9. Limb development

The process of development of limbs is well understood as the genes controlling the growth, patterning and signaling pathways for its development is well conserved from Drosophila to mammals. The developing limb is divided into a proximal stylopod, middle zeugopod and distal autopod. In humans the stylopod becomes the arm or thigh, the zeugopod becomes the forearm or leg and the autopod changes into the hand or foot. The limb buds appear on Day 26 for upper limb and 1 or 2 days later for lower limb. The limb bud to begin with consists of a mesenchyme core as an extension from the parietal layer of lateral plate mesoderm which leads to the formation of bones and connective tissues and it is covered with a layer of cubical ectoderm. The positioning of the limb buds depend on the correct functioning of HOX genes, which control the expression of FGF8, and the latter determines the limb type, i.e., fore or hind limb. The apical

Figure 9. Molecular regulation of limb development. Fibroblast growth factor 10 and AER initiate limb growth. These are induced by bone morphogenetic protein S (RA is retinoic acid, ZPA is zone of polarizing activity, AER is apical endodermal ridge).

ectodermal ridge (AER), which is a localized area of thickening on the surface ectoderm covering the limb bud, produces growth signals such as FGF4 and FGF8, which result in growth in proximal-distal axis. A collection of cells at the posterior margin of limb bud near body wall also undergoes proliferation to form the zone of polarizing activity which produce morphogen retinoic acid which in turn expresses SHH (Sonic Hedge Hog) which plays a huge role in determining the anteroposterior axis for limb development (Figure 9). Bones of the limb are derived by endochondral ossification of the parietal layer of the lateral plate mesoderm which is determined by HOX genes (HOXA and HOXD), which regulate the type, and shape of the bones. Digits are formed due to organized apoptosis of the AER into separate ridges and ultimately by interdigital apoptosis. HOXD13 mutations result in synpolydactyly, which is a dominantly inherited condition. Hand foot genital syndromes results from mutations of HOXA13 and presents hypospadias in males and bilateral thumb and great toe hypoplasia [6]. Dorsalization of the limb bud is under the control of genes like radical fringe and Wnt7a. Holt Oram syndrome an autosomal dominant inheritance is due to mutation of T-box 5 gene and is characterized by forelimb abnormalities and cardiac defects thus T-box genes determine the limb identity by activating FGFs. Another gene is the PitX, which contribute in forming hind limb characteristics [6].

#### 10. Head and neck formation

genitalia does not fuse and develop into normal female genitals. Normal sexual differentiation is completed by 14 weeks though the testis descends at a much later part of pregnancy [1].

Different cells in an embryo express different sets of genes at different times. A common set of genes is present in all cells at all times and is called as the "House-keeping genes." These genes carry out the normal functions of a cell. Speciality genes are the special genes that enable a cell to carry out special functions. The genes responsible for embryonic body plan Homeobox

The gene expression is regulated by three sets of proteins called as the promoter, silencer and enhancer proteins. These proteins can modify the gene expression and define a particular cell type. There are also signaling proteins that facilitate the development of adjacent areas in a

The SRY gene on the Y chromosome produces testis determining factor, which in turn activate the SOX9, and steroidogenesis factor (SF1) and it stimulates development of Sertoli and Leydig cells. WNT4 is the master gene for ovarian development. It inhibits SOX9 and up regulates DAX1. WNT4 along with other genes promotes formation of cortical cords and causes regres-

The extracellular growth factors regulate cell division and differentiation by the process of signal transduction and thus mutations in genes, which produce these factors and receptors, have been implicated in cancer development and also in developmental anomalies. The Fibroblast growth factor receptors (FGR) play an important role in embryogenesis. Mutations in FGR have been associated with two groups of disorders of skeletal system, which broadly include the craniosynostosis syndromes and achondroplasias. Genes such as Fibrillin-I and Elastin code for proteins that are needed for arranging microfibrils in the matrix and thus the

The process of development of limbs is well understood as the genes controlling the growth, patterning and signaling pathways for its development is well conserved from Drosophila to mammals. The developing limb is divided into a proximal stylopod, middle zeugopod and distal autopod. In humans the stylopod becomes the arm or thigh, the zeugopod becomes the forearm or leg and the autopod changes into the hand or foot. The limb buds appear on Day 26 for upper limb and 1 or 2 days later for lower limb. The limb bud to begin with consists of a mesenchyme core as an extension from the parietal layer of lateral plate mesoderm which leads to the formation of bones and connective tissues and it is covered with a layer of cubical ectoderm. The positioning of the limb buds depend on the correct functioning of HOX genes, which control the expression of FGF8, and the latter determines the limb type, i.e., fore or hind limb. The apical

sion of medullary cords and prevents the tunica albuginea to develop [2].

mutations are associated with Marfans' syndrome [3].

