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 CONTEMPORARY ENDOCRINOLOGY ™

Developmental
Endocrinology
From Research
to Clinical Practice

Edited by
Erica A. Eugster, MD
Ora Hirsch Pescovitz, MD

HUMANA PRESS
Contents i

DEVELOPMENTAL ENDOCRINOLOGY
ii Contents

CONTEMPORARY ENDOCRINOLOGY
P. Michael Conn, SERIES EDITOR
Developmental Endocrinology: From Research The IGF System: Molecular Biology, Physiol-
to Clinical Practice, edited by ERICA A. ogy, and Clinical Applications, edited by
EUGSTER AND ORA HIRSCH PESCOVITZ, 2002 RON G. ROSENFELD AND CHARLES T.
Challenging Cases in Endocrinology, edited by ROBERTS, JR., 1999
MARK E. MOLITCH, MD, 2002 Neurosteroids: A New Regulatory Function in
Selective Estrogen Receptor Modulators: the Nervous System, edited by ETIENNE-
Research and Clinical Applications, EMILE BAULIEU, MICHAEL SCHUMACHER, AND
PAUL ROBEL, 1999
edited by ANDREA MANNI AND MICHAEL F.
Autoimmune Endocrinopathies, edited by
VERDERAME, 2002
ROBERT VOLPÉ, 1999
Transgenics in Endocrinology, edited by MARTIN
Hormone Resistance Syndromes, edited by J.
MATZUK, CHESTER W. BROWN, AND LARRY JAMESON, 1999
T. RAJENDRA KUMAR, 2001 Hormone Replacement Therapy, edited by A.
Assisted Fertilization and Nuclear Transfer in WAYNE MEIKLE, 1999
Mammals, edited by DON P. WOLF AND Insulin Resistance: The Metabolic Syndrome X, edited
MARY ZELINSKI-WOOTEN, 2001 by GERALD M. REAVEN AND AMI LAWS, 1999
Adrenal Disorders, edited by ANDREW N. Endocrinology of Breast Cancer, edited by
MARGIORIS AND GEORGE P. CHROUSOS, 2001 ANDREA MANNI, 1999
Endocrine Oncology, edited by STEPHEN P. Molecular and Cellular Pediatric Endocrinology,
ETHIER, 2000 edited by STUART HANDWERGER, 1999
Endocrinology of the Lung: Development and Gastrointestinal Endocrinology, edited by
Surfactant Synthesis, edited by CAROLE R. GEORGE H. GREELEY, JR., 1999
MENDELSON, 2000 The Endocrinology of Pregnancy, edited by
Sports Endocrinology, edited by MICHELLE P. FULLER W. BAZER, 1998
WARREN AND NAAMA W. CONSTANTINI, 2000 Clinical Management of Diabetic Neuropathy,
Gene Engineering in Endocrinology, edited by edited by ARISTIDIS VEVES, 1998
G Proteins, Receptors, and Disease, edited by
MARGARET A. SHUPNIK, 2000
ALLEN M. SPIEGEL, 1998
Endocrinology of Aging, edited by JOHN E. MORLEY
Natriuretic Peptides in Health and Disease, edited by
AND LUCRETIA VAN DEN BERG, 2000
WILLIS K. SAMSON AND ELLIS R. LEVIN, 1997
Human Growth Hormone: Research and Clinical Endocrinology of Critical Disease, edited by K.
Practice, edited by ROY G. SMITH AND PATRICK OBER, 1997
MICHAEL O. THORNER, 2000 Diseases of the Pituitary: Diagnosis and Treatment,
Hormones and the Heart in Health and edited by MARGARET E. WIERMAN, 1997
Disease, edited by LEONARD SHARE, 1999 Diseases of the Thyroid, edited by LEWIS E.
Menopause: Endocrinology and Management, BRAVERMAN, 1997
edited by DAVID B. SEIFER AND ELIZABETH A. Endocrinology of the Vasculature, edited by
KENNARD, 1999 JAMES R. SOWERS, 1996
Contents iii

DEVELOPMENTAL
ENDOCRINOLOGY
FROM RESEARCH TO CLINICAL PRACTICE

Edited by
ERICA A. EUGSTER, MD
Riley Hospital for Children,
Indiana University School of Medicine,
Indianapolis, IN
ORA HIRSCH PESCOVITZ, MD
Riley Hospital for Children,
Indiana University School of Medicine,
Indianapolis, IN

HUMANA PRESS
TOTOWA, NEW JERSEY
iv Contents

© 2002 Humana Press Inc.

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For additional copies, pricing for bulk purchases, and/or information about other Humana titles,
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Fax: 973-256-8341; E-mail: [email protected] or visit our website at http://humanapress.com

All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or
by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission
from the Publisher.

Due diligence has been taken by the publishers, editors, and authors of this book to assure the accuracy of the
information published and to describe generally accepted practices. The contributors herein have carefully checked to
ensure that the drug selections and dosages set forth in this text are accurate and in accord with the standards accepted
at the time of publication. Notwithstanding, as new research, changes in government regulations, and knowledge from
clinical experience relating to drug therapy and drug reactions constantly occurs, the reader is advised to check the
product information provided by the manufacturer of each drug for any change in dosages or for additional warnings
and contraindications. This is of utmost importance when the recommended drug herein is a new or infrequently used
drug. It is the responsibility of the treating physician to determine dosages and treatment strategies for individual
patients. Further it is the responsibility of the health care provider to ascertain the Food and Drug Administration
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Library of Congress Cataloging-in-Publication Data

Developmental Endocrinology : from research to clinical practice / edited by Erica A.

Eugster, Ora Hirsch Pescovitz.
p. cm. -- (Contemporary endocrinology)
Includes bibliographical references and index.
ISBN 0-89603-860-2 (alk. paper)
1. Endocrinology, Developmental. I. Eugster, Erica A. II. Pescovitz, Ora Hirsch. III.
Contemporary endocrinology (Totowa, N.J.)

QP187.6 .D484 2002

612'4--dc 21
2001051948
PREFACE
Contents v

From the complex molecular control of endocrine cell differentiation to the amazing
physiology of normal growth and puberty, developmental processes represent an integral
and recurrent theme within the field of Endocrinology. Our goal in the creation of this
book was to incorporate the latest scientific information regarding the development of
endocrine systems into a larger context in which molecular genetics is combined with
state-of-the-art understanding of endocrine physiology and optimal management of en-
docrine diseases. Each section is organized according to the chronologic development
of the human organism, from the fetal/prenatal period through childhood, adolescence,
and in some cases, into adulthood. In parallel with this sequence is a consistent progres-
sion of topics, which begins with a focus on molecular genetic aspects of endocrine
development and concludes with chapters devoted to the diagnosis and treatment of
endocrine disorders. Our hope is that the material contained herein will provide critical
information for basic researchers regarding clinical applications of laboratory investiga-
tion, and will likewise benefit practitioners by elucidating the underlying pathophysiol-
ogy of the many endocrinopathies encountered in the clinical setting. Ultimately, we aim
to promote the collaborative translational efforts that are essential to the dual objectives
of advancing knowledge of human biology and improving our ability to care for our
patients. We wish to thank our authors for their invaluable contributions to this project.
Lastly, this book is dedicated with utmost love and gratitude to our husbands and to our
children, Aliza, Alex, Ari, Ariana, Naomi, Sophia, and Lydia: May you always follow
your dreams!
Erica A. Eugster, MD
Ora Hirsch Pescovitz, MD

v
vi Contents
C
Contents
ONTENTS
vii

Preface ........................................................................................................ v
Contributors .............................................................................................. ix
Part I HYPOTHALAMUS/PITUITARY
1 Transcriptional Control of the Development and Function
of the Hypothalamic-Pituitary Axis ............................................. 3
Gretchen E. Parker, Kyle W. Sloop, and Simon J. Rhodes
Part II GROWTH
2 Molecular Mutations in the Human Growth Hormone Axis ......... 43
Zvi Laron
3 Normal and Abnormal Growth ....................................................... 77
Emily C. Walvoord and Erica A. Eugster
4 Adult Growth Hormone Deficiency ............................................. 105
Zehra Haider and James W. Edmondson
Part III THYROID
5 Molecular Mechanisms of Thyroid Gland Development:
Insights from Clinical Studies and from Mutant Mice ............ 123
Guy Van Vliet
6 Fetal-Perinatal Thyroid Physiology .............................................. 135
Delbert A. Fisher
7 Thyroid Disorders in Children and Adolescents .......................... 151
Scott A. Rivkees
Part IV CALCIUM AND BONE
8 Genetic Control of Parathyroid Gland Development
and Molecular Insights into Hypoparathyroidism ................... 181
Michael A. Levine
9 Bone Physiology ........................................................................... 193
Salvatore Minisola and Lorraine A. Fitzpatrick
10 Pediatric Bone Disease ................................................................. 217
Linda Anne DiMeglio
Part V GONADAL DEVELOPMENT
11 Transcriptional Development
of the Hypothalamic-Pituitary-Gonadal Axis .......................... 243
Sally Radovick, Helen H. Kim, Diane E. J. Stafford,
Andrew Wolfe, and Marjorie Zakaria

vii
viii Contents

12 Sexual Differentiation ................................................................... 261

Tamara S. Hannon and John S. Fuqua
13 Prenatal Androgens
and Sexual Differentiation of Behavior ................................... 293
Sheri A. Berenbaum
14 Normal and Precocious Puberty ................................................... 313
Joan DiMartino-Nardi
15 Delayed Puberty ............................................................................ 331
Steven G. Waguespack and Ora Hirsch Pescovitz
Part VI ADRENAL
16 Molecular Development
of the Hypothalamic-Pituitary-Adrenal (HPA) Axis ............... 359
Sophia P. Tsakiri, George P. Chrousos,
and Andrew N. Margioris
17 Molecular Genetics of Adrenal Disease ....................................... 381
Perrin C. White
Index .............................................................................................. 403
C
Contents
ONTRIBUTORS
ix

SHERI A. BERENBAUM, PhD, Department of Psychology, Pennsylvania State University,

