. Rosenfeld, Eya protein phosphatase activity regulates chromosome 2q22–q23, J. Med. Genet. 35 (1998) 617–623. Six1–Dach–Eya transcriptional effects in mammalian organogenesis, Nature 426 [74] A. Brendolan, E. Ferretti, V. Salsi, K. Moses, S. Quaggin, F. Blasi, M.L. Cleary, L. (2003) 247–254. Selleri, A Pbx1-dependent genetic and transcriptional network regulates spleen [51] X. Li, V. Perissi, F. Liu, D.W. Rose, M.G. Rosenfeld, Tissue-specific regulation ontogeny, Development 132 (2005) 3113–3126. of retinal and pituitary precursor cell proliferation, Science 297 (2002) [75] N. Rave-Harel, M.L. Givens, S.B. Nelson, H.A. Duong, D. Coss, M.E. Clark, S.B. Hall, 1180–1183. M.P. Kamps, P.L. Mellon, TALE homeodomain proteins regulate gonadotropin- [52] C.A. Bentley, M.P. Zidehsarai, J.C. Grindley, A.F. Parlow, S. Barth-Hall, V.J. Roberts, Pax6 releasing hormone gene expression independently and via interactions with Oct- is implicated in murine pituitary endocrine function, Endocrine 10 (1999) 171–177. 1, J. Biol. Chem. 279 (2004) 30287–30297. [53] T.R. Burglin, Guidebook to the Homeobox Genes, Oxford University Press, Inc., [76] S. Nishimoto, E. Nishida, Fibroblast growth factor 13 is essential for neural New York, 1994. differentiation in Xenopus early embryonic development, J. Biol. Chem. 282 [54] S.W. Davis, S.A. Camper, Noggin regulates Bmp4 activity during pituitary (2007) 24255–24261. induction, Dev. Biol. 305 (2007) 145–160. [77] S. Zhang, Y. Lin, P. Itaranta, A. Yagi, S. Vainio, Expression of Sprouty genes 1, 2 and 4 [55] M. Treier, A.S. Gleiberman, S.M. O'Connell, D.P. Szeto, J.A. McMahon, A.P. during mouse organogenesis, Mech. Dev. 109 (2001) 367–370. McMahon, M.G. Rosenfeld, Multistep signaling requirements for pituitary [78] N. Hacohen, S. Kramer, D. Sutherland, Y. Hiromi, M.A. Krasnow, sprouty encodes organogenesis in vivo, Genes. Dev. 12 (1998) 1691–1704. a novel antagonist of FGF signaling that patterns apical branching of the Dro- [56] K.P. McNatty, J.L. Juengel, T. Wilson, S.M. Galloway, G.H. Davis, N.L. Hudson, C.L. sophila airways, Cell 92 (1998) 253–263. Moeller, M. Cranfield, K.L. Reader, M.P. Laitinen, N.P. Groome, H.R. Sawyer, O. [79] G. Minowada, L.A. Jarvis, C.L. Chi, A. Neubuser, X. Sun, N. Hacohen, M.A. Ritvos, Oocyte-derived growth factors and ovulation rate in sheep, Reprod. Suppl. Krasnow, G.R. Martin, Vertebrate Sprouty genes are induced by FGF signaling 61 (2003) 339–351. and can cause chondrodysplasia when overexpressed, Development 126 (1999) [57] L.E. Olson, J. Tollkuhn, C. Scafoglio, A. Krones, J. Zhang, K.A. Ohgi, W. Wu, M.M. 4465–4475. Taketo, R. Kemler, R. Grosschedl, D. Rose, X. Li, M.G. Rosenfeld, Homeodomain- [80] P. Carninci, T. Shiraki, Y. Mizuno, M. Muramatsu, Y. Hayashizaki, Extra-long first- mediated beta-catenin-dependent switching events dictate cell-lineage determi- strand cDNA synthesis, Biotechniques 32 (2002) 984–985. nation, Cell 125 (2006) 593–605. [81] P. Carninci, A. Westover, Y. Nishiyama, T. Ohsumi, M. Itoh, S. Nagaoka, N. Sasaki, [58] H.M. Pogoda, S. von der Hardt, W. Herzog, C. Kramer, H. Schwarz, M. Hammersch- Y. Okazaki, M. Muramatsu, C. Schneider, Y. Hayashizaki, High efficiency selection midt, The proneural gene ascl1a is required for endocrine differentiation and cell of full-length cDNA by improved biotinylated cap trapper, DNA Res 4 (1997) survival in the zebrafish adenohypophysis, Development 133 (2006) 1079–1089. 