Normal Development of Pituitary and Hypothalamus Utilizing Mouse Model

S t e v e n M a y h e r P a g e | 1
Normal Development of the Pituitary and Hypothalamus Utilizing the Mouse Model
The pituitary gland and hypothalamus are major components of the endocrine system,
which produces and secretes hormones to regulate the activity of cells and organs. During
mammalian organogenesis, these two glands co-develop and are linked to one another
throughout life. Along the hypothalamic-pituitary axis, pituitary cells respond to external signals
that activate transcription factors to produce distinct cell types that have unique characteristics
during development of the embryo. The hypothalamus acts as the central regulator and
coordinates signals arising from the environment and brain to control the development and
function of the pituitary gland (Kioussi et al., 1999). The pituitary and hypothalamus of mice will
be examined in literature to determine normal development of these two organs.
The pituitary gland in mice is made up of three lobes designated as anterior, intermediate
and posterior lobes. The adenohypophysis, primarily the glandular portion, consists of the
anterior and intermediate lobes while the neurohypophysis comprises the posterior lobe and
contains the terminals of hypothalamic magnocellular neurons (Kioussi et al., 1999). The
magnocellular neurons are part of the visual system and are specialized for transmitting coarse-
grain and movement information from the retina to the brain (Thomson et al., 2015). The anterior
portion of the pituitary coordinates responses in-between the hypothalamus and endocrine organs
and is comprised of a variety of hormone-producing cell types. The intermediate lobe’s main
function is the production of melanocyte-stimulating hormones and endorphins while the
posterior lobe is stimulated by the hypothalamus to release vasopressin and oxytocin (Burrows et
al., 1999). Overall, the pituitary gland is delineated as the body's master gland and regulates
growth, reproduction, metabolism and response to stress through the secretion of hormones
(Davis et al., 2013).

The pituitary gland is generated from the neural and surface ectoderm. The neural
ectoderm forms the posterior lobe from the ventral diencephalon while the surface ectoderm
gives rise to Rathke's pouch, which is the predecessor of the anterior and intermediate lobes
(Davis et al., 2013). In the mouse, the anterior and intermediate lobes make their initial
appearance at embryonic day 8 (E8) with the oral (surface) ectoderm becoming thicker and
turning inside out to produce Rathke's pouch. Separation of Rathke's pouch from the surface
ectoderm occurs by E12.5. On the anterior side of Rathke’s pouch, cells proliferate to form the
anterior lobe, and cells on the opposing side form the intermediate lobe. At the same time, the
posterior lobe is developing from neural ectoderm. During development, the pituitary and
hypothalamus physically remain close together. By E16.5, the anterior pituitary’s various cell
types have already differentiated and begun to produce hormones (Burrows et al., 1999). Once
fully developed in the human, the pituitary gland is a six mm ovoid structure that is located
above the sphenoid sinus, which is a cavity in the sphenoid bone that opens into the nasal
passages (Miller-Keane, 2003) and right below the optic chiasm, the place where parts of each
optic nerve cross over (Mosby, 2009). It is connected to the hypothalamus by a stalk and has a
complicated blood supply, which includes hypothalamic veins that drain into the pituitary gland
(Atkinson, 2010).
The pituitary is characterized by a network organization in the anterior lobe and is made
of both endocrine and non-endocrine cells. In the anterior pituitary gland, specialized cell types
are differentiated during embryogenesis through an outburst of transcription factors and
signaling pathways (Le Tissier et al., 2012). In particular, transcription factor Sox2, which is an
indicator of many stem or precursor cells, is expressed in Rathke's pouch and the primitive
pituitary gland (Kioussi et al., 1999). In the adult pituitary, Sox2 cells are mostly located in the

pituitary cleft, which is a split or fissure in the pituitary (American Heritage Dictionary, 2007),
and are a group of pituitary stem cells (one of the non-endocrine cell types) from which
differentiated cell lineages can arise (Mollard et al., 2012). Folliculostellate cells, another non-
endocrine type, help coordinate hormone release by the propagation of electrical signals
(Mollard et al., 2012).
During development of the anterior pituitary, five types of endocrine cells are formed.
Through GFP staining, it is possible to determine the development and localization of the various
cell types. Somatotrophs secrete growth hormone (GH) and are detected in mice at E15.5.
During embryogenesis, some somatotrophs have been shown to migrate at E18.5. In the
postnatal period, there is a regeneration of somatotrophs to maintain a cellular continuity of
somatotroph clusters, which is later modified in male mice by increased cell clustering from 40
to 70 days of age during post-pubertal growth where GH is needed (Le Tissier et al., 2012).
Lactotroph cells secrete prolactin (PRL) and develop last in the sequence of pituitary hormone
cell proliferation. Not many lactotrophs preside in the developing embryo, but studies have
shown that cells which later develop into lactotrophs are present at an earlier stage. Their
distribution in the anterior pituitary is not even. However, in female mice, the organization of
lactotrophs becomes more apparent when prolactin is required for the mammary glands to
become active (Le Tissier et al., 2012). Gonadotrophs secrete luteinizing and follicle stimulating
hormone (LH and FSH) and are first detected in mice at E16.5. These cells cluster by E18.5 and
concentrate themselves in the central mediolateral section of the anterior pituitary, with some
cells locating themselves in lateral regions (Le Tissier et al., 2012). Corticotrophs secrete
adrenocorticotrophic hormone (ACTH) and are the first endocrine cell type to differentiate. They
can be detected by E13.5 in mice and localize themselves to the ventral surface of the pituitary