University Park, PA
GEORGE P. CHROUSOS, MD, Pediatric and Reproductive Endocrinology Branch,
National Institute of Child Health and Human Development, National Institutes of
Health, Bethesda, MD
JOAN DIMARTINO-NARDI, MD, Division of Pediatric Endocrinology, Montefiore
Medical Center, Bronx, NY
LINDA ANNE DIMEGLIO, MD, Section of Pediatric Endocrinology and Diabetology,
James Whitcomb Riley Hospital for Children, Indiana University School of
Medicine, Indianapolis, IN
JAMES W. EDMONDSON, MD, Section of Endocrinology, Indiana University,
Indianapolis, IN
ERICA A. EUGSTER, MD, Section of Pediatric Endocrinology and Diabetology, James
Whitcomb Riley Hospital for Children, Indiana University School of Medicine,
Indianapolis, IN
DELBERT A. FISHER, MD, Quest Diagnostic Nichols Institute, San Juan Capistrano, CA
LORRAINE A. FITZPATRICK, MD, Division of Endocrinology, Metabolism and Nutrition,
Mayo Clinic and Mayo Foundation, Rochester, MN
JOHN S. FUQUA, MD, Section of Pediatric Endocrinology and Diabetology, James
Whitcomb Riley Hospital for Children, Indiana University School of Medicine,
Indianapolis, IN
ZEHRA HAIDER, MD, Section of Endocrinology, Indiana University, Indianapolis, IN
TAMARA S. HANNON, MD, Section of Pediatric Endocrinology and Diabetology, James
Whitcomb Riley Hospital for Children, Indiana University School of Medicine,
Indianapolis, IN
HELEN H. KIM, MD, Department of Obstetrics and Gynecology and Section of
Pediatric Endocrinology, The University of Chicago Children’s Hospital, Chicago,
IL
ZVI LARON, MD, Endocrinology and Diabetes Research Unit, Schneider Children’s
Medical Center, Tel Aviv University, Tel Aviv, Israel
MICHAEL A. LEVINE, MD, Division of Pediatric Endocrinology, Johns Hopkins School
of Medicine, Baltimore, MD
ANDREW N. MARGIORIS, MD, Department of Clinical Chemistry, University of Crete
School of Medicine, Crete, Greece
SALVATORE MINISOLA, MD, Dipartimento di Scienze Cliniche, Università degli Studi di
Roma “La Sapienza”, Rome, Italy
GRETCHEN E. PARKER, PhD, Department of Biology, Indiana University-Purdue
University, Indianapolis, IN
ORA HIRSCH PESCOVITZ, MD, Section of Pediatric Endocrinology and Diabetology,
James Whitcomb Riley Hospital for Children, Indiana University School of
Medicine, Indianapolis, IN
SALLY RADOVICK, MD, Section of Pediatric Endocrinology, The University of Chicago
Children’s Hospital, Chicago, IL
ix
x Contents
Contributors

SIMON J. RHODES, PhD, Department of Biology, Indiana University-Purdue University,

Indianapolis, IN
SCOTT A. RIVKEES, MD, Section of Pediatric Endocrinology, Yale University, New
Haven, CT
KYLE W. SLOOP, PhD, Department of Biology, Indiana University-Purdue University,
Indianapolis, IN
DIANE E. J. STAFFORD, MD, Department of Obstetrics and Gynecology and Section of
Pediatric Endocrinology, The University of Chicago Children’s Hospital, Chicago,
IL
SOPHIA P. TSAKIRI, MD, Department of Pediatrics, Venizelion Hospital, Heraklien,
Crete, Greece
GUY VAN VLIET, MD, Endocrinology Service, Sainte-Justine Hospital; Department of
Pediatrics, University of Montreal, Montréal, Quebec, Canada
STEVEN G. WAGUESPACK, MD, Sections of Pediatric Endocrinology and Diabetology
and Endocrinology, Indiana University School of Medicine, Indianapolis, IN
EMILY C. WALVOORD, MD, Section of Pediatric Endocrinology and Diabetology, James
Whitcomb Riley Hospital for Children, Indiana University School of Medicine,
Indianapolis, IN
PERRIN C. WHITE, MD, Division of Pediatric Endocrinology, University of Texas
Southwestern Medical Center, Dallas, TX
ANDREW WOLFE, PhD, Department of Obstetrics and Gynecology and Section of
Pediatric Endocrinology, The University of Chicago Children’s Hospital, Chicago,
IL
MARJORIE ZAKARIA, MD, Department of Obstetrics and Gynecology and Section of
Pediatric Endocrinology, The University of Chicago Children’s Hospital, Chicago,
IL
Contents xi
Chapter 1 / Hypothalamic-Pituitary Axis 1

I HYPOTHALAMUS/PITUITARY
2 Part I / Hypothalamus/Pituitary
Chapter 1 / Hypothalamic-Pituitary Axis 3

1 Transcriptional Control
of the Development and Function
of the Hypothalamic-Pituitary Axis

Gretchen E. Parker, PhD, Kyle W. Sloop, PhD,

and Simon J. Rhodes, PhD
CONTENTS
INTRODUCTION
TRANSCRIPTIONAL REGULATION OF ENDOCRINE HYPOTHALAMUS
DEVELOPMENT
TRANSCRIPTIONAL REGULATION OF PITUITARY DEVELOPMENT
PITUITARY TRANSCRIPTION FACTORS IN HUMAN DISEASE
ALTERNATE FORMS OF HYPOTHALAMUS-PITUITARY TRANSCRIPTION
FACTORS
REGULATION OF HYPOTHALAMUS-PITUITARY TRANSCRIPTION FACTOR
FUNCTION
COMBINATIONS OF HYPOTHALAMUS-PITUITARY TRANSCRIPTION
FACTORS IN THE SPECIFICATION OF CELL TYPES
CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES

INTRODUCTION
Function of the Hypothalamic-Pituitary Axes and Their Hormone Products
The hypothalamic-pituitary (H-P) axis regulates many aspects of mammalian endo-
crine physiology by the controlled secretion of hormones. The hypothalamus is part of
the diencephalon at the base of the brain beneath the third ventricle (1). It is physically
located posterior to the optic chiasm and rostral to the mammillary bodies. The pituitary
gland (or hypophysis) is a small organ located beneath the hypothalamus that weighs
about 0.5 g in humans (2). The posterior pituitary lobe (or neurohypophysis or pars
nervosa) develops directly from the brain (see below) and is connected to the hypothala-
mus by the infundibulum (or pituitary stalk). The anterior lobe (or adenohypophysis or

From: Contemporary Endocrinology: Developmental Endocrinology: From Research to Clinical Practice

Edited by: E. A. Eugster and O. H. Pescovitz © Humana Press Inc., Totowa, NJ

3
4 Part I / Hypothalamus/Pituitary

Fig. 1. Development of the hormone-secreting cell types of the hypothalamus. (A) Schematic dia-
gram of a sagittal section of the ventral diencephalon region of the brain. Hypothalamic nuclei are
shaded in black. POA, preoptic area; SON, supraoptic nucleus; OC, optic chiasm; PVN, paraventric-
ular nucleus; aPV, anterior periventrucular nucleus; DM, dorsomedial nucleus; VM, ventromedial
nucleus; ARN, arcuate nucleus; PN, posterior nucleus; MB, mammillary body; ap, anterior pituitary;
pp, posterior pituitary. (B) The development of hypothalamic nuclei is dependent on the actions of the
indicated transcription factors. Neurons in the differentiated nuclei then secrete the indicated hormones.

pars distalis) and intermediate lobe (or pars intermedia) of the pituitary have distinct
embryological origins. In the mature gland, these structures are fused to the posterior
pituitary, and some cells of the anterior pituitary proliferate up and around the pituitary stalk
to form the pars tuberalis. The intermediate lobe is a poorly developed structure in adult
humans and is absent in birds (2). A vascular link called the portal system arises from the
median eminence at the base of the hypothalamus and provides a connection for the pas-
sage of neurosecretory hormones from the hypothalamus to the anterior pituitary gland.
THE HYPOTHALAMUS
The hypothalamus can be subdivided into three groups of nuclei of neural cells: the
supraoptic group, the tuberal (or middle) group, and the mammillary (or posterior) group
of nuclei. Within the supraoptic group, the paraventricular (PVN) and supraoptic (SON)
nuclei project their axons to the posterior lobe of the pituitary as the hypothalamic-
hypophyseal tract (Fig. 1). Together, the large, highly vascularized cells of the PVN and
SON are termed the magnocellular secretory system. These cells produce arginine vaso-
pressin (AVP, also called antidiuretic hormone), and oxytocin (OT). OT and AVP are pep-
tide hormones that are packaged in vesicles and then transported to the posterior pituitary
for release. In the kidney, AVP promotes water retention; within the vasculature and
heart, it activates blood pressure-sensitive receptors; and in the liver, it has glycogenolytic
effects. Other actions of AVP include regulation of the secretion of pituitary hormones
Chapter 1 / Hypothalamic-Pituitary Axis 5

such as adrenocorticotropin (ACTH). OT induces uterine contraction during the later

stages of pregnancy and promotes milk ejection during suckling. OT also has been pro-
posed to have roles in mating and maternal behavior. Within the PVN, smaller cells form
the parvocellular neurosecretory system. This system delivers neurohormones via the
hypophyseal-portal system to control anterior pituitary function. Parvocellular neurons
positioned in the PVN secrete corticotropin-releasing hormone (CRH), gonadotropin-
releasing hormone (GnRH or LHRH), and thyrotropin-releasing hormone (TRH), which
regulate pituitary corticotropes, gonadotropes, and thyrotropes, respectively. Other sig-
naling molecules released by the parvocellular system into the median eminence include
dopamine (DA), which acts at the pituitary as a prolactin (PRL) inhibitory factor.
The tuberal group of hypothalamic nuclei includes the ventromedial (VM), dorsomedial
(DM), and arcuate (ARN) nuclei (Fig. 1). These nuclei also are important in pituitary
regulation. Somatostatin (SS, or growth hormone-inhibiting hormone) and growth hor-
mone-releasing hormone (GHRH, or growth hormone-releasing factor) are synthesized
and released from the ARN. The ARN also is an autonomous generator of reproductive
timing and rhythms.
The mammillary group of nuclei is less well-defined in their nuclear structure. Neurons
in this region produce neurotransmitters such as gamma-aminobutyric acid, histamine,
and also neuropeptides such as galanin, which has many roles within the body, including
modulation of pituitary lactotrope function (3).
THE PITUITARY GLAND
The pituitary gland secretes polypeptide hormones that regulate physiological func-
tions including growth, the stress response, reproductive activity, metabolism, and lacta-
tion. The anterior pituitary hormones are released from five distinct cell types, and these
peptide or protein hormones serve as characteristic, defining markers for each cell type
(Fig. 2). The five cell types (and their respective hormone products) are corticotropes
(producing ACTH by proteolytic processing of the product of the proopiomelanocortin
[POMC] gene); gonadotropes (follicle-stimulating hormone [FSH] and luteinizing hor-
mone [LH]); thyrotropes (thyroid-stimulating hormone [TSH]); somatotropes (growth
hormone [GH]); and lactotropes (PRL). FSH, LH, and TSH are polypeptide heterodimers
consisting of a common subunit, alpha glycoprotein (αGSU), and a distinct β subunit
(FSHβ, LHβ, TSHβ). The anterior/intermediate pituitary also contains nonhormone-
secreting cells known as folliculo-stellate cells that have glial-like properties (4). The func-
tion and developmental origin of these cells is unclear, but some studies suggest that they
arise in the region of Rathke’s pouch (the primordial structure of the anterior/interme-
diate pituitary) closest to the posterior lobe (5). In the intermediate pituitary, melanotrope
cells secrete alpha-melanocyte stimulating hormone (α-MSH). As described earlier, the
posterior lobe of the pituitary receives the peptide hormones AVP and OT directly from
hypothalamic nuclei of the axons of the hypothalamic-hypophyseal tract. In addition, the
posterior lobe contains modified glial cells known as pituicytes (reviewed in ref. 6).

