Chapter CD

Molecular Control of the

Development of Some
Organs
THIS CHAPTER IS FOR POSTGRADUATE STUDENTS ONLY
AND IS MEANT ONLY FOR REFERENCE.
In Chapter CD3 we have considered the principles that govern molecular biology. In this
Chapter we will consider the molecular control of the development of some structures.

Molecular Control for Establishment of Body Axes

The appearance of the primitive streak defines the left and right sides of the embryo. Soon after
its appearance, the primitive node and streak express the fibroblast growth factor 8 (FGF-8).
This induces the expression of the nodal gene on the left side of the disc. After the induction
of the neural tube the FGF-8 gene induces the nodal and Lefty 2 genes, (in the lateral plate
mesoderm) on the left side only. These genes then regulate the expression of PITX2, which is
a homeobox containing the transcription factor important for establishing left sidedness. The
sonic hedgehox (SHH) gene, which is expressed in the notochord, prevents the expression of
left sided genes on the right side, by acting as a midline barrier. The expression of the snail
gene on right side establishes right sidedness. Because of handed asymmetry, some organs in
the body lie on the right side and some on left side (stomach, heart, spleen on the left side and
the liver on the right).

Molecular Control of Gastrulation

Several signaling molecules are expressed in the primitive streak. These are chordin, crypto,
Vg1, brachyury and nodal.
The dorsal lip of the blastopore is considered to be the primary organizer. The induction
of this organizer is due to the signalling molecules of the Wnt family (Wnt-3), the paired type
homeobox transcription factor MIX and members of the fibroblast growth factor family (FGF).
A homeobox gene goosecoid is expressed specifically in the primitive node. This is the first
zygotic gene to be expressed in humans. It is similar to the fruit fly genes goosberry and bicoid.

Human Embryology

The different sub-regions of the primitive streak give rise to mesoderm and endoderm for
the regions of the head, the trunk and the tail. The cells of the epiblast invaginate into the
primitive streak, and the primitive node, and migrate in different directions. This migration
takes place between epiblast and hypoblast.

Organising centres for the head, the trunk and the tail are present along the cranio-caudal
axis of the primitive streak. Their arrangement is genetically controlled. This is achieved by the
expression of a gene called Cripto, a gene belonging to the EGF family of growth factors.

The organization of the head region is under the control of the gene Lim-1. The knockout
of gene Lim 1 produces an animal without a head. Other genes, which help in the formation
of the head region are Otx-1 and Otx-2, acting along with HNF-3 (hepato-nuclear factors). The
expression of the nodal gene of the TGF family is also necessary for the development of cranial
structures.
The trunk-organizing centre controls the development of paraxial and lateral plate
mesoderm of the neck, of the thorax and of the abdomen. The formation of trunk paraxial
mesoderm is under the control of T-box genes (Tbx-6 and brachyury). The fibroblast growth
factor-9 (FGF-9) appears to play a role in the formation of intermediate mesoderm. The tailorganizing centre forms the mesoderm of the sacral region. Genes responsible for formation
of the mesoderm of this region, and of the caudal portion of the neural tube are brachyury,
Wnt-5a and Wnt-5b. A mutation of Brachyury is responsible for caudal dysplasia or caudal
dysgenesis.

Formation of Notochord
Cells of the primitive node express HNF-3B (hepatic nuclear factor-3B) that is important for
the formation of the notochord. This molecule is also required for initiation of the functions of
the notochord. The notochord fails to develop in the absence of this transcription factor.

Molecular Control of the Formation of Somites

The formation of a somite from the homogenous strip of paraxial mesoderm involves
expression of many genes.
The somites are formed by cyclic expression of segmentation genes like c-hairy
(homologue of pair rule gene) and members of the Notch and WNT signaling pathways. Each
cycle of somite formation lasts a few minutes to a few hours. At the start of the cycle c-hairy
is expressed throughout the non-segmented paraxial mesoderm but at the end of the cycle
its expression is concentrated near the posterior edge of a new somite. Similarly, the lunatic
fringe molecule of the Notch signaling pathway is expressed in the presomite mesoderm (in
a cycle of 90 minutes). Once the somite is formed its concentration decreases. Formation of
boundaries between somites involves retinoic acid and FGF8 genes.
The specific segmental identity of future somites is determined by the expression of a
unique sequence of HOX genes.