gland. By E15.5, the corticotrophs increase their surface area and reach into the center of the
gland, where they maintain this organized structure in the postnatal state and into adulthood (Le
Tissier et al., 2012). As of 2012, the thyrotrope network of cells, which secretes thyroid
stimulating hormone (TSH), had not yet been visualized through laboratory techniques (Mollard
et al., 2012).
Various signaling pathways are involved in the development of the pituitary gland and
were analyzed on genetically engineered mice. An overlap of bone morphogenetic protein
(BMP) and fibroblast growth factors (FGF) are expressed in the ventral diencephalon, where it
folds over to form the infundibulum, the stalk that connects the pituitary gland and hypothalamus
(Davis et al., 2013). In particular, FGF signaling induces the gene which encodes factor Lhx3/P-
Lim. This signaling pathway is necessary to induce further development of Rathke’s pouch.
Studies have shown that if the gene encoding for FGF10 or FGF receptor type 2 is deleted, the
pituitary gland fails to proliferate (Scully & Rosenfeld, 2002). The role of BMPs and FGFs in the
specification of cell types within the anterior lobe of the pituitary are being explored. FGF8 and
FGF10 are expressed in the infundibulum at E10.5 in mice while BMP is expressed on the
ventral section and nearby mesoderm. Proposals have been made which specify that
counteracting gradients of FGF and BMP signaling regulate the development of specific
endocrine cell types dependent on location of progenitor cells relative to the gradient. (Davis et
al., 2013). As a result of the signaling gradients, there is a positional commitment of cell type
lineages from a dorsal to ventral sequence by E11.5 (Scully & Rosenfeld, 2002).
The pituitary gland continues to undergo extensive alterations after birth in order to
respond to stress and damage. Tissue maintenance is required in the adult to supply new cells to
replace those which perish due to normal cell death. Gleiberman et al. (2008) proposed that in

the mature pituitary gland, there are nestin-expressing adult stem cells that assist in the process
of tissue maintenance. By using genetic inducible fate mapping, they were able to demonstrate
that adult pituitary stem cells produce all of the pituitary endocrine cell types. To identify
anterior pituitary stem cells, Gleiberman et al. (2008) utilized transgenic mice and nestin-GFP to
determine the genetic lineage. Expression of the nestin gene has been discovered in several stem
cell types that have the ability to become tissue-specific, making it ideal to utilize as a marker.
GFP-positive cells were present in the pituitary gland’s intermediate and anterior lobes of mice
between 3 to 4 weeks old, mostly located in the region of the lumen between the anterior and
intermediate lobes (Gleiberman et al., 2008). A marker known as Lhx3, which is key in all six
pituitary cell lines, was found to be expressed in GFP-positive cells. This finding suggests a
histogenetic relationship to the endocrine cell types (Gleiberman et al., 2008). However, the
nestin-GFP cells did not contain markers that would lead to terminal differentiation. Through
analyzing various stages of nestin-GFP expression, the development of pituitary cells was traced
during embryonic and perinatal periods. At the end of embryogenesis and right after birth, the
number of GFP-expressing cells increased gradually. Although these cells were positive for
Lhx3 and other markers, they were not expressing markers to promote terminal differentiation.
These observations suggest that nestin-GFP cells are unlike the embryonic precursor cells that
are present during embryonic development. Gleiberman et al. (2008) used a lineage analysis
technique to show that GFP-positive cells were in the rostral tip of the newborn pituitary gland,
which degenerates after birth. It is after the first postnatal week that a second wave of growth of
GFP-positive cells occurs in the intermediate and anterior lobes to produce differentiated cells
that are similar in phenotype but originate from a different source. The data in this research