Embryology of the Hypothalamus-Pituitary Axis

The developmental programs that guide organogenesis of the hypothalamus and pitui-
tary are highly interdependent processes. Inductive signals pass between the two devel-
oping structures, and these signals prime the transcriptional cascades that subsequently
6 Part I / Hypothalamus/Pituitary

Fig. 2. Development of the hormone-secreting cell types of the mammalian pituitary gland. The
pituitary arises as a result of interactions between the infundibulum (inf), a process of the ventral
diencephalon (dien), and Rathke’s pouch (rp). Rathke’s pouch arises from the oral ectoderm at the
most anterior end of the anterior neural ridge (ANR) and eventually is separated from the oral cavity
by the developing sphenoid bone (sb). Pouch development occurs in distinct steps: first a rudimen-
tary pouch is formed, followed by the establishment of the definitive pouch. Transcription factors and
secreted protein signaling molecules that are important in development of the differentiated cell types
of the anterior (ap) and intermediate lobes of the pituitary (the derivatives of Rathke’s pouch) are
shown. In the anatomical drawings Rathke’s pouch and its derivatives are lightly shaded. The infun-
dibulum and the resulting posterior pituitary (pp) are darkly shaded.

control specification of cell types within the two organs (see Transcriptional Regulation
of Endcrine Hypothalamus Development). In humans, the hypothalamus is the first region
of the brain to differentiate (7). The hypothalamus is neuroectodermal in origin and
develops from the floor of the diencephalon. By the 6th wk of human development, char-
acteristic nuclei of the hypothalamus and the mammillary bodies can be discerned (7).
In contrast to the hypothalamus, the mature pituitary gland is a composite organ. Like
other endocrine glands, such as the adrenals and the thyroid, the tissues of the anterior/
intermediate and posterior lobes have distinct embryological origins. The posterior pitui-
tary lobe is neuroectodermal in origin and originates from the base of the diencephalon
as an extension of the infundibulum (Fig. 2). The anterior and intermediate lobes arise
from a pocket known as Rathke’s pouch that develops in the roof of the stomodeum, the
Chapter 1 / Hypothalamic-Pituitary Axis 7

primitive oral cavity (8). Rathke’s pouch is conventionally described as being derived
from the oral ectoderm (8). Some studies, however, indicate that the endocrine cells of
the eventual anterior/intermediate pituitary are committed very early during embryogen-
esis as part of the anterior neural ridge (ANR) of the neural plate (7,9–17). Rathke’s
pouch can be clearly observed by the 4th wk of human gestation (corresponding to approx
embryonic days 2, 9, and 11 of chick, mouse, and rat development, respectively [7,17
18]). The diverticulum of the diencephalon that becomes the infundibulum becomes
distinct at about the 6th wk of human development (18). After contacting the diencepha-
lon, the pouch gradually moves in a caudal direction (18). The pouch then is surrounded
by proliferating mesodermal tissue and pinches off from the oral cavity after the 7th wk
and associates with the developing posterior lobe (18). The pituitary is morphologically
developed by about the 13th wk of human development (18). The order of the appearance
of the hormone-secreting cell types of the anterior pituitary is similar in most mammals,
including humans, rodents (reviewed in refs. 19,20), and swine (see ref. 21, and references
therein). In humans, ACTH- and TSHβ-secreting cells can be detected by the 9th wk of
gestation, followed by the appearance of GH- and FSHβ-positive cells by 13 wk, and the
observation of LHβ- and PRL-containing cells by the 21st wk of pregnancy (7,18).
Experiments using amphibian, chick, and rodent tissues indicate that the cell-cell
contact relationship between the infundibulum (ventral diencephalon) and the develop-
ing Rathke’s pouch is critical to correct pituitary-cell differentiation (22–26). Similar
inductive processes are found in the development of other embryonic structures such as
during lens formation in the developing eye (reviewed in ref. 27). Recent studies have
begun to delineate the extrinsic and intrinsic signaling events that coordinate the earliest
stages of pituitary organogenesis (28–32). Protein signaling molecules such as bone
morphogenetic protein 4 (BMP4), Wnt5a, and fibroblast growth factor 8 (FGF8) are
expressed in the ventral diencephalon and exert important effects on the ectodermal tissue
of Rathke’s pouch (Fig. 2 and see p. 25). Sonic hedgehog (Shh) appears to be a boundary
signal: it is expressed in the oral ectoderm but is excluded from the region from which
Rathke’s pouch originates (29). Within the developing pouch, BMP2 and Wnt4 are crucial
intrinsic signals that guide the ordered, positional development of pituitary cell types
(29,31,32). Studies of anencephalic human fetuses (which lack the hypothalamus) demon-
strate the importance of brain-derived signals for the development of corticotropes, gona-
dotropes, and somatotropes, but also suggest that some of the other pituitary cell types
can differentiate in the absence of the brain (33–35).
During rodent embryogenesis, protein signaling pathways promote the development of
Rathke’s pouch-derived pituitary cell types in a distinct stratified pattern (31,32). The dor-
sal side of Rathke’s pouch becomes the intermediate pituitary lobe that contains the melano-
tropes. The anterior lobe develops by the proliferation and differentiation of the cells of
the most ventral side of the pouch. In the developing anterior lobe, the gonadotropes form
closest to the ventral BMP2 signal; thyrotropes arise in the central part of the lobe; and
somatotropes and lactotropes (which have a common precursor; see Fig. 2) emerge in the
most dorsal side of the anterior lobe (31,32). The corticotropes also are located on the
dorsal side of the anterior lobe portion of the developing gland but are concentrated towards
the rostral tip (31,32). A transient population of thyrotropes, known as the rostral thyro-
tropes, is found in the rostral region of the developing anterior lobe (36; Fig. 2; and see
p. 17). It is interesting to note that the stratification of the secretory cells of the embryonic
anterior lobe does not persist in most adult mammalian pituitaries: in the mature gland
8 Part I / Hypothalamus/Pituitary

the cells are mostly homogeneously mixed (2). By contrast, individual cell types are
located in distinct zones in the pituitary glands of amphibians, birds, reptiles, and in many
teleost fish (2).
Over the past 12 yr, many gene regulatory proteins (transcription factors) that are
important in the development of H-P endocrine tissues have been identified. The role of
gene regulation in the development of the anterior pituitary has been intensively studied.
The development of the five physiologically important anterior cell types in a precise
order from a common origin (Fig. 2) make the anterior pituitary an excellent model sys-
tem for the analysis of the transcriptional mechanisms that regulate the commitment and
subsequent differentiation of specific phenotypes during mammalian organogenesis.
Recently, however, transcription factors with roles in hypothalamic development have
been identified and studied. This article is intended to provide an introduction to the H-P
developmental transcription factors and to discuss their functions, control mechanisms,
and involvement in human endocrine diseases.

TRANSCRIPTIONAL REGULATION
OF ENDOCRINE HYPOTHALAMUS DEVELOPMENT
Table 1 lists the properties of transcription factors that have been demonstrated to play
roles in hypothalamic development. Some of these factors also are discussed in more
detail below.

Thyroid-Specific Enhancer-Binding Protein

Thyroid-specific enhancer-binding protein (T/ebp, also known as TTF-1/Titf1/Nkx2.1)
is a Nk-2 class homeodomain transcription factor that originally was identified as a
regulator of thyroid gland development and later demonstrated to be an important tran-
scription factor in the lung (see ref. 55, and references therein). Recent analyses of mice
with disrupted T/ebp genes have demonstrated the importance of this factor in hypothal-
amic development and have revealed signaling relationships between the hypothalamus
and the primordial pituitary gland. Whereas T/ebp heterozygous mice develop normally,
mice with ablated T/ebp genes are stillborn, lack lung parenchyma, and have no thyroid
gland (55). In addition, these mice lack the ARN, and the mammillary body and supra-
mammillary nucleus of the posterior hypothalamus. Further, the entire pituitary gland is
missing (55). This result was unanticipated because T/ebp is not expressed in the devel-
oping anterior pituitary (55). However, the diencephalon, which expresses T/ebp during
development, is misformed in these animals, and recent experiments have demonstrated
that expression of T/ebp is required for the hypothalamic production of FGF8, an essential
early signal in pituitary-gland induction (28). These studies illustrate the importance of
T/ebp and the diencephalon in the development of Rathke’s pouch. The related Nkx2.2 and
Dlx genes also are expressed in the developing hypothalamus, suggesting that this class
of homeodomain genes is important in the differentiation of cells within this tissue (56).

POU Domain Transcription Factors

The POU domain class of transcriptional regulatory proteins was named for its origi-
nal members: Pit-1, Oct-1/2, and the C. elegans factor unc-86 (reviewed in ref. 57). POU
proteins are a subfamily of the homeodomain superfamily of transcription factors and
contain a structurally related DNA binding domain, known as the POU domain. The POU
 Chapter 1 / Hypothalamic-Pituitary Axis
Table 1
Transcription Factors Important in Development of the Mammalian Hypothalamus
Factor Class Mutant phenotype/Expression pattern References
Brn-2 POU-HD Lethal; PVN, SON affected-loss of AVP, OT, CRH 37,38
c-Ets-1 Ets Expressed during angiogenesis in H-P development 39
DAX-1 Nuclear receptor-like Sex-reversal; congenital adrenal hypoplasia and hypogonadotropic 40–43
hypogonadism. Expressed in developing hypothalamus.
Gsh-1 HD Dwarfed, sexually infantile, absence of GHRH in arcuate nucleus 44
Mf3 (Fkh5/Hfh-e5.1) Winged helix/forkhead Variable phenotype-some lethality. Growth defects, unable to eject milk. 45
Nhlh2 (NSCL2) bHLH Hypogonadal, progressive obesity; male infertility 46
9

Otp Paired-like HD PVN, SON, aPV, ARN affected-loss of TRH, CRH, SS, OT, AVP 47
SF-1 (Ad4BP/FtzF1) Nuclear receptor Lethal; lack adrenals and gonads. Hypothalamus and pituitary defects. 48–51
Sex reversal in humans.
Sim1 bHLH PAS Lethal; PVN, SON, aPV affected-loss of AVP, OT, TRH, SS, CRH 52
Loss of Brn-2
Six3 HD Human patients with mutations have holoprosencephaly 53,54
Expressed in anterior neural plate
T/ebp NK-2 HD Lethal; lack lung parenchyma, thyroid, pituitary. Lack premammillary, ARN, 55
(Ttf-1/Titf1/Nkx2.1) mammillary body, supramammillary nuclei of hypothalamus
HD, homeodomain; bHLH, basic helix-loop-helix.