Chapter CD 4 Molecular Control of the Development of Some Organs

Fig. CD-4.1:Somatomere and somites are

arranged on either side of the notochord.

Unsegmented paraxial mesoderm grows
caudally while new somites are formed at its
cranial end.

Molecular Control of the Formation of Blood and of Blood Vessels

The fibroblast growth factor 2 (FGF2) binds to its receptors (FGFR) located on mesenchymal
cells where blood vessels and blood has to form. Under the influence of FGF2, the mesenchymal
cells get converted into hemangioblasts. The notochord secretes the factor sonic hedgehog.
This factor causes the surrounding mesenchyme to secrete VEGF (vascular endothelial growth
factor). Under the influence of VEGF, hemangioblasts now form precursors of blood and
blood vessels. The cells in the centre of blood island form precursors of all types of blood cells.
The peripheral cells of blood island, form endothelial cells of developing blood vessels (both
arteries and veins). The maturation of blood vessels is under the control of two more growth
factors i.e., PDGF (platelets derived growth factor) and TGF (transforming growth factor).

Skin
The proliferation of basal epidermal cells is under the control of many growth factors. Growth
factors that stimulate mitosis are epidermal growth factor (EGF), fibroblast growth factor

Human Embryology

(FGF), interleukin-1 and insulin like growth factors. However, some factors may inhibit this
proliferation. These are transforming growth factors and interferons.

Molecular Control of Hair Formation

The formation of a hair follicle is the result of interaction between epidermis and underlying
mesenchyme. At the site of the formation of a hair the mesenchyme express the FGF-5 that
influences the epidermis to form a downgrowth. The epidermal downgrowth now begins
to express Sonic hedgehog (Shh) that stimulates the formation of a hair follicle. Hoxc-13
expressed in hair follicles helps in keratinization of hair. (Note that at this site the function of
Hox is different from functions expressed during the development of axial structures.)
The factors BMP-2 and BMP-4 are expressed in intervals between epidermal down
growths. They inhibit the production of more epidermal downgrowths. This is how the spacing
between hair is regulated.
The EDA gene provides instructions for making a protein called ectodysplasin A. This
protein is part of a signaling pathway that plays an important role in the development of
ectodermal appendages (hair, teeth and sweat glands) before birth. Ectodysplasin-A has an
important role to play in ectodermal-mesodermal interactions during embryonic development.
Defects in the molecular structure of this protein inhibit the action of enzymes necessary for
normal development of ectoderm. More than 60 mutations have been identified in the EDA
gene. The gene is located on the long (q) arm of the X-chromosome between positions 12 and
13.1 (Xq12-q13.1). The hypohydrotic ED is inherited as an X-linked recessive trait.

Molecular Control of the Formation of the Vertebral Column

Shh is expressed by the notochord. It influences the expression of Pax-1. Pax-1 acts on the
sclerotome causing it to form the centrum (body) of the vertebra.. The roof plate of the neural
tube expresses Pax-9 and Msx-1 and Msx-2 that act on mesenchymal cells of the sclerotome to
form the neural arch.
The formation of a segmented vertebral column, along the cranio-caudal axis of the
embryo, is controlled by expression of homeobox containing genes. Most of the vertebrae are
formed under the influence of a unique combination of many Hox genes. For example the first
lumbar vertebra is formed by the expression of genes Hoxc-9, Hoxd-8, Hoxa -10 and Hoxd-9.
The imbalance in the expression of the Hox genes results in variations in vertebral structure or
vertebral number.

The factor Pax-1 is expressed during the formation of an intervertebral disc. Its absence
leads to fusion of adjacent vertebrae.

The Sternum
When the fusion of the two sternal bars is faulty, the body of the sternum shows a partial or
even a complete midline cleft. This is due to mutation in the Hoxb-2 and Hoxb-4 genes. Minor
degrees of non-fusion may result in a bifid xiphoid process or in midline foramina. Transverse

Chapter CD 4 Molecular Control of the Development of Some Organs

clefts may also occur. Malformation of xiphoid process is due to mutation in the genes Hoxc-4
and Hoxa-5.