confirms that tissue maintenance of the adult pituitary gland occurs through nestin-positive
pituitary stem cells (Gleiberman et al., 2008).
The hypothalamus is one of the smallest parts of the brain at a weight of 4 grams.
It is symmetrical on each side of the brain with the third ventricle in the midline forming a
boundary. The hypothalamus is divided from rostral to caudal into thirds and surrounded by
blood vessels. (Saper & Lowell, 2014). Contained within the hypothalamus are numerous
neuronal circuits. Axons of the magnocellular neurosecretory system ascend from the
hypothalamus and terminate in the pituitary gland’s posterior lobe (Kioussi et al., 1998). The
rostral section of the hypothalamus lies above the optic chiasm and includes various nuclei in the
preoptic area. The middle section is the tubular hypothalamus, which is where the pituitary stalk
rises from the ventral surface. The posterior section includes mammillary bodies and the areas
above them (Saper & Lowell). Initially in the developing embryo, the hypothalamus is located at
the most rostral region of the neural tube and later assumes a more caudal and ventral position
(Braverman & Cooper, 2012).
The hypothalamus is integrative in that this gland brings together various sensory inputs
that are necessary to make important decisions concerning basic life functions. There are three
sets of endocrine outputs that control the autonomic nervous system: magnocellular system,
parvicellular system and autonomic innervation (Saper & Lowell, 2014). The magnocellular
system transmits coarse-grain information about movement while the parvicellular system
transmits fine-grain highly detailed information. The autonomic innervation is the part of the
nervous system that regulates vital involuntary functions of the body, including heart muscles,
smooth muscles and the glands. The magnocellular and parvicellular systems are the main
pathways that lead from the retina to the brain in the visual system (Thomson, Shillcock &

McDonald, 2015). The hypothalamus controls thermoregulation, feeding and energy metabolism,
sleep and wakefulness and social responses (Saper & Lowell, 2014).
The tissue of the hypothalamus arises from the most rostro-ventral part of embryonic
prosencephalic neuroepithelium, also known as the ventral diencephalon (Braverman & Cooper,
2012). The most rostral component, the preoptic area, develops from the telencephalon (Saper &
Lowell, 2014). In the ventral section, the infundibulum evaginates and forms multiple structures,
including the posterior pituitary gland and the pituitary stalk, which connects the hypothalamus
to the pituitary (Braverman & Cooper, 2012). Once fully developed, the hypothalamus becomes
a network of neuronal circuits that are capable of regulating vital body functions (Kioussi et al.,
1998).
Immunohistochemical studies in both rodents and humans have shown that the
hypothalamus is essentially produced by three neurogenic waves. The first neurons to be
produced are in the peripheral region known as the lateral zone. The second neurogenic wave
forms the core of the hypothalamus or intermediate zone. The last neurogenic wave produces
neurons of the midline zone, which connect with the retina, pituitary and autonomic centers
(Maggi et al., 2015). In mice, early tissue growth is first noted at embryonic day 9.5. From
E12.5-14.5, neuroblasts start to produce specific neurohormones as they migrate towards the
hypothalamic nuclei, which is their final destination. At E16-18, ciliated and non-ciliated
ependymal cells take their place on the 3rd ventricle wall. During the perinatal period and right
after birth, tanycytes are generated and seem to act as adult neural precursor cells and assist in
feeding and chemoception (Maggi et al., 2015).
The signal pathways in the development of the hypothalamus are not as widely known as
that of the pituitary gland. However, nodal signals have been shown to be required for posterior-

ventral development of the hypothalamus. Hedgehog signaling diminishes the development of
this hypothalamic region and promotes development of the anterior-dorsal section (Mathieu et
al., 2002). In order to identify genes expressed in a developing mouse hypothalamus, Shimogori
et al. (2010) conducted microarray analysis at 12 different points in development. Next they
conducted developmental in situ hybridization (ISH) for 1,045 genes which were expressed
during hypothalamic neurogenesis. In the process of ISH, markers were identified in the
hypothalamic nuclei and were used to construct a well-defined molecular atlas of the developing
hypothalamus. To illustrate the usefulness of this data, Shimogori et al. (2010) utilized these
markers to examine the phenotype of mice in which selective deletion of Shh from the
hypothalamic neuroepithelium was performed. The results of this investigation demonstrated that
Shh is vital for patterning in the anterior hypothalamus. From E10 to E16 in mice, identification
of genes whose expression were enhanced during neurogenesis was completed. The prethalamic-
hypothalamic border was determined and further studies were conducted to identify the location
of Shh expression. Shh was selectively removed from the hypothalamic basal plate where
subsequently these mice showed a diminishing area located in the anteriotuberal section of the
hypothalamus and the ventral telencephalic neuroepithelium by E12.5. In conjunction with
various other data, it was demonstrated that Shh is crucial for differentiation of the anterior and
tuberal hypothalamus (Shimogori et al., 2010).
The pituitary gland and hypothalamus are important parts of the endocrine system and are
involved in key regulatory functions in the developing embryo as well as in postnatal life and
throughout adulthood. The two begin to co-develop at very early stages in embryogenesis and
work together to control basic life and body functions, such as growth, reproduction,
metabolism, feeding, sleep and wakefulness and social responses through secretion of hormones.

Along the hypothalamic-pituitary axis, developing cells respond to external signals that trigger
transcription factors which determine the final unique characteristics of each cell type. Together,
the pituitary and hypothalamus are crucial for homeostasis and sustained life.

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Normal Development of Pituitary and Hypothalamus Utilizing Mouse Model

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