9
10 Part I / Hypothalamus/Pituitary

domain is a bipartite DNA-interacting structure consisting of the POU-specific domain

joined by a linker region to a homeodomain, the POU homeodomain. The POU-specific
domain adopts a helical structure reminiscent of the lambda and 434 bacteriophage
repressor molecules, and the POU homeodomain is configured in a helix-turn-helix
structure that is similar to classical homeodomains such as those within the Drosophila
engrailed and antennapedia proteins (58). The POU proteins form a large family of con-
served factors that have been demonstrated to play critical roles in the development of
many tissues. They are classified into subgroups based on their structural properties (59).
BRN-2
Brn-2 is a class III POU domain transcription factor. This class of POU proteins is
expressed predominantly in the central nervous system (CNS). Brn-2 gene knockout
mice die within 10 d of birth and lack expression of AVP, OT, and CRH in the PVN and
SON of the hypothalamus (37,38). These data imply that Brn-2 is necessary for the
terminal differentiation and/or survival of magnocellar neurosecretory and parvocellar
neurons of the PVN and SON. In the absence of Brn-2 gene function, the posterior
pituitary develops normally at first, but later, the pituicytes disappear, and there is a
complete loss of the posterior lobe, suggesting that magnocellar axons produce trophic
factors needed for pituicyte development (37,38). The anterior and intermediate pituitary
lobes of the Brn-2 null mouse form normally, implying that the signals required for the
proper development of these structures arise from the undifferentiated diencephalon and
not the terminally differentiated cells of the hypothalamus. Brn-2 heterozygotes are
anatomically normal but express half of the normal levels of AVP, OT, and CRH, sug-
gesting that Brn-2 may regulate expression of these genes (37,38; Fig. 1).
OTHER POU PROTEINS
Oct-2, a POU protein first described in the hematopoietic system, may be important
in the hypothalamic neuroendocrine tissues as a regulator of puberty through activation
of the transforming growth factor-alpha (TGF-α) gene (60). Another POU factor, Tst-1/
Oct-6/SCIP, which is known to be required for peripheral nerve-cell differentiation myeli-
nation, recently has been shown to be a repressor of the GnRH gene promoter (61). Tst-1,
therefore, may play a role in the control of GnRH neuron activity.

Single-Minded
Several orthologs of the Drosophila single-minded gene have been described in mam-
mals. These include Sim1, a gene that encodes Sim1, a basic helix-loop-helix (bHLH)-
PAS transcription factor expressed during development in the PVN, SON, and anterior
periventricular (aPV) nuclei of the hypothalamus (52). Mice with deleted Sim1 genes die
shortly after birth and lack OT, AVP, TRH, CRH, and SS production in the developing
PVN, SON, and aPV nuclei (52). Further, these nuclei gradually lose expression of Brn-2,
implying that Sim1 functions upstream of Brn-2, which subsequently directs the terminal
differentiation of neuroendocrine lineages within the hypothalamus (52; Fig. 1).

Orthopedia
Orthopedia (Otp), a homeodomain transcription factor, is expressed in neurons that
give rise to the PVN, SON, aPV, and ARN hypothalamic nuclei (47). Except in the ARN,
Otp is temporally and spatially co-expressed with Sim1. Mice lacking the Otp gene die
Chapter 1 / Hypothalamic-Pituitary Axis 11

soon after birth and have reduced neuroendocrine cell proliferation and migration (47).
In these mutants, the parvocellular and magnocellular neurons of the PVN, SON, aPV,
and ARN fail to terminally differentiate: axonal outgrowth is absent, and TRH, CRH, SS,
OT, and AVP transcription does not occur (47). As in the Sim1 mutant, the Otp mutant
animals gradually lose expression of Brn-2, which implies that both genes are important
in control of the Brn-2 gene (Fig. 1). These data, together with the studies described
earlier, suggest that the aPV, PVN, and SON cell types are controlled developmentally
by Otp, Brn-2, and Sim1. In addition, Otp also regulates development of the ARN.

Gsh-1
Gsh-1 is a homeobox gene that is expressed in the neural tube, hindbrain, telencephalon,
mesencephalon, and diencephalon of the embryonic CNS (62). Homozygous Gsh-1
mutant mice display hypothalamic defects that cause secondary pituitary disease (44).
The mutants have hypoplastic pituitaries and are dwarfed, sexually infantile, and short-
lived. Pituitary GH, PRL, and LH levels are reduced. Gsh-1 appears to be essential for
GHRH gene expression in the ARN of the hypothalamus and binds to elements within the
GHRH promoter (44; Fig. 1). Cell lines derived from Gsh-1 -/- hypothalamus tissue have
been used as tools to probe DNA arrays and identify potential target genes of Gsh-1 (63).

Nhlh2
Nhlh2 (NSCL2) is a bHLH transcription factor expressed in the developing hypothala-
mus and in the embryonic and adult pituitary gland (46). Nhlh2 knockout male mice are
hypogonadal, infertile, have decreased levels of FSH and testosterone, have defects in
spermatogenesis, and feature loss of sexual behavior (46). Mutant females also are hypo-
gonadal, but their reproductive systems do develop, and the mice are fertile after expo-
sure to males. Both sexes of Nhlh2 mutant mice have progressive adult obesity. Therefore,
Nhlh2 expression appears to be required at multiple levels of the H-P axes with important
roles in the onset of puberty and the regulation of body-weight metabolism. Transgenic
mouse experiments involving misexpression of the closely related bHLH factor, NSCL1,
result in abnormal brain development (64), suggesting that other members of this class
of proteins also are important in brain embryogenesis.

Steroidogenic Factor-1
Steroidogenic factor-1 (SF-1, also known as Adrenal 4-binding protein) is encoded
by the Ftz-F1 gene (65). SF-1 is an orphan nuclear receptor transcription factor that is
expressed during development in the ventral diencephalon and in adrenal and gonadal
tissues. Ftz-F1 null mice die by postnatal day 8 and lack adrenals and gonads (48). The
mutant mice also exhibit altered structure of the VM nucleus of the hypothalamus (66).
In addition, SF-1 mutants lack pituitary gonadotropins (49), and SF-1 has been demon-
strated to be an activator of the αGSU gene promoter (49,67). SF-1, therefore, is required
for the development of steroidogenic organs and for all levels of the reproductive axis
formation. A recent report has described a mutation of the human FTZ-F1 gene that
causes XY sex reversal and adrenal failure (51). The actions of SF-1 and of other tran-
scription factors important in sex determination, such as Dax-1 (see below) and Wilm’s
tumor 1 (WT1), appear to be functionally interdependent (68,69).
12 Part I / Hypothalamus/Pituitary

DAX-1
The DAX-1 gene encodes a nuclear receptor-related protein and is located in the dosage
sensitive sex reversal (DSS) region of the X chromosome (see ref. 43, and references
therein). DAX-1 may serve as an “antitestis” gene because XY patients with DSS region
duplications exhibit male-to-female sex reversal (43). Human patients with DAX-1 gene
mutations also may develop X-linked congenital adrenal hypoplasia and hypogonado-
tropic hypogonadism (40,41,70). In mice, Dax-1 is expressed during adrenal and gonadal
differentiation and in the developing hypothalamus, suggesting a regulatory role in these
tissues (42).

Mf3
The Mf3 gene, which encodes a winged helix/forkhead transcription factor also known
as Fkh5/HFH-e5.1, is expressed in the developing hypothalamus. Mice lacking the Mf3
gene have variable phenotypes, suggesting that the gene may be necessary at several
stages of embryonic and postnatal development (45). Some mutant embryos have an
open neural tube in the diencephalon and midbrain region and die in utero; others display
a severe reduction of the posterior body axis and also die in utero. Surviving mutants have
a normal phenotype at birth but postnatally exhibit growth retardation, and one-third die
before weaning. In these animals, GH and TSH levels are normal (45). Surviving knock-
out animals are fertile, but the females cannot eject milk during suckling. This defect can
be corrected by injections of OT. These studies suggest that Mf3 is necessary for hypothal-
amic development and may play a role in postnatal growth and lactation.

Other Transcription Factors in the Developing Hypothalamus

SINE OCULIS-3
Sine oculis-3 (Six3) is a homeobox gene related to the Drosophila sine oculis gene. In
the mouse, the Six3 gene is expressed at the most anterior border of the neural plate and
later in the developing retina, lens, hypothalamus, and in Rathke’s pouch (53,71,72).
Mutations in the homeodomain of human SIX3 cause holoprosencephaly, demonstrating
that SIX3 is involved in the development of head midline structures by regulating gene
expression in the developing anterior neural plate (54).
C-E TS1

The c-ets1 proto-oncogene encodes an Ets domain transcription factor that is expressed
in the H-P system and has been proposed to play a role in angiogenesis in the hypothala-
mus, pituitary, and placenta (39).

TRANSCRIPTIONAL REGULATION OF PITUITARY DEVELOPMENT

Table 2 lists the properties of transcription factors that have been demonstrated to play
roles in mammalian pituitary development. Some of these factors also are important in
hypothalamic development and have been discussed earlier; others are discussed in more
detail below.
The Ptx Family
Three members of the Ptx family of bicoid-related homeodomain transcription factors
have been described in mammals to date (reviewed in refs. 103,104). These factors play
 Table 2

Chapter 1 / Hypothalamic-Pituitary Axis

Transcription Factors Important in Pituitary Gland Development
Factor Class Mutant phenotype/Expression pattern/Role References
Brn-4 POU-HD Ventrally induced factor during pituitary development 31
DAX-1 Nuclear receptor-like Sex-reversal; congenital adrenal hypoplasia and hypogonadotropic hypogonadism.
Expressed in developing hypothalamus 40–43
Egr-1 Zinc finger Reduced growth, female infertility, LHβ deficiency 73,74
(Krox24/NGFIA/Zif268)
ER alpha Nuclear receptor PRL and gonadotropin gene expression affected. Lactotrope growth reduced 75
GATA2 Zinc finger Regulation of gonadotrope and thyrotrope cell differentiation 31,76
Isl-1 LIM-HD Expressed in Rathke’s Pouch 77
Isl-2 LIM-HD Expressed in Rathke’s Pouch 78
Lhx3 (P-Lim/LIM3) LIM-HD Hypoplastic anterior pituitary; no differentiation 79–81
Loss of GH, PRL, TSH, LH, FSH
Lhx4 (Gsh4) LIM-HD Hypoplastic anterior pituitary; some differentiation 80
Msx1 HD Expressed in thyrotropes; can activate the αGSU gene 82
NeuroD1 (Beta2) bHLH Important in POMC gene regulation 83
Nhlh2 (NSCL2) bHLH Hypogonadal, progressive obesity; male infertility 46
13

