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

Th e appearance of the primitive streak defi nes the left and right sides of the embryo. Soon after its appearance, the primitive node and streak express the fi broblast growth factor 8 (FGF-8). Th is 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. Th ese genes then regulate the expression of PITX2, which is a homeobox containing the transcription factor important for establishing left sidedness. Th e 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. Th e 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. Th ese are chordin, crypto, Vg1, brachyury and nodal. Th e dorsal lip of the blastopore is considered to be the primary organizer. Th e 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 fi broblast growth factor family (FGF). A homeobox gene goosecoid is expressed specifi cally in the primitive node. Th is is the fi rst zygotic gene to be expressed in humans. It is similar to the fruit fl y genes goosberry and bicoid.

Molecular Control of the Development of Some Organs

Chapter CD 4

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 tail-organizing 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

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

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.

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 FGF-10. 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 FGF-8. 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 BMP-2, 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

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.

Fig. CD-4.2

Various genes expressed during the development of the face and palateRegion 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 FGF-8, BMP-4/7 FGF-4/2, Dlx-5/6, Dlx-3/7

Lateral palatal process Shh, ECF, Max-1, BMP-4/2

Fusion of palatal process with nasal process

TGF-B3

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 enamel knot. The type of the tooth to be formed is strictly under genetic

Fig. CD-4.3: Future teeth are formed at the sites where Fgf-8 activates Pax-9 expression. No teeth are formed at sites where Pax-

9 is inhibited by Bmp-1/2.

Chapter CD 4 – Molecular Control of the Development of Some Organs

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

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.

Human Embryology

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

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.

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 midbrain–hindbrain 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 BF-1, 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.