Molecular Control of Limb Development

Control of Limb Bud Formation
The lateral plate mesoderm, present at the site of the formation of a limb bud, expresses FGF10. The absence of FGF-10 (in FGF-10 knockout mice) results in failure of the limb to form.
This view is supported by the experimental finding that if FGF-10 is made to express at some
other site in the body it leads to the formation of an extra limb. Another stimulus known to
form a limb bud is retinoic acid.

FGF-10 secreted by mesoderm acts on overlying ectoderm to influence it to express FGF8. The interaction of primordia of the ectoderm and the mesoderm of the limb bud, provides
sufficient information for formation of the limb.

The early mesoderm expresses the molecules Tbx-4, and Tbx-5. These determine whether
the limb bud will give rise to the forelimb or to the hindlimb. Tbx-5 is expressed only in the
forelimb, while Tbx-4 is expressed only in the hindlimb.

Control of Axis Formation in a Limb Bud

The posterior region of a limb bud (along the future antero-posterior axis) acts as a signaling
centre for formation of the limb. This signaling centre is called the zone of polarizing activity
(ZPA). The signal expressed by this centre is Shh.
ZPA stimulates production of retinoic acid. This leads to expression of Shh which controls
the development of the limb along the anteroposterior axis. ZPA also control the activities of
AER (apical ectodermal ridge).
AER controls and stimulates the outgrowth of a limb (along a proximo-distal axis) by
secreting the FGF family (FGF-2, FGF-4 and FGF-8). The mesenchyme underlying the AER
expresses Msx-1, which helps in the proximo-distal growth of mesenchyme. Homeobox
containing genes (Hoxd-9 to Hoxd-13 and a few Hoxa genes) are involved in patterning of the
limb along the proximo-distal axis. Mutation of Hox genes may result in abnormal formation
of the bones of a limb.

The dorso-ventral axis is organized by expression of Wnt-7 in the dorsal ectoderm, while
the ventral ectoderm expresses En-1 (Fig. CD 10.4).

Control of Formation of Digits

At the time of formation of the digits, at the apex of limb, the AER begins to break up. AER
only persists at the sites of digital rays. In between the digits it starts regressing (by cell death)
leading to formation of inter-digital spaces. Cell death occurs due to expression of BMP2, BMP-4, BMP-7, Msx-1 and Msx-2. Absence of BMP leads to the non-separation of digits
resulting in syndactyly.

Human Embryology

Molecular Control of the Development of the Face

It should be noted that all processes from which the face develops (frontonasal,
maxillary, mandibular) consist of a covering of ectoderm beneath which there is mesoderm
(mesenchyme). The mesenchyme of the upper face is derived from neural crest cells of the
hindbrain region. BMP signaling is necessary to form the edge of the neural crest. The same
signaling then regulates the expression of WNT, which help in the migration of neural crest
cells into the first pharyngeal arch.

The genetic control of the early development of the face is guided as per sequential events
given below.
1. The frontonasal process is formed by synthesis of retinoid acid in ectodermal cells
covering the forebrain. Retinoic acid is responsible for the maintenance of the signals of
the fibroblast growth factor 8 (FGF-8) , and sonic hedgehog (Shh) signals.
2. Shh and FGF-8 molecules now stimulate neural crest cells to proliferate in the frontonasal
process.
3. After the fifth week of development, the proliferation of the frontonasal process slows
down. The maxillary process, the mandibular process, and the nasal processes (medial
and lateral) start growing rapidly. Growth of all these processes results from interactions
between the overlying ectoderm and underlying mesoderm. Here again the active
signaling molecules in the ectoderm are FGF-8 and Shh. These signals stimulate growth of
the mesenchyme.
4. The growth of the maxillary process is due to establishment of a signaling center in the
mandibular arch. FGF- 8 is the molecular signal for formation of the maxillary process.
5. Thereafter, the homeobox containing MSX-1 gene is expressed in the mesenchyme of all
the facial processes.
6. The transcription factor Otx-2 is expressed in the first arch (maxillary and mandibular
processes). This gene characterises the precursors of the first arch. (It should be noted
that the HOX genes are not expressed in the first arch. However, they are expressed in
all the other pharyngeal arches.)
7. Further development of the mandibular process is strictly under genetic control. The
medial region of the mandibular process responds to local epithelial signals FGF-2 and
FGF-4 and stimulates growth of the underlying mesenchyme. These signals are mediated
through Msx-1 factors. Growth of the lateral region of the mandibular process is due to
FGF-8 signals. These signals are mediated by bone morphogenetic proteins BMP-4 and
BMP-7 that are produced in the lateral regions of the mandibular process.