Nzf-1 (MyT1) Zinc finger Expressed in the pituitary: can regulate the Pit-1 gene 84
Otx1 Bicoid-related HD Transient dwarfism, hypogonadism. Can activate the GH, αGSU, LHβ, FSHβ genes 85
Pax6 HD Establishes early boundary between dorsal and ventral pituitary cell types 32
P-Frk1 Winged-helix Expressed in early pituitary 29,31
Pit-1 (GHF-1/POU1F1) POU-HD Dwarfism, hypothyroidism, hypoplastic anterior pituitary 86
Loss of GH, PRL, and TSH
Prop-1 Paired-like HD Dwarfism, hypoplastic anterior pituitary Loss of GH, PRL, and TSH 87
Ptx1 (P-Otx/Pitx1/Otlx1) Bicoid-related HD Lethal, pituitary and limb defects 88,89
Ptx2 (RIEG/Pitx2/Otlx2) Bicoid-related HD Lethal; regulates lung asymmetry, cardiac positioning, and pituitary and tooth 90–95
morphogenesis
RAR Nuclear receptor Involved in Pit-1 and GH gene regulation 20,96
Rpx (Hesx1) Paired-like HD Anterior defects; septooptic dysplasia 97,98
SF-1 (Ad4BP/FtzF1) Nuclear receptor Lethal; lack adrenals and gonads. Hypothalamus and pituitary defects 49–51
Sex reversal in humans.
Six3 Sine oculis HD Expressed in the developing pituitary 53
T3R Nuclear receptor Affects pituitary-thyroid axis, growth, and bone maturation 99
TEF PAR bZIP Can activate the TSHβ gene 100
Zn16 (Zn15/Zfp15) Zinc finger Can activate the GH gene 101,102

13
HD, homeodomain; T3R, thyroid hormone receptor; RAR, retinoic acid receptor.
14 Part I / Hypothalamus/Pituitary

critical roles in many aspects of mammalian development, including pituitary organo-

genesis. Ptx1 (also known as P-OTX/Pitx1/Otlx1) was identified as an activator of the
POMC gene (105) and as an interaction partner of Pit-1 (106). Ptx1 is expressed early in
pituitary development (107,108) and appears to play a broad role in the transcriptional
activation of many pituitary hormone genes (105,106,109–111). Ptx1 also is proposed
to be an upstream regulator of the Lhx3 LIM homeodomain gene (109), a critical pituitary
transcription factor (see p. 14,15). Mice with ablated Ptx1 genes exhibit defects in both
hindlimb and pituitary development (88,89).
The Ptx2 gene also is expressed early in pituitary development (112). Targeted disrup-
tion of the Ptx2 gene (the Ptx2 protein also is known as Pitx2/RIEG/Otlx2/solurshin)
demonstrated that it is essential for proper left-right asymmetry, cardiac positioning, and
pituitary, tooth, and craniofacial development (90–95).

Egr-1
Egr-1 is a zinc finger-transcription factor that also is known as Krox24/NGFIA/Zif268.
An analysis of Egr-1 gene mutant mice demonstrated that the males were fertile, but
females were infertile due to reduced levels of LHβ (73). This study also demonstrated
the abilities of Egr-1 and SF-1 to activate the LHβ gene (73). An independent study of
mice homozygous for an Egr-1 gene mutation described mutant animals with reduced
body size and sterility in both males and females (74). This phenotype was related to
defects in the anterior pituitary of both sexes and in the ovary (74). In the pituitary, two
cell lineages expressing Egr-1 are affected differentially by the mutation: somatotropes
display abnormal cytological features and are decreased in number, consistent with the
reduced GH content observed in Egr-1 null animals (74). By contrast, gonadotropes are
normal in number but fail to synthesize LHβ (74).
Egr-1 also may play a role in hypothalamic signaling to the pituitary. For example, the
effects of GnRH-induced signals from the hypothalamus on transcription of gonadotro-
pin genes are not well-understood. GnRH is a stimulator of Egr-1 but not Ptx1 or SF-1
expression (113). Egr-1 interacts directly with Ptx1 and with SF-1, leading to an augmen-
tation of Ptx1/SF-1-induced LHβ gene transcription (113). Egr-1, therefore, may be a
central mediator of GnRH-induced signals for transcriptional activation of the LHβ gene.

LIM Homeodomain Transcription Factors

Many LIM homeodomain transcription factors, including Lhx2 (also known as LH-2),
Lhx3 (P-Lim/LIM3), Lhx4 (Gsh4), Isl-1, and Isl-2, are expressed during pituitary develop-
ment. The LIM homeodomain proteins regulate many aspects of mammalian organogene-
sis and development (reviewed in ref. 114). Several of these factors have been demonstrated
to be essential for the establishment of Rathke’s pouch and the subsequent differentiation
of the specialized trophic hormone-secreting cell types of the pituitary. These transcrip-
tion factors contain two cysteine-rich, zinc finger-like LIM motifs that mediate protein-
protein interactions with other transcription factors and regulatory proteins. In addition,
proteins in this class possess a characteristic DNA-binding homeodomain.
Lhx3 is transiently expressed in the embryonic neural cord and brainstem and then is
detected during pituitary organogenesis, first appearing in the forming Rathke’s pouch
and persisting in the pituitary throughout adulthood (79,80,115–119). Cross-species com-
parisons of Lhx3 protein sequences reveals conservation of the LIM and homeodomain
Chapter 1 / Hypothalamic-Pituitary Axis 15

regions and of a motif in the carboxyl-termini of these orthologs known as the Lhx3/LIM3-
specific domain (118–121). Lhx3 can activate pituitary trophic hormone genes, including
the PRL, TSHβ, αGSU, and Pit-1 genes (117–119,122–124). Another LIM homeodomain
factor, Lhx2, also can induce transcription from the αGSU gene (125). Both Lhx2 and
Lhx3 specifically bind to the pituitary glycoprotein basal element within the proximal
region of the αGSU promoter. In transgenic mice, this element is required to correctly
restrict expression of the αGSU gene to pituitary gonadotropes and thyrotropes (126). In
activation of other promoters, such as the PRL and Pit-1 gene promoters, Lhx3 has been
shown to transcriptionally synergize with pituitary transcription factors such as Pit-1 and
Ptx1 (117–119,122–124; Fig. 3).
Mice with disrupted Lhx3 genes are stillborn or die soon after birth and lack the anterior
and intermediate lobes of the pituitary gland (79). In these animals, pituitary develop-
ment is arrested after the initial formation of Rathke’s pouch, and most of the differen-
tiated hormone-secreting cells are missing; a few corticotrope cells can be detected
(79,80). Lhx3, therefore, is critical for both early structural events and for the specifica-
tion and proliferation of the lactotrope, somatotrope, gonadotrope, and thyrotrope pitu-
itary-cell lineages (79,80). The structurally-related Lhx4 LIM homeodomain factor also
is required for complete development of Rathke’s pouch; but unlike Lhx3, this factor is
not essential for the determination and specification of differentiated pituitary cell types
(80). Studies of mice lacking both Lhx3 and Lhx4 genes have revealed that the formation
of Rathke’s pouch is a multistep process requiring LIM homeodomain gene function.
Lhx3/Lhx4 null mice do not develop a definitive Rathke’s pouch but rather form a rudi-
mentary pouch structure (80). For this early step, there is redundancy of genetic control:
either Lhx3 or Lhx4 is required during this early stage of pituitary development (80).

Pit-1
Biochemical analyses of the GH and PRL gene promoters suggested that a common
pituitary-specific trans-acting factor bound to important A/T-rich elements within the
promoters and was critical for activation of these genes (reviewed in ref. 20). The subse-
quent cloning of this transcription factor (known as Pit-1/ or GHF-1; the human gene locus
is classified as POU1F1) revealed that the protein contained a DNA-binding homeo-
domain. Several studies have reported expression of the Pit-1 RNA in all five anterior
pituitary-cell types (127,128), but the Pit-1 protein appears to be restricted to the thyro-
trope, somatotrope, and lactotrope lineages. Consistent with this, the functions ascribed
to Pit-1 involve genes expressed in these three cell types. Pit-1 has been shown to activate
anterior pituitary genes, including those encoding GH, PRL, TSHβ, the GHRH receptor,
and the thyroid hormone receptor beta type 2 promoter (reviewed in ref. 20). In addition,
Pit-1 positively autoregulates the Pit-1 gene (reviewed in refs. 19,20), providing a mecha-
nism for the maintenance and stability of committed Pit-1-dependent anterior pituitary-
cell lineages. Pit-1 target genes possess clusters of characteristic Pit-1 binding sites that
tend to conform to an A/T-rich consensus sequence (reviewed in ref. 20).
Pit-1 is a founder member of the POU domain family of developmental regulatory trans-
cription factors. Both the POU-specific and POU homeodomain subdomains of Pit-1 are
required for high-affinity binding of Pit-1 dimers to DNA sites. The amino terminus of
Pit-1 contains the major trans-activation domain, but the POU-specific domain also appears
to contribute to Pit-1 trans-activation function (reviewed in ref. 20). High-resolution
16 Part I / Hypothalamus/Pituitary

Fig. 3. Synergistic actions of pituitary transcription factors and their cofactors. The diagram depicts
examples of reported cooperative effects of various transcription factors in activation of anterior
pituitary target genes. Symbols denote ligands for nuclear receptors.

X-ray analysis of the Pit-1 POU domain bound to a DNA element revealed that the mole-
cule binds as a homodimer in an unusual conformation (58). These data provide structural
explanations for the molecular basis of disease-causing mutations in PIT-1 (see p. 22)
and also provide insights into the mechanism by which Pit-1 can synergistically induce
transcription from pituitary genes in cooperation with a variety of other types of transcrip-
tion factors, including nuclear receptors, homeodomain factors, and LIM homeodomain
proteins (Fig. 3).
Chapter 1 / Hypothalamic-Pituitary Axis 17

Fig. 4. Mutations of the PIT-1 anterior pituitary transcription factor associated with compound pitui-
tary diseases in humans and animal models. A schematic representation of the domains of the PIT-1
protein is shown. The functions of domains are indicated. Numbers denote the locations of mutations,
and the nature of each mutation is described in the table below the protein diagram.

The importance of Pit-1 in the development of the thyrotrope, somatotrope, and

lactotrope pituitary lineages was confirmed by the molecular analysis of naturally-occur-
ring strains of dwarf mice (reviewed in refs. 19,20). The Snell (dw) and Jackson (dw J )
dwarf mice carry recessive genetic defects that segregate with chromosome 16 and lack
the thyrotrope, somatotrope, and lactotrope cell types and their respective hormone
products. Li and colleagues (86) mapped the Pit-1 gene to mouse chromosome 16 and
then demonstrated that the Snell mouse Pit-1 gene contains a single point mutation that
disables the POU DNA binding domain (Fig. 4) and that the Jackson Pit-1 gene is
rearranged and does not produce a functional protein.
The demonstration that the Pit-1-defective Snell dwarf mouse lacked thyrotrope cells
suggested that functional Pit-1 was required for the proliferation and/or maintenance of
this cell type (86). In support of this hypothesis, Pit-1 has been shown to transcriptionally
activate the TSHβ gene, and modulators of TSHβ gene activity have been demonstrated
to act through Pit-1-binding sites within the gene (reviewed in ref. 20). However, the
detection of TSHβ transcripts in the developing pituitary before Pit-1 gene activation
indicated that Pit-1 was not required for the appearance of differentiated thyrotrope cells
(127). This puzzle was solved by an analysis of TSHβ gene expression during pituitary
development in wild-type and Snell mice that revealed that there are two spatially and
temporally distinct populations of thyrotrope cells (36; Fig. 2). The first population is
found in the rostral tip of the developing pituitary (a region that eventually contributes
to the pars tuberalis) and is independent of Pit-1 activity. The rostral thyrotropes are
detected in both normal and Pit-1-defective animals during the early stages of pituitary
development and have been reported to disappear at around the time of birth (36). The
18 Part I / Hypothalamus/Pituitary

second thyrotrope population appears at the time of Pit-1 gene activation and co-localizes
with Pit-1-positive cells. These caudomedial thyrotropes are absent in the Pit-1-defective
Snell dwarf mouse, demonstrating their dependence on Pit-1. The Pit-1-dependent thyro-
tropes form the thyrotropes of the adult animal. More recent experiments have described
a population of Pit-1-independent thyrotropes that is associated with the pars tuberalis and
that persists in the adult gland (146). The mechanism that controls the initial appearance
of the Pit-1-independent population(s) of cells is unclear. Thyrotrope embryonic factor
(TEF; ref. 100), a PAR leucine zipper transcription factor that is expressed in the developing
pituitary and that can potently activate TSHβ promoter reporter genes, is a possible candi-
date for involvement in the initial activation of these thyrotropes (Fig. 2).