61–66. [59] F. Vinals, J. Reiriz, S. Ambrosio, R. Bartrons, J.L. Rosa, F. Ventura, BMP-2 decreases [82] T. Hirozane-Kishikawa, T. Shiraki, K. Waki, M. Nakamura, T. Arakawa, J. Kawai, M. Mash1 stability by increasing Id1 expression, Embo. J. 23 (2004) 3527–3537. Fagiolini, T.K. Hensch, Y. Hayashizaki, P. Carninci, Subtraction of cap-trapped full- [60] C.S. Moreno, C.O. Evans, X. Zhan, M. Okor, D.M. Desiderio, N.M. Oyesiku, Novel length cDNA libraries to select rare transcripts, Biotechniques 35 (2003) 510–516 molecular signaling and classification of human clinically nonfunctional pituitary 518. adenomas identified by gene expression profiling and proteomic analyses, Cancer. [83] A. Alexa, J. Rahnenfuhrer, T. Lengauer, Improved scoring of functional groups from Res. 65 (2005) 10214–10222. gene expression data by decorrelating GO graph structure, Bioinformatics 22 [61] A.W. Mould, R. Duncan, M. Serewko-Auret, K.A. Loffler, C. Biondi, M. Gartside, G.F. (2006) 1600–1607. Kay, N.K. Hayward, Global expression profiling of murine MEN1-associated [84] L.J. Cushman, D.E. Watkins-Chow, M.L. Brinkmeier, L.T. Raetzman, A.L. Radak, R.V. tumors reveals a regulatory role for menin in transcription, cell cycle and Lloyd, S.A. Camper, Persistent Prop1 expression delays gonadotrope differentiation chromatin remodelling, Int. J. Cancer. 121 (2007) 776–783. and enhances pituitary tumor susceptibility, Hum. Mol. Genet. 10 (2001) [62] K.H. Ruebel, A.A. Leontovich, L. Jin, G.A. Stilling, H. Zhang, X. Qian, N. Nakamura, 1141–1153. B.W. Scheithauer, K. Kovacs, R.V. Lloyd, Patterns of gene expression in pituitary [85] A.H. Vesper, L.T. Raetzman, S.A. Camper, Role of prophet of Pit1 (PROP1) in carcinomas and adenomas analyzed by high-density oligonucleotide arrays, gonadotrope differentiation and puberty, Endocrinology 147 (2006) 1654–1663. reverse transcriptase-quantitative PCR, and protein expression, Endocrine 29 [86] V.D. Sarapura, W.M. Wood, W.W. Woodmansee, D.J. Haakinson, J.M. Dowding, (2006) 435–444. D.F. Gordon, E.C. Ridgway, Pituitary tumors arising from glycoprotein hormone [63] C.H. Asbreuk, H.S. van Schaick, J.J. Cox, M.P. Smidt, J.P. Burbach, Survey for paired- alpha-subunit-deficient mice contain transcription factors and receptors like homeodomain gene expression in the hypothalamus: restricted expression present in thyrotropes, Pituitary 9 (2006) 11–18. patterns of Rx, Alx4 and goosecoid, Neuroscience 114 (2002) 883–889. [87] S.W. Davis, M.A. Potok, M.L. Brinkmeier, P. Carninci, R.H. Lyons, J.W. MacDonald, [64] J.S. Bailey, N. Rave-Harel, S.M. McGillivray, D. Coss, P.L. Mellon, Activin regulation of M.T.Fleming,A.H.Mortensen,N.Egashira,D.Ghosh,K.P.Steel,R.Y.Osamura,Y. the follicle-stimulating hormone beta-subunit gene involves Smads and the TALE Hayashizaki, S.A. Camper, Genetics in Growth, and Pituitary Tumorigenesis 10th homeodomain proteins Pbx1 and Prep1, Mol. Endocrinol. 18 (2004) 1158–1170. KIGS/KIMS Expert Meeting on Growth Hormone and Growth Disorders [65] G.V. Ermakova, E.A. Solovieva, N.Y. Martynova, A.G. Zaraisky, The homeodomain Florence, Italy, April 2008, in: D.B. Dunger, V. Popovic-Brkic (Eds.), Genetics, factor Xanf represses expression of genes in the presumptive rostral forebrain that Gene Expression and Bioinformatics of the Pituitary Gland, Hormone Research, specify more caudal brain regions, Dev. Biol. 307 (2007) 483–497. vol. 71, 2009, (Suppl 2).