The development of the mandibular arch (in proximal to distal direction) depends upon
the expression of the Dlx group of transcription factors.

Dlx-1 and Dlx-2 are expressed most proximally in the mandibular process. Dlx-5 and Dlx-6
are expressed more proximally and Dlx-3 and Dlx-7 are expressed most distally.

Dlx-1 and Dlx-2 are also expressed in the maxillary process.

Chapter CD 4 Molecular Control of the Development of Some Organs

Fig. CD-4.2
Various genes expressed during the development of the face and palate
Region

Early phase

Later phase

Frontonasal process

Retinoic acid, FGF-8, Shh

Msx-1

Maxillary process

FGF-8, Msx-1

Dlx-1/2

Mandibular process

FGF-8

Mandible

Msx-1, Otx-2

Lateral palatal process

Shh, ECF, Max-1, BMP-4/2

Fusion of palatal process

TGF-B3

FGF-8, BMP-4/7
FGF-4/2, Dlx-5/6, Dlx-3/7

with nasal process

Molecular Control of Formation of the Palate

The two lateral palatine processes are formed as shelf-like outgrowths from the maxillary
processes (in the 6th week of development). The growth of these processes depends upon the
interaction between ectoderm and mesenchyme. The following genes play an important role
in the development of the palate.

The mesenchyme of the palatal shelf expresses Msx-1 that stimulates BMP-4 signaling in
the mesenchyme.

This leads to expression of Shh signaling in the apical ectoderm.

Shh further induces BMP-2 signaling in the underlying mesenchyme.

Both BMP-2 and BMP-4 stimulate mesenchymal proliferation leading to the growth of the
shelf like palate.
The Epidermal growth factor (EGF) stimulates glycosaminoglycan production within the
palatal shelves.

As the right and the left palatal shelves start fusing with each other in the midline, they
are covered by epithelium. Some of these fused midline epithelial cells soon disappear by
the process of apoptosis, while some other cells transform themselves from epithelial to
mesenchymal cells. This transformation of cells is mediated by the release of transforming
growth factor (TGF-B3 ). It is well known that TGF-B3 is expressed in the epithelium just before
fusion of palatal processes. Mutation of the TGF-B3 gene leads to formation of isolated cleft
palate.

Molecular Control of Development of Teeth

Each tooth has a distinct morphology and a specific location. Determination of both the factors
is under strict genetic control. The following description discusses the molecular control of the
formation of a tooth during various stages of its development.

Human Embryology

Initiation of the formation of a tooth begins with signals originating in the epithelium of
the dental lamina. These signals induce neural crest mesenchyme (underlying the epithelium)
to form a tooth.
The transcription factor gene Lef-1 (lymphoid enhancer factor-1) stimulates the
ectoderm of the dental lamina to secrete factor FGF-8. (The absence of Lef-1 results in the
arrest of bud initiation. Expression of Lef-1 at any abnormal place in the oral epithelium
results in ectopic tooth formation.)
FGF-8 reaches the underlying mesenchyme where it induces the mesenchyme to express
the Pax-9 gene. Pax-9 defines the location of a tooth germ.

Expression of the gene Pax-9 is important for further development of the tooth. Absence or
mutation of Pax-9 leads to the arrest of all teeth development at the bud stage.

In addition to Pax-9 another transcription factor gene induced by the effect of FGF-8 is
Msx-1.

BMP-4 and BMP-2 are also expressed by surface epithelium. These genes are able to inhibit
the expression of Pax-9.
Tooth germs develop only in those areas where Pax-9 expression is induced in the
mesenchyme by FGF-8 expression in the overlying epithelium. A tooth germ does not develop
in those areas where BMP-4/2 signaling inhibits Pax-9 inducing activity of FGF-8.
At the bud stage the following genes send signals to stimulate the mesenchyme of the
tooth bud. These genes are BMP-4, Shh and FGF-8. Shh is an important signaling gene for
initiation of tooth development. Gli genes are downstream mediators of Shh expression.