Prophet of Pit-1
Prophet of Pit-1 (Prop-1) is a paired class homeodomain transcription factor that is
specifically expressed in the pituitary gland (87). In the adult, expression continues at low
levels (21). Mutations in the Prop-1 gene cause compound pituitary diseases in human
patients (see below). This factor was identified by the positional cloning of the gene that
is defective in the Ames dwarf mouse (87; Fig. 5). Similar to the Pit-1-defective Snell and
Jackson mice, in Ames mice the lactotrope, thyrotrope, and somatotrope cell types are
absent or occur in low numbers. Ames mice also exhibit reduced secretion of the gona-
dotropin pituitary hormones (164). Analyses of these animal models during development
demonstrated that Prop-1 is genetically epistatic to Pit-1. For example, the size of the
nascent Ames pituitary is reduced earlier during development compared to the Snell; Pit-1
expression is deficient in the Ames dwarf; and the three Pit-1-dependent cell lineages fail
to proliferate and differentiate in the Ames pituitary (87,165–167). In addition, Prop-1
appears to be required for the repression of early pituitary transcription-factor genes,
including the gene encoding Rpx/Hesx1 (168). Little is known about the direct transcrip-
tional targets of Prop-1, although there are potential sites within Pit-1 gene regulatory
regions that may mediate Prop-1 activities (87).

Rpx/Hesx1
Rpx/Hesx1 is a paired-like homeobox transcription factor (169,170). Rpx is expressed
in the early developing mouse embryo, being later expressed in the neural plate, and
finally is restricted to Rathke’s pouch (170). The Rpx gene is down regulated when the
mature hormone-secreting cells of the pituitary begin to differentiate (170). Rpx, there-
fore, is an early marker of pituitary organogenesis and may be important for the deter-
mination of the pouch where it is controlled by other pituitary regulatory genes. For
example, whereas Lhx3 is necessary for maintaining the expression of Rpx in the first
stages of pituitary formation (80), Prop-1 is required to repress Rpx gene prior to pitu-
itary-cell differentiation (168). Mice lacking Rpx exhibit variable anterior CNS defects
and pituitary dysplasia (97). Human patients with mutations in RPX display septo-optic
dysplasia (see p. 20). Therefore, Rpx/RPX plays a role in forebrain, midline, and pituitary
development in mouse and human.

Pax6
Pax6 is an evolutionarily conserved paired-class homeodomain transcription factor
that is expressed in the developing CNS (reviewed in ref. 171). During human embryo-
Chapter 1 / Hypothalamic-Pituitary Axis 19

Fig. 5. Mutations of the PROP-1 anterior pituitary transcription factor identified as causing com-
bined pituitary hormone deficiency in human patients and in rodent models. A schematic represen-
tation of the domains of the PROP-1 gene and protein are shown. Exons are labeled in Roman
numerals. The functions of protein domains are indicated. Numbers denote the locations of muta-
tions, and the nature of each mutation is described in the table below the protein diagram.

genesis, PAX6 is expressed in the ventricular zone of telencephalon and diencephalon,

in the ventricular and ventral intermediate zones of the medulla oblongata, in the spinal
cord, in the optic cup, in the optic stalk, and in the prospective corneal epithelium (172).
Expression also is detected in the human infundibulum and Rathke’s pouch (172). The
role of Pax6 in eye development is well-established: this function is strikingly conserved
from flies to humans (reviewed in ref. 171). Analyses of mice carrying Pax6 mutations
also indicate important roles for this gene in ocular, neural, pancreatic, and pituitary
development (32,173,174). In the developing pituitary, the transient dorsal expression of
Pax6 is essential for establishing a boundary between the dorsal and ventral cell types,
perhaps by confining the ventral actions of BMP2 (32).
20 Part I / Hypothalamus/Pituitary

NeuroD1
NeuroD1 (also known as Beta2) is a bHLH transcription factor. Mice homozygous for
a deletion at the NeuroD1 locus fail to develop a granule-cell layer within the dentate
gyrus, one of the principal structures of the hippocampal formation, and exhibit sponta-
neous seizures (175). It has been reported that transcription of the POMC gene in pitu-
itary corticotrope cells depends on the synergistic actions of Ptx1 and bHLH heterodimers
containing NeuroD1 (83). Direct interactions occur between the bHLH of NeuroD1 and
the homeodomain of Ptx1 (110).

Estrogen Receptor
The actions of estrogen are critical to hypothalamic and pituitary function at many
times. Estrogen regulates the synthesis and secretion of several pituitary hormones dur-
ing the reproductive cycle. Of the two estrogen receptors, ER alpha and ER beta, the alpha
receptor appears to be the predominant receptor expressed in the pituitary gland (176).
A recent analysis of mice lacking the ER alpha gene indicated that ER alpha is involved
in PRL and gonadotropin gene transcription and plays an important role in lactotrope cell
growth but is not necessary for specification of lactotrope cell phenotype (75).

PITUITARY TRANSCRIPTION FACTORS IN HUMAN DISEASE

PTX2/RIEG
The human PTX2/RIEG gene contains 4 exons and maps to chromosome 4 at position
4q24-q25 (177). Rieger syndrome is an autosomal dominant disorder that often features
eye, dental, craniofacial, umbilical, limb, and pituitary developmental defects (see ref.
177, and references therein). Six mutations have been identified in the PITX2/RIEG gene
in patients with Rieger syndrome (177; Fig. 6). These include two splicing mutations,
three homeodomain mutations, and one mutation that introduces a premature stop codon
in the carboxyl terminus of the molecule. However, these particular patients did not
display abnormal pituitary function (177). Mutations in PTX2/RIEG also have been found
in patients with iridogoniodysgenesis syndrome (179), autosomal dominant iris hypopla-
sia type 2 (180), and Peter’s anomaly (178; Fig. 6). Molecular characterization of some of
these mutations indicates that the Thr68Pro and Leu54Gln defects reduce the ability of
PTX2/RIEG to induce transcription. These mutations appear to impair the protein-pro-
tein interaction and protein stability characteristics of the molecule, respectively (181).

RPX/HESX1
RPX/HESX1 is one of the earliest factors expressed in the embryonic pituitary gland
(169,170). Interestingly, Hesx1 knockout mice possess developmental abnormalities of
the corpus callosum, the anterior and hippocampal commissures, and the septum pellu-
cidum (97). This phenotype is similar to that seen in humans with septo-optic dysplasia.
The human HESX1 gene contains 4 exons and maps to chromosome 3p21.2-p21.1 (97).
A brother and sister with septo-optic dysplasia have been shown to be homozygous for
an Arg160Cys missense mutation in homeodomain of RPX/HESX (97; Fig. 7). Septo-
optic dysplasia in these patients is characterized by agenesis of the corpus callosum and
panhypopituitarism and is inherited in an autosomal recessive fashion (97), although
patients with dominant mutations may exist (98). Molecular characterization of the
Chapter 1 / Hypothalamic-Pituitary Axis 21

Fig. 6. Mutations of the PTX2/RIEG transcription factor associated with Rieger’s syndrome. A
schematic representation of the domains of the PTX2/RIEG gene and protein are shown. Exons are
labeled in Roman numerals. Numbers denote the locations of mutations, and the nature of each
mutation is described in the table below the protein diagram.

Fig. 7. Mutations of the RPX/HESX1 transcription factor associated with septo-optic dysplasia and
pituitary diseases. A schematic representation of the domains of the protein is shown. The functions
of domains are indicated. Numbers denote the locations of mutations, and the nature of each muta-
tion is described in the table below the protein diagram.

mutant protein indicates that the mutation inhibits the ability of RPX/HESX1 to bind
DNA (97).
LHX3
Two human LHX3 isoforms exist, and these factors may regulate transcription of
different target genes (119). The human LHX3 gene contains 7 exons and maps to chro-
mosome 9q34.3 (81,182). Humans with mutations in LHX3 are growth retarded and have
combined pituitary hormone deficiency (CPHD), featuring deficiencies of the anterior
pituitary hormones with the exception of ACTH (81). Interestingly, they also have ele-
vated and anteverted shoulders that appear as a stubby neck associated with severe restric-
tion of rotation of the cervical spine (81). Patients with a Tyr116Cys mutation have severe
pituitary hypoplasia (81; Fig. 8). However, one patient with an intragenic deletion in
22 Part I / Hypothalamus/Pituitary

Fig. 8. Mutations of the LHX3 transcription factor associated with compound pituitary disease. A
schematic representation of the domains of the two LHX3 protein isoforms is shown. The functions
of domains of the LHX3 molecule are indicated. Numbers denote the locations of mutations, and
the nature of each mutation is described in the table below the protein diagram.

LHX3 that results in a truncated protein lacking the DNA-binding homeodomain pos-
sesses an enlarged anterior pituitary gland (81), a structure similar to that found in some
patients with PROP-1 mutations (162). To date, only patients with homozygous muta-
tions in LHX3 have been identified. CPHD associated with posterior pituitary ectopia
does not appear to be caused by mutations in LHX3 (183).

