Identification of members of the Wnt signaling pathway in the
embryonic pituitary gland
Kristin R. Douglas,
Michelle L. Brinkmeier,
Jennifer A. Kennell,
Tabitha A. Harrison,
Athena I. Patrianakos,
Bradley S. Sprecher,
Mary Anne Potok,
Robert H. Lyons, Jr.,
Ormond A. MacDougald,
Sally A. Camper
Department of Human Genetics, University of Michigan Medical School, 4301 MSRBIII, 1500 W. Medical Center Dr.,
Ann Arbor, Michigan 48109-0638, USA
University of Michigan Medical School, Cellular & Molecular Biology Program, Ann Arbor, Michigan 48109-0638, USA
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0638, USA
Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0638, USA
Received: 20 April, 2001 / Accepted: 16 July 2001
Abstract. Prop1 is one of several transcription factors important
for the development of the pituitary gland. Downstream targets of
PROP1 and other critical pituitary transcription factors remain
largely unknown. We have generated a partial expression profile of
the developing pituitary gland containing over 350 transcripts,
using cDNA subtractive hybridization between Prop1
wild-type embryonic pituitary gland primordia. Numerous classes
of genes including transcription factors, membrane associated mol-
ecules, and cell cycle regulators were identified in this study. Of
the transcripts, 34% do not have sequence similarity to known
genes, but are similar to ESTs, and 4% represent novel sequences.
Pituitary gland expression of a number of clones was verified
using in situ hybridization.
Several members of the Wnt signaling pathway were identified
in the developing pituitary gland. The frizzled2 receptor, Apc,
␤-catenin, groucho, and a novel isoform of TCF4 (officially named
Tcf7l2) were identified in developing pituitary libraries. Three N-
terminal alternatively spliced Tcf7l2 isoforms are reported here,
each of which lacks a DNA-binding domain. Functional studies
indicate that these isoforms can act as endogenous inhibitors of
Wnt signaling in some contexts.
This is the first report of Tcf7l2 and Fzd2 expression in the
developing pituitary. These molecules may be important in medi-
ating Wnt signaling during pituitary ontogeny. We expect other
transcripts from these libraries to be involved in pituitary gland
The rodent pituitary gland is composed of the anterior, intermedi-
ate, and posterior lobes. The anterior lobe contains five hormone-
producing cell types that secrete hormones necessary for growth,
fertility, lactation, response to physiological stress, and thyroid
function. The major secretory product of the intermediate lobe is
melanocyte-stimulating hormone; oxytocin and vasopressin are
produced in the posterior lobe.
The anlage of the pituitary gland begins to develop at embry-
onic day 9.0 (e9.0) in response to inductive signals provided by
surrounding tissues (Treier and Rosenfeld 1996; Burrows et al.
1999; Sheng and Westphal 1999). Over the next several days,
signals from the ventral diencephalon and juxtaposed mesenchy-
mal cells induce spatially restricted patterns of transcription factor
expression in the nascent pituitary. Stratified transcription factor
domains are translated into distinct regions of cell differentiation
within the gland. Several critical genes regulating pituitary gland
development have been identified through the characterization of
hereditary mouse and human pituitary endocrine deficiencies. The
pituitary transcription factors Pitx2, Lhx3, Hesx1, and Prop1 are
each essential for pituitary gland development; however, their
downstream targets remain largely unknown.
Differential gene expression analysis is invaluable for gaining
a better understanding of the molecular events regulating pituitary
gland development. The RIKEN group (http://genome.rtc.riken.
go.jp) released a large transcriptome from adult male pituitary
(Aizawa et al. 2000). Although this is a rich resource for the
identification of genes important for adult pituitary function, em-
bryo-specific genes such as Hesx1 and Prop1 are not represented.
We demonstrated the feasibility of establishing an expression pro-
file of the developing organ using differential display to compare
transcripts present at e12.5 and e14.5 in the pituitary primordium.
This is a period of intense cell proliferation (Carbajo-Perez et al.
1989) preceding the differentiation of four of the five mature cell
types and a time when the effects of mutations in Pitx2, Lhx3,
Hesx1, and Prop1 are evident. We reported a limited transcrip-
tome, including two validated, novel, differential transcripts
(Douglas and Camper 2000). Here, we describe a library of pitu-
itary transcripts not previously reported that are expressed at e14.5.
This library was generated by using cDNA subtractive hybridiza-
tion comparing normal pituitary primodia with Prop1
Analysis of both libraries reveals that numerous members of
the Wnt signaling pathway are expressed in the developing pitu-
itary gland. Several Wnt genes, including Wnt4, Wnt5a, and
Wnt10a, are known to be expressed in or adjacent to the develop-
ing gland (Wang and Shackleford 1996; Treier et al. 1998), and
Wnt4 has been shown to have a functional role in pituitary gland
development (Treier et al. 1998). We identified a novel TCF4
isoform (the Tcf4 gene is officially named Tcf7l2), frizzled2, ad-
enomatosis polyposis coli (Apc), beta-catenin, and groucho in the
developing pituitary. The presence of several members of the Wnt
signaling pathway in these pituitary libraries supports the impor-
tance of Wnt signaling in the development of the pituitary gland.
Materials and methods
Mice and genotyping.
Swiss Webster mice were purchased from
Taconic (Germantown, N.Y.), CD1 stocks were obtained from Charles
River Laboratories (Wilmington, Mass.), and Prop1
stocks were ob-
tained from A. Bartke (Southern Illinois University at Carbondale, Ill).
Correspondence to: S.A. Camper; E-mail: email@example.com
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have been assigned the accession numbers: BE692747–
BE693109; BG487405; BG487404; AF363722–AF363726.
Mammalian Genome 12, 843–851 (2001).
© Springer-Verlag New York Inc. 2001