As a result of these signals received from the epithelium of the dental lamina the underlying
mesenchyme expresses the following genes, Msx-1 and 2, BMP-4, EGR-1 (early growth
response-1), tenascin and syndecan. Msx 1 is required for BMP-4 expression. The expression
of BMP-4 in the bud mesenchyme is required to maintain BMP-2 and SHH expression in the
epithelium. Mutation of the
MSX-1 gene may result in the
loss of SHH expression. Shh
signaling has a major role in
the bud up to the cap transition
stage of development.
The
enamel
knot
formation directs the next phase
of tooth development. It is
responsible for the formation of
tooth cusps that later give each
individual teeth its characteristic
surface. Mesenchymal BMP-4
activates p21/Msx-2/ Bmp-2
in the epithelium to form the
Fig. CD-4.3:Future teeth are formed at the sites where Fgf-8 enamel knot.
The type of the tooth to be
activates Pax-9 expression. No teeth are formed at sites where Pax-
9 is inhibited by Bmp-1/2.
formed is strictly under genetic

Chapter CD 4 Molecular Control of the Development of Some Organs

Fig. CD-4.4: Msx1 gene is responsible for formation of incisors, while Dlx and Barx-1 are responsible for
molars. B. The overlapping expression of Msx and Dlx genes determines the formation of
canines and pre-molars.

control. Msx-1 and Msx-2 homeobox genes are expressed in the region of future incisors. Dlx-1
and Dlx-2 genes are expressed in ecto-mesenchyme where future molars develop. The gene
Barx-1 acts along with Dlx-1 and Dlx-2 to develop molars. It is believed that the formation of
canine and premolar teeth in the human results due to overlapping expression of Msx and Dlx
genes.

Thus Msx genes can be designated as incisor genes and Dlx and Barx-1 as molar genes.
The transcription product of Msx-1 is active during later stages of tooth development
(possibly regulating the differentiation of ameloblasts and odontoblasts).

Molecular Control of the Development of the Gut

The molecular control of the development of the gut tube is already well established in the fourth
week of intrauterine life. Various transcription factors are expressed in different regions of gut
to initially differentiate it into oesophagus and stomach (Sox-2); duodenum (Pax-1 ; small and
large intestine (Cdx-1 and 2). Further differentiation of the gut occurs due to expression of the
signaling molecule Shh in the endoderm of the entire gut tube. The expression of Shh leads to
orderly expression of homeobox containing genes (HOX genes) in gut associated mesoderm.
Once the fate of gut mesoderm is specified it interacts with endoderm (that also expresses
the HOX genes) to form various parts of gut. Mutation of these genes results in the common
structural malformation of the gut tube (Fig. CD 4.5).

Molecular Control of the Development of the Liver and the Biliary

Passages
In the third week of intrauterine life an endodermal hepatic diverticulum arises from the
floor of the foregut due to expression of FGF2 and BMP signaling. The FGF2 is secreted by

Human Embryology

Fig. CD-4.5: Schematic diagram showing molecular control of gut-formation. The initial specification of

various regions of gut is achieved by expression of transcription factors Sox, Pax and Cdx. Further patterning
of gut is due to interaction between gut endoderm (which expresses Shh) and gut associated mesoderm
(which expresses HOX genes). The orderly expression of HOX genes in both mesoderm and endoderm leads
to the pattern of the gut.

the neighbouring cardiac mesoderm and BMPs from the septum transversum. Both these
molecules act on gut endoderm to express liver specific genes. They convert endoderm into
precursors of hepatic epithelium. The differentiation of hepatocytes and the biliary cell lineage
is now regulated by HNF3 and 4.

Molecular Control of the Development of Pancreas

The formation of the ventral pancreatic bud is induced by visceral mesoderm, while the
formation of the dorsal pancreas is induced (from dorsal gut endoderm) by activin and
FGF signals arising from the notochord. These signals inhibit the activity of Shh in the dorsal
endoderm. Shh activity of dorsal endoderm has to be repressed to enable differentiation
of the pancreas. The pancreatic progenitor cells now express the transcription factor Pdx-1

Chapter CD 4 Molecular Control of the Development of Some Organs

(pancreatic and duodenal homeobox 1 gene) in the future pancreatic buds. Pdx-1 mutation
leads to failure of formation of pancreatic buds.