PIT-1
As described earlier, Pit-1 is necessary for the specification of the anterior pituitary-
cell types that produce GH, PRL, and TSH, and gene defects in Pit-1 have been shown to
cause hypopituitarism in the Jackson and Snell dwarf mice. The human PIT-1 (POU1F1)
gene contains 6 exons and is located on chromosome 3 at position 3p11 (184). Mutations
in the PIT-1 gene in humans lead to the development of a hypoplastic anterior pituitary
gland and CPHD with postnatal growth failure. Patients with PIT-1 mutations commonly
exhibit loss of circulating GH and PRL and low or absent TSH levels that lead to the
development of central hypothyroidism. Similar to the phenotype of the Jackson and
Snell mice, these patients possess normal gonadotrope and corticotrope function. Muta-
tions have been identified in each of the regions of the PIT-1 gene that encode the func-
tional domains of the factor. Homozygous (recessive mutation), compound heterozygous
(recessive), and heterozygous (dominant) patients have been described (Fig. 4). Two
autosomal dominant mutations have been found in the amino terminal transcriptional
activation domain, several autosomal recessive mutations have been identified in the
POU specific domain, and both dominant and recessive mutations have been character-
ized in the POU homeodomain (Fig. 4).
Mutations in PIT-1 impair its ability to induce transcription by several mechanisms.
For example, the Ala158Pro (132) and Pro239Ser (137) mutations reduce the ability of
the molecule to induce transcription of pituitary target genes. Nonsense mutations such
as Arg172Ter (133) and Glu250Ter (138) result in shortened proteins that do not contain
the DNA-binding POU homeodomain. Interestingly, the common Arg271Trp mutation
Chapter 1 / Hypothalamic-Pituitary Axis 23

may exert a dominant negative effect by enabling PIT-1 to bind certain DNA elements
better than the wild-type protein (130,139,140,185). In addition, the Arg271Trp mutation
may affect dimerization of the molecule in a site-specific DNA-binding manner (58). CPHD
also occurs in heterozygous patients carrying the Pro14Leu (129) or Pro24Leu (130)
mutations, but it is as yet unclear how these mutations impair PIT-1 and cause pituitary
disease. Recently, the Lys216Glu mutation has been shown to be defective in cooperating
with the retinoic acid receptor in induction of the Pit-1 gene via the distal enhancer (186).

PROP-1
Prop-1 is necessary for the initial determination of the Pit-1-dependent pituitary-cell
types (87). The finding that a mutation in Prop-1 (Ser83Pro) is the genetic defect respon-
sible for hypopituitarism and pituitary hypoplasia in the Ames dwarf mouse (87; Fig. 5)
enabled further molecular analyses of human pituitary disease. The human PROP-1 gene
contains 3 exons and maps to the distal end of chromosome 5q (147,156,161,187). Muta-
tions in PROP-1 also cause CPHD. Similar to CPHD patients with PIT-1 mutations, these
patients typically have hypoplastic anterior pituitary glands and deficiencies of GH, PRL,
and TSH, however, they also may display low or absent levels of the circulating gonado-
tropins, LH and FSH, and many patients do not spontaneously enter puberty or experi-
ence pubertal delay (147,156,157). CPHD in patients with PROP-1 gene defects appears
to be more variable compared to the phenotype of patients with PIT-1 mutations. This
variability may be a function of the severity of the specific type of PROP-1 mutation, but
phenotypic differences in the timing of hormone loss in individuals with the same mutation
also have been described (163). In addition, a few PROP-1 mutant patients have normal-
sized or enlarged pituitaries rather than hypoplastic glands (151,160,162). Interestingly,
some older individuals with PROP-1 mutations develop adrenal insufficiency later in life
as a result of ACTH deficiency (175,162,163), while others do not (152).
Several different PROP-1 mutations cause CPHD, and most of these affect the homeo-
domain of the molecule (Fig. 5). Homozygous and compound heterozygous patients with
PROP-1 mutations have been identified (156). To date, dominant-negative PROP-1 muta-
tions have not been described. Nucleotide deletions at three different positions that dis-
rupt the reading frame and lead to a premature stop codon resulting in a truncated protein
without a complete DNA-binding homeodomain have been described (Fig. 5). One of
these deletions, the 301-302delAG, is found in what appears to be a mutational “hot spot”
region and is the most common PROP-1 genetic defect yet identified (Fig. 5). Similar to
the Ser83Pro Prop-1 mutation in the Ames mouse, the Phe88Ser, Phe117Ile, and Arg120Cys
mutations in human PROP-1 decrease its ability to bind DNA (154,156).

ALTERNATE FORMS
OF HYPOTHALAMUS-PITUITARY TRANSCRIPTION FACTORS
The production of more than one protein isoform from a single gene provides an increased
capacity for regulatory transcription factors and may allow higher levels of control in
complex regulatory mechanisms. Recent studies have demonstrated that many H-P tran-
scription factor genes encode more than one protein isoform. The structural differences
between the various isoforms vary, ranging from isoforms with amino acid sequences
missing to isoforms with distinct domain structures. The focus of most investigations of
the variant forms of H-P transcription factors has been to determine whether the alternate
24 Part I / Hypothalamus/Pituitary

proteins possess distinct transcriptional properties from the “wild-type” form. Indeed,
functional differences in activation of trophic hormone target genes, for example, have
been noted (see below). The biological relevance of such observations is, however, only
likely to be significant if the variant isoforms are expressed at levels high enough to exert
their unique effects, or if they are expressed in distinct locations or at discrete times from
the “common” or “wild-type” form. Of course, if the variant isoform exhibits entirely
separate activities from the wild-type form, then such corollaries may not apply.
Variant forms of the Rpx/Hesx1, Ptx1, and Ptx2 early pituitary transcription factors have
been reported (97,103,111,112), and the Ptx2 isoforms appear to have different activities
in assays using amphibian model systems (188). By contrast, experiments using pituitary-
hormone promoter-reporter genes have suggested similar activities for Ptx1 and Ptx2
isoforms (111). Although isoforms of the Prop-1 pituitary transcription factor protein
have not been described, the human and porcine PROP-1 genes produce alternate mes-
senger RNAs that are found in the adult pituitary at similar levels to the wild-type RNA (21;
Sloop and Rhodes, unpublished observations). To date, however, the characterized vari-
ant RNAs do not appear to encode functional proteins, and their role therefore is unclear.
The Pit-1 gene produces multiple alternate protein isoforms (reviewed in refs. 19,20).
These forms, which were originally characterized in rodents, may be expressed at much
lower levels than the wild-type form (which is sometimes called Pit-1/Pit-1α/GHF-1).
One variant, Pit-1β/GHF-2/Pit-1a, contains an insertion of 26 amino acids in the amino
terminal trans-activation domain of the protein. A second Pit-1 splice product (Pit-1T)
results from the insertion of 14 amino acids at the same position as the Pit-1β insertion.
Pit-1T has been speculated to have a role in transcription of the TSHβ promoter. A third
class of Pit-1 isoform results from a splice choice that excludes exon 4 coding informa-
tion, removing most of the POU-specific domain. Variant forms of Pit-1 also are detected
in primates such as humans and rhesus monkeys (189). Interestingly, multiple forms of
Pit-1 also are found in nonmammalian species, such as birds and fish, suggesting that the
alternate forms may have been adapted to specialized roles during evolution (190,191).
In addition, the mammalian and nonmammalian Pit-1 molecules have provided valuable
models for analysis of the functions of Pit-1 protein domains: using “natural mutagen-
esis” to test the roles of specific amino acid sequences (191,192). In addition to alternate
Pit-1 forms that are generated from differently spliced RNAs, alternative translation
initiation-site usage results in two forms of the major Pit-1/Pit-1α molecule (193).
As described earlier, the Lhx3 LIM homeodomain transcription factor gene is critical
to both the early development of Rathke’s pouch and for the later commitment and dif-
ferentiation events that mediate the establishment of the hormone-secreting cell types of
the anterior and intermediate lobes of the pituitary gland. The mouse Lhx3 gene has been
demonstrated to produce two isoforms, Lhx3a and Lhx3b (117,194). These isoforms
share common LIM domains and homeodomain sequences but possess alternate amino
terminal sequences that are encoded by alternate exons (182,194; Fig. 8). In contrast
to Pit-1, the Lhx3 genes of nonmammalian species (often known as LIM3 class genes)
do not appear to encode multiple protein isoforms. The Lhx3 gene may use distinct pro-
moters to generate RNAs encoding the two isoforms (182,194). Analysis of the human
LHX3 isoforms has revealed that LHX3a is a more potent activator of pituitary-gene
promoters such as those from the αGSU and TSHβ genes (119). The different trans-acti-
vation abilities of LHX3a and LHX3b correlate with observed differences in DNA bind-
ing affinities for binding sites within these genes, consistent with the b-specific domain
Chapter 1 / Hypothalamic-Pituitary Axis 25

serving a repressive function on this type of DNA-recognition element (119). These expe-
riments suggest that the two isoforms may serve distinct roles during pituitary develop-
ment. In accord with this hypothesis, the two forms display distinct expression patterns,
both in the timing of their appearance and in their levels of expression in various pituitary
cell types (119,194; Sloop and Rhodes, unpublished observations).

REGULATION OF HYPOTHALAMUS-PITUITARY
TRANSCRIPTION FACTOR FUNCTION
Inductive Events and Extracellular Signals
As described earlier, recent studies have begun to delineate the signaling events between
the infundibulum (ventral diencephalon) and Rathke’s pouch that coordinate the earliest
stages of pituitary organogenesis. Protein-signaling molecules such as BMP4, BMP2,
FGF8, and Shh appear to be critical to the activation of the transcription factors that
regulate H-P development (28–30). For example, BMP4 appears to be required for the
early expression of the Isl-1 LIM homeodomain gene in the primordial Rathke’s pouch.
Later, FGF8 signals cause repression of Isl-1 expression and activation of the Lhx3 gene.
Expression of the T/ebp homeodomain transcription factor in the ventral diencephalon
is a critical step in these early events. In mice lacking the T/ebp gene, pituitary develop-
ment is arrested (28,55). It is clear that the further elucidation of the extracellular-signal-
ing pathways that activate H-P transcription-factor programs will be an important focus
of future research in this field.

Post-Translational Modification
Control of protein activity by post-translational modification is a common regulatory
mechanism in eukaryotic cells. Modifications include the removal or addition of phos-
phate groups; the addition of carbohydrate moieties; the addition of lipid, methyl, acetyl,
sulfate, or isoprenoid groups; and proteolytic processing. Little is known about the possi-
ble involvement of post-translational modification in the regulation of most H-P tran-
scription factors. However, the role of phosphorylation in the activities of Pit-1 has been
extensively studied. Signaling pathways have been demonstrated to exert their actions on
pituitary hormone genes through Pit-1 DNA recognition elements (reviewed in ref. 20).
Pit-1 is phosphorylated at three positions by enzymes including protein kinase A (PKA)
and protein kinase C (PKC). Phosphorylation can modulate binding to specific pituitary
hormone target-gene sequences (195). However, other studies have suggested that the
basal trans-activation function of Pit-1 may be independent of the phosphorylation status
of the protein (196,197). Phosphorylation may serve to repress Pit-1 function by lowering
its DNA binding affinity or by reducing stability of the protein. For example, activin, an
endocrine growth factor, represses pituitary somatotrope proliferation and GH biosyn-
thesis and secretion by increasing Pit-1 phosphorylation and decreasing its stability (198).
In another study, a cell cycle-regulated kinase was observed to inhibit Pit-1 DNA binding
(199). A mutation near one of the defined Pit-1 phosphorylation sites may interfere with
human PIT-1 phosphorylation causing altered PIT-1 function and pituitary disease (200).
In summary, the precise role of Pit-1 phosphorylation is unclear. Pit-1 certainly is a key
mediator of hormone gene responses to cyclic AMP and growth factors; however, these
effects may be mediated by regulation of Pit-1 interaction with cofactor proteins that
serve coregulatory functions for these pathways (186,201, and see p. 26).
26 Part I / Hypothalamus/Pituitary