The pancreatic progenitor cells now differentiate into exocrine and endocrine pancreatic
cells. The surrounding mesoderm secretes signaling molecules FGFs and follistatin, which act
on some progenitor cells (through the activation of the notch receptor system) to make them
differentiate into exocrine cells of the pancreas.
Other progenitor cells, that are not activated by the notch receptor system, do not
differentiate into the exocrine pancreas. These cells express transcription factors neurogenin
-3 and Isl-1 to become endocrine precursor cells. These precursor cells then differentiate into
two different types of cells. The cells of one group differentiate into alpha and gamma cells,
while the cells of the other group differentiate into beta and delta cells. Alpha and gamma
cells are formed under the influence of Pax-6 and Nkx2.2, while beta and delta cells are
formed under the influence of Pax-4 and Nkx2.2.

Molecular Control of Development of Respiratory System

TBX is expressed in the endoderm of the respiratory diverticulum under the influence of
retinoic acid. TBX is the master gene for the formation of the respiratory system. Once the lung
bud is formed the further growth of bud is due to expression of FGF-10, which is produced
by mesenchyme at the tip of the growing lung bud. FGF-10 stimulates the proliferation of
epithelium at the tip of the bud. However, the branching of the bud is initiated by BMP-4 and
Shh. FGF-10 mutation results in failure of budding in the developing lung.

Molecular Control of the Development of the Heart

Signals for formation of the heart tube reach the cardiogenic area. These signals originate in
the anterior endoderm. The crescent and Cerberus are secreted by endoderm. They act as
inhibitors to WNT proteins expressed by the neural tube. (WNT proteins are expressed by the
neural tube and act as inhibitors to the formation of the heart tube. Hence, their expression has
to be first inhibited by crescent and Cerberus molecules).

The secretion of BMPs from the endoderm causes expression of transcription factors
NKX2.5 and FGF8. NKX2.5 is the master gene for heart development.

Once the heart tube is formed its folding (looping) depends upon the expression of genes
nodal and lefty. These genes further induce the expression of transcription factor PITX2.
PITX2 regulates the deposition of extracellular matrix on the left side. This helps in looping of
the heart tube.

Secretion of retinoic acid, by mesoderm surrounding the venous part of the heart tube,
leads to the formation of atria and of the sinus venosus. Retinoic acid is an important molecule
in heart development. Its absence or overdose can cause malformations of the heart.
NKX2.5 regulates the expression of HAND1 for expansion and differentiation of the left
ventricle, while expression of HAND2 is responsible for differentiation of the right ventricle.

Transcription factor TBX5 helps in the formation of interatrial and interventricular septa.

Human Embryology

Neural Crest and Spiral Septum

The neural crest cells contribute to the formation of the spiral septum (in the truncus arteriosus
and in the outflow tracts of the right and left ventricles). These neural crest cells migrate from
the region of the hindbrain to the developing heart. They pass through pharyngeal arches 3,
4 and 6. Failure of such migration of neural crest cells leads to congenital malformations of
the outflow tract of the heart e.g., pulmonary stenosis, teratology of Fallot, persistent truncus
arteriosus and transposition of great vessels. As the neural crest cells also contribute to the
formation of various parts of the head and neck congenital anomalies of the heart, and of the
head and neck, are often associated with each other.

Molecular Control of the Development of the Kidney

The development of the kidney depends on interaction between the ureteric bud epithelium
and the surrounding mesenchyme of the metanephric blastema.
WT1 is a transcription factor that is secreted by the mesenchyme of the metanephric
blastema just before the ureteric bud comes in contact with it. It prepares the mesenchyme for
interaction with the epithelium of the ureteric bud. WT1 also regulates the production of HGF
(hepatocyte growth factor) and GDNF (glia derived neurotrophic factor) by the mesenchyme.
The gene WT1 is located on the long arm of chromosome number 11 (11p13) whose mutation
causes cancer of the kidney (Wilms tumour) in fetal life or in early childhood.

Molecules expressed by mesenchyme cause the ureteric bud to bifurcate when it comes in
contact with the blastema. The molecules HGF and GDNF help in branching and growth of the
ureteric bud. Mutation of the GDNF gene causes renal agenesis.
The epithelium of the ureteric bud interacts with mesenchyme by expressing FGF2
(fibroblast growth factor 2), LIF (leukemia inhibitory factor) and BMP7 (bone morphogenetic
protein 7). These help in growth of the mesenchyme of the metanephric blastema.
Due to this interaction between the ureteric bud and the metanephric mesenchyme,
there is condensation of mesenchyme around the tips of the ureteric buds. This is achieved
by expression of genes WNT6 and WNT9B by the epithelium of the ureteric bud. These two
molecules promote the secretion of PAX2 by the mesenchyme, which helps in the condensation
of mesenchyme. Renal agenesis may occur due to mutation in PAX2 gene.

The mesenchymal condensations now transform into small groups of epithelial cells. These
cells later convert into the tubules of nephrons, due to secretion of WNT4 in the metanephric
mesenchyme.

The conversion of mesenchymal cells into epithelium is also associated with the formation
of a basal lamina by production of type IV collagen and laminin by the extracellular matrix. The
cell to cell lateral adhesion in the epithelium is achieved by production of adhesion molecules
L-CAM. These stromal inductive signals are regulated by expression of the BF-2 gene.

Molecular Control of Differentiation of Genital Organs

From the account of the development of the gonads and genitalia, it is seen that these organs
are derived from the same primordia in both sexes. The male and female genital systems are

Chapter CD 4 Molecular Control of the Development of Some Organs

identical until the beginning of seventh week of intrauterine life. The factors that determine
whether these organs will develop as in the male, or as in the female are as follows:
1. The most important factor is the chromosomal sex of the individual, which is determined
at the time of fertilization. We have already seen that individuals with two X-chromosomes
are female, while those with one X-chromosome and one Y-chromosome are male.
2. The Y-chromosome bears a gene (SRY gene, present on short arm) that is responsible
for production of a testis determining factor. This factor plays a vital role in causing the
developing gonad to become a testis. Apart from a direct action on the gonad, this factor
influences other genes (SOX-9) that play a role in the process. Under the influence of these
genes, Sertoli cells are formed from cells of the sex cords and Leydig cells are formed from
mesenchymal cells of the gonadal ridge.
3. Once the testis is formed, the interstitial (Leydig) cells in it begin to produce testosterone
(under the influence of gonadotropins formed in the placenta). This testosterone influences
the differentiation of genital ducts and external genitalia. By the end of eighteenth week
fetal Leydig cells disappear to reappear only at the time of puberty.
4. Supporting cells in the fetal testis (Sertoli cells) produce a Mullerian inhibiting
substance. This substance causes regression of paramesonephric ducts. The sertoli cells
also secrete an androgen binding factor that helps in the formation of spermatozoa from
spermatogonia.
As the Y-chromosome is missing
in a female fetus, none of the processes
described above take place. The ovary is
formed under the influence of the WNT4
gene expressed in the gonadal ridge. The
oestrogens (derived from maternal and
placental sources) influence the formation
of internal and external genital organs.

Molecular Control of Differentiation

of Motor and Sensory Neurons in
the Spinal Cord
The differentiation of motor neurons, in the
ventral part, and of sensory neurons in the
dorsal part of the spinal cord, is under the
control of two different sets of molecules
(Fig. CD 4.6).

Dorsal (sensory) Part of Spinal Cord

Differentiation of the dorsal portion of
spinal cord is initiated by secretion of growth
factors BMP- 4/7 by the ectoderm overlying

Fig. CD-4.6: Differentiation of neurons (motor and

sensory) in the spinal cord.

Human Embryology

the neural tube. Thereafter, the roof plate of the neural tube itself starts secretion of BMP-4/5/7.
Under the influence of these factors, the dorsal portion of neural tube now activates PAX-3/7,
that ultimately controls the formation of sensory neurons.

Ventral (motor) Part of the Spinal Cord

The ventral part of the neural tube differentiates under the influence of Shh (Sonic Hedgehog),
which is secreted by the notochord, and by the floor plate of the neural tube. Shh then activates
the expression of transcription factors NKX6.1 and PAX-6, which regulate the differentiation of
motor neurons in the ventral part of spinal cord.