Transcription Factor Cofactors: Regulation and Execution of Activity

It is evident that most transcription factors (even those that are expressed in tissue- or
cell-specific fashion) do not function in isolation; rather, they work in combination with
other transcription factors and cofactor/corepressor proteins to regulate the rate of RNA
polymerase activity at a particular target-gene promoter. Over the past five years, critical
aspects of the biochemical mechanisms by which transcription factors activate and repress
target genes have been determined. These studies, which primarily began with studies of
nuclear receptors, have now demonstrated that many types of transcription factors recruit
coactivator and corepressor proteins to their transcriptional complexes (reviewed in refs.
202–204; Fig. 9). The coactivators acetylate chromatin histone proteins, either directly
or by the recruitment of additional factors with histone acetyltransferase (HAT) enzyme
activities. By contrast, corepressor factors cause histone deacetylation by the recruitment
of deacetylase (HDAC) enzymes. HATs acetylate lysine residues of chromatin histone
proteins resulting in greater accessibility of chromatin regions containing transcription
factor binding sites, thereby promoting gene activation. Histone deactylases catalyze the
removal of acetyl groups from the lysine residues, promoting tighter assembly of chro-
matin complexes and therefore reduced levels of transcription.
Some coregulatory proteins, such as the GRIP1 and SRC-1 nuclear receptor coactiva-
tors, appear to serve as ligand-dependent coactivators for specific types of transcription
factors such as the nuclear receptors (205,206). As described earlier, nuclear receptors,
especially those with hormone ligands such as the estrogen receptors, appear to play criti-
cal roles in the establishment and control of H-P cell types. Other transcription factor
cofactors, such as the CREB-binding protein and p300 “cointegrator” proteins, the p/CAF
and p/CIP coactivator proteins, and the N-CoR/SMRT and mSIN3 corepressor proteins,
interact with many types of transcription factors (201,207–213). The co-integrator factors
may serve multiple roles, acting as bridging molecules in transcriptional complexes and
also directly providing histone-modifying activities (214–216). Co-integrator, coactiva-
tor, and corepressor factors have been demonstrated to interact with important H-P devel-
opmental transcription factors, including Pit-1 and Rpx/Hesx1 (186,201,217). These
studies also provide important insights into the mechanism of how H-P transcription fac-
tors mediate intracellular responses to hormone and growth-factor signals resulting in
altered expression of tissue-specific genes, such as the GH and TSHβ genes. Intriguingly,
some nuclear-receptor coactivators may even act as RNAs, rather than as proteins (218).
It will be interesting to see if any coactivators of non-nuclear receptor H-P transcription
factors function by similar biochemical mechanisms. Such discoveries might allow the
design of nucleic acid-based therapies for diseases of the H-P axis.
LIM homeodomain class transcription factors, including the Lhx3, Lhx4, and Isl-1
proteins, appear to interact with multiple regulatory cofactor proteins. The LIM domains
of the molecules mediate the cofactor interactions. One extensively studied class of LIM
protein cofactors is the nuclear LIM interactor proteins (NLI, also known as Ldb/CLIM/
CHIP). The NLI proteins are broadly expressed nuclear factors that can mediate higher
order complexes involving one or more type of LIM homeodomain proteins and can
modulate LIM protein activity and cellular location (reviewed in refs. 114,219). Interes-
tingly, a recent report has demonstrated that the NLI class of factors may regulate a broader
range of target molecules, including homeodomain proteins (220). This finding has
important implications for H-P development where many of the key regulatory transcrip-
Chapter 1 / Hypothalamic-Pituitary Axis 27

Fig. 9. Chromatin-modifying cofactor proteins may mediate the actions of many H-P transcription
factors. In the absence of ligands or positive influences, H-P transcription factors (TF, including
both nuclear receptor and non-nuclear receptor factors) may interact with DNA binding elements
of repressed or silent target genes as complexes with corepressor and histone deacetylase (HDAC)
proteins. In the presence of ligands, hormones, signals (represented by the symbols on the left of the
figure), or positive regulatory factors, the corepressor and HDAC molecules are released and replaced
by coactivators, cointegrators, and histone acetylases (HATs) leading to gene activation.

tion factors are homeodomain class DNA binding proteins (reviewed in refs. 221,222).
Some cofactor proteins may be more selective in their interactions. For example, the SLB
molecule appears to interact with specific LIM homeodomain transcription factors, includ-
ing Lhx3 and Lhx4, and may serve a repressive function for PRL gene expression (123).
The mechanism of action of many cofactors is unknown. However, recent experiments
have demonstrated that factors such as RLIM, an inhibitory protein that interacts with
Lhx3, may recruit HDAC-associated protein complexes, thereby creating the chromatin
environment that does not support transcription as described above (223). Further, another
LIM-interacting cofactor, MRG1, is a positive regulator of αGSU promoter reporter
28 Part I / Hypothalamus/Pituitary

genes and has been demonstrated by Glenn and Maurer (124) to interact with the p300
cointegrator molecule and with the TATA element-binding protein, TBP.

Regulation of Intranuclear Location

A further level of regulation of H-P transcription factor function may be achieved by
the functional partitioning of these factors within the nucleus. The nucleus is a complex
structure that includes components such as the nucleoplasm, chromatin, and the nuclear
matrix. After entering the nucleus, transcription factors may undergo additional traffick-
ing, including targeting to the nuclear matrix. The nuclear matrix is a proteinaceous
substructure that resists nuclease digestion and high salt extraction (224). The nuclear
matrix is composed of proteins such as the nuclear mitotic apparatus protein, NuMA (225).
It has been proposed that transcriptionally active genes are associated with the nuclear
matrix, and multiple proteins involved in gene regulation (including HATs) have been
demonstrated to be associated with the matrix. The nuclear matrix may provide a func-
tional scaffold for chromatin and has been proposed to mediate the actions of both
extranuclear and extracellular regulatory signals that result in altered gene expression
(reviewed in ref. 226). Recent studies have demonstrated that the pituitary transcription
factors Pit-1 and Lhx3 are associated with the nuclear matrix (227,228). Intriguingly,
these two transcription factors cooperate in synergistic activation of the PRL, TSHβ, and
Pit-1 pituitary-specific genes (117–119), and it has been speculated that the interaction
of these two factors with the nuclear matrix may contribute to the transcriptional activation
of pituitary target genes (228). Consistent with this hypothesis, another homeodomain pro-
tein, Oct-1, also is present within the nuclear-matrix fraction, and this interaction has
been proposed to play a role in regulation of the TSHβ gene (229).

COMBINATIONS OF HYPOTHALAMUS-PITUITARY
TRANSCRIPTION FACTORS IN THE SPECIFICATION OF CELL TYPES
Although some H-P-specific transcription factors have been identified, it is clear that
the commitment and differentiation pathways that regulate terminal H-P cell type specifi-
cation involve interactions of both tissue-specific and ubiquitous or widespread transcrip-
tion factors of many kinds (Figs. 1 and 2). For example, in considering the determination
of the Pit-1-dependent anterior pituitary-cell lineages, the transcription factor “equations”
that specificy the thyrotrope, somatotrope, and lactotrope phenotypes must include Pit-1
plus other factors, such as nuclear receptors. Such combinatorial codes mediate the acti-
vation of the trophic hormone genes and the other cell-specific gene products that char-
acterize these cells, such as receptors for hypothalamic peptides. Consistent with this
model, synergy between H-P transcription factors has been observed in studies of many
H-P genes (Fig. 3). For example, Pit-1 has been observed to synergize with the retinoic
acid receptor (RAR) in autoregulation of its own gene (96); synergism has been reported
between the thyroid hormone receptor (T3R) and the zinc-finger protein Zn15/Zn16 and
Pit-1 in the rat GH gene promoter (reviewed in ref. 20); and between the estrogen receptor
and Pit-1 in regulation of the rat PRL gene distal enhancer (reviewed in refs. 20,230). The
Lhx3 LIM-homeodomain factor also appears to be important in the development of these
cells, and synergy has been demonstrated between Lhx3 and Pit-1 in induction of the Pit-1,
PRL, and TSHβ genes (117–119,123,228). The outcome of interactions between tran-
scription factors may be dependent on (or may determine) the differentiated phenotype
Chapter 1 / Hypothalamic-Pituitary Axis 29

of particular H-P cell types. For example, recent studies have demonstrated that Pit-1 can
synergize with the GATA2 zinc-finger protein in activation of the TSHβ gene in thyro-
tropes (31,76). Within this cell type, Pit-1 also may repress gonadotrope-specific genes
in a DNA-independent fashion (31). Further, in the gonadotrope, GATA2 may repress the
Pit-1 gene and participate in the induction of gonadotrope-specific genes (31). The
restriction of terminal H-P differentiated cell types, therefore, likely requires both induc-
tive and repressive activities of transcription factors and may involve complex protein/
protein interactions, some of which may not be DNA-dependent.
The biochemical mechanism underlying H-P transcription factor synergy is not clear,
although direct protein-protein interaction between partners often has been observed (31,
76,106,110,117,118,122,230). Further, these transcription factor combinations may pro-
mote the recruitment of coregulatory proteins to transcriptional complexes, as described
earlier. An increased comprehension of the mechanisms that drive such transcriptional
synergy will be vital to understanding the processes that regulate the establishment of the
specialized endocrine cells of the hypothalamus and pituitary.

CONCLUSIONS
Following the pivotal identification and cloning of Pit-1 in the late 1980s, substantial
progress has been made in our understanding of the tissue-specific transcriptional mecha-
nisms that regulate the development and function of the hypothalamus and pituitary. As
summarized in this article, many transcription factors that are expressed in developing
H-P tissues have been cloned. The roles of some of these factors have been elucidated
by experiments with transgenic and knockout mice; by the analysis of the naturally
occurring mutant animals; and by the molecular diagnosis of patients with H-P diseases.
In addition, elegant transgenic experiments have provided insights into the developmen-
tal lineages of specific terminal, differentiated cell types. Together, these investigations
have allowed us to generate a preliminary “road map” that outlines some of the molecular
and cellular decisions that occur during H-P organ development. This map will provide
a foundation for the significant questions that must next be answered. These include: 1)
What are the precise transcriptional codes that program the establishment of each of the
specialized cell types of the mature hypothalamus and pituitary (including determination
of the functions of alternate and modified forms of H-P transcription factors)? 2) What
are the extracellular signaling events that activate and repress H-P transcription factors?
3) What are the intracellular pathways that modulate H-P transcription factor function? and
4) How do H-P transcription factors exert their effects at the chromatin level? Solutions
to these questions will provide tools that will permit future gene or small molecule ther-
apies that specifically target transcriptional regulatory pathways in the treatment of H-P
diseases. Further, the central role of the H-P axes in controlling growth, reproduction, and
metabolism will make similar protocols useful in the animal agriculture and aquaculture
industries.
ACKNOWLEDGMENTS
The authors apologize to colleagues whose work was not cited due to space constraints.
Research in the laboratory of SJR is supported by grants from the National Science
Foundation and the U.S. Department of Agriculture National Research Initiative Com-
petitive Grants Program.
30 Part I / Hypothalamus/Pituitary

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