Molecular Control of Development of Brain

The dorsal ectoderm, which overlies the notochord, forms the neural plate under the influence
of neural inducers noggin and chordin. These inducers are secreted by prechordal mesoderm,
by the notochord and by the primitive node. They act by inhibiting BMP-4. Homeobox genes
expressed in notochord, the prechordal plate and the neural plate subdivide the neural plate
into forebrain, midbrain and hindbrain regions. The future forebrain and midbrain regions
of the neural plate now express Otx-2. The induction of the hindbrain and spinal cord is due
to expression of Wnt-3 and FGF. Gbx-2 is expressed in the part of the neural plate designed
to form the hindbrain and the spinal cord (Fig. CD 4.7). The midbrainhindbrain border is
defined by Otx-2 and Gbx-2. Retinoic acid regulates the expression of homeobox genes, which
organise the hindbrain into segments along the cranio-caudal axis.

Fig. CD-4.7: Signals from the prechordal plate act on future forebrain and midbrain regions of the neural

plate to express Otx-2. Similarly signals from the notochord act on the hindbrain and future spinal cord
regions of the neural plate to express Gbx-2. The isthmus appears at the junction of the future midbrain
and hindbrain.

Chapter CD 4 Molecular Control of the Development of Some Organs

Forebrain and Midbrain

As stated above, after formation of the neural plate, the transcription factor Otx-2 is expressed
by forebrain and midbrain regions, and is responsible for designating these areas. Under the
influence of the signaling molecule FGF-8 an organizing center (anterior neural ridge, ANR)
appears at the cranial border of the neural plate (Fig. CD 4.7). FGF-8 induces the expression of
BF1, which controls the development of the telencephalon and of the optic vesicle (retina).
Within the forebrain the expression of transcription factor NKX2,1 marks the border between
alar and basal laminae. In the alar lamina EMX1,2 and Pax-6 are expressed to specify the
identity of the forebrain areas. Expression of the Dlx gene in the ventral region leads to the
formation of the basal ganglia.
The signaling molecule Shh is secreted by the prechordal plate and the notochord. In
the forebrain and midbrain areas, Shh controls the patterning of the ventral portion of the
brain. Shh induces the expression of Nkx2, and Nkx1 that controls the development of the
hypothalamus. In the absence of Shh, signaling of the tissues of the ventral forebrain does
not take place properly. This may leads to midline fusion of the optic vesicles and a general
reduction of the growth of the middle part of the face. These defects may lead to conditions
known as holoprosencephaly and cyclopia.

At the junction of the midbrain and the hindbrain there is the presence of a circumferential
band called the isthmus. At the isthmus, an organizing center appears under the influence of
FGF-8. This is called isthmic organizer. This organizer induces the expression of Wnt-1 just in
front of the region of FGF-8 expression. Both, FGF8 and Wnt-1 now induce the expression of
EN1, and EN2, Pax-2 and Pax-5. EN1 and EN2 are expressed both anterior and posterior to the
organizing center of the isthmus. EN1 controls the development of the tectum of the midbrain,

Fig. CD-4.8: FGF-8 of the isthmic organizer induces the secretion of Wnt-1 in the midbrain. In turn this
induces expression of EN1 and 2 in the midbrain and in the first rhombomere (of hindbrain). Shh is secreted
by the ANR and by the ventral portion of the neural tube. HOX genes are responsible for formation of seven
rhombomeres in the hindbrain.

Human Embryology

and of the cerebellum; while EN 2 controls the development of the cerebellum only. Like
the forebrain, the patterning of the midbrain in a dorso-ventral axis is regulated by ventrally
secreted Shh.

Hindbrain
In the human, the hindbrain shows seven segments, which are called rhombomeres. The
segmentation is due to action of segmentation genes. The HOX genes are mainly involved in the
segmentation of the hindbrain. These genes are involved in overlapping patterns. HOX genes
are not only involved in segmentation of the hindbrain but also give identity to each segment.
They also provide identity to cranial nerves and to the derivatives of the pharyngeal arch that
arise from a specific rhombomere. HOX proteins are not present in the first rhombomere as
the presence of FGF-8 is antagonist to HOX proteins. FGF-8 is confined to anterior part of
rhombomere 1, which gives origin to the cerebellum.