9. Limb development

8. Gene expression during development

genes, proto-oncogenes and PAX genes.

84 Genetic Diversity and Disease Susceptibility

particular organ.

The most important feature in development of head and neck is the formation of pharyngeal arches resembling the gills of the fish. These arches are bars of mesenchyme tissue appearing at fourth or fifth week of development with an outer covering of ectoderm and inner layer of endoderm at the cranial most part of the primitive gut. The arches are separated from each other by ectodermal clefts and form out pouching in the lateral wall of developing pharynx as endodermal pouches. The mesoderm which lies in the center of the arches form the arch arteries and musculature and the skeletal elements are formed from the neural crest cells, which migrate early from hindbrain region. The FGF signaling plays an important role in determining the migration and fate of neural crest cells in the arches, endodermal pouch formation and differentiation [7]. Though it was previously thought that the neural crest cells are important for pharyngeal arch development but nowadays it is shown that the endoderm of the pharyngeal apparatus acts as the principal organizer and thus it is proved that neural crest cells are not necessary for pharyngeal arch development. The out pocketing of the endoderm is significant for pharyngeal arch development and regulated by TBX1 and RA signaling [8]. The most well-known condition due to faulty development of third and fourth pouches is the DiGeorge syndrome which results due to submicroscopic chromosome deletion 22q11 and loss of approximately 30 genes. Treacher Collins is hypoplasia of the maxilla and mandible with coloboma of the lower eyelid, cleft palate, hearing impairment has an autosomal dominant inheritance affecting the first arch and results due to mutation in TCOF1 gene [1].

transcription factor NKX2 which is a homolog of the gene tinman that regulates heart development in Drosophila, is induced in the endoderm overlying the splanchnopleuric mesoderm for human heart development. NKX2 is considered as the master gene for human heart development and its expression is brought about by BMP activity and WNT inhibition. The venous portion of the heart is specified and formed under influence of retinoic acid and cardiac

Developmental Genetics and Preimplantation Genetic Diagnosis

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The cephalocaudal and lateral folding of the embryo gives rise to formation of primitive gut tube which is endoderm lined yolk sac cavity incorporated into the embryo by fourth week of development. The retinoic acid (RA) concentration varies in this primitive gut and it is the highest in colon region and least in that part of this gut tube which give rise to the pharynx. This variation of RA concentrations causes expression of certain transcription factors, which leads to the specification of the gut tube to develop the parts. SOX2 is transcription factor for development of esophagus and stomach, PDX1 is for duodenum development, CDXC is for small intestine, and CDXA is for large intestine and rectum. The interaction of the epithelial and underlying mesenchyme components of the gut follows the initial patterning and brought about by sonic hedgehog expression throughout the gut. The transition of the foregut to midgut and thus hind gut derivatives and the change in the epithelial characteristics are brought about by nested expressions of HOX genes which are activated by sonic hedgehog to bring about an orchestrated development of the gut. The secretion of FGFs from cardiac mesoderm and BMPs from the septum transversum leads to the hepatic development. The FGFs inhibits the activity of inhibitors and thus specifies the development of hepatic endoderm by a negative feedback mechanism as suppressing the inhibitors brings about the induction. The sonic hedgehog expression in gut endoderm, which is destined to form dorsal pancreatic bud, is suppressed in the gut endoderm by FGF2 and activin. The ventral bud is induced by visceral mesoderm, which up regulates expression of pancreatic and duodenal homeobox gene (PDX). Two types of PAX genes specify the endocrine cells. PAX4 and PAX6 genes specify the insulin, somatostatin, and pancreatic polypeptide secreting cells whereas PAX 6 genes specify

The urogenital system develops from a common mesodermal ridge, i.e., the intermediate mesoderm. The intermediate mesoderm gives rise to three overlapping kidney systems the pronephros, the mesonephros and the metanephros. The metanephros, which appears by fifth week, gives rise to the definitive kidney in humans. The mesonephric duct of the intermediate mesoderm gives rise to an ureteric bud which forms the collecting part of the kidneys the renal pelvis, calyces, 1–3 million collecting tubules. The interaction of this ureteric bud from the mesonephric duct with the mesenchyme of the metanephric blastema is the key factor in determining the initiation of kidney development. WT1 is the transcription factor expressed by the mesenchyme of metanephros, which increases the sensitivity of the metanephros to interact with the ureteric bud and respond to it. The branching and growth of ureteric bud is under control of production of Glial derived neurotrophic factor (GDNF) and Hepatocyte growth factor produced by the mesenchyme of metanephros which bind to the receptor on

looping is due to expression of PITX2 in lateral plate mesoderm.

the glucagon secreting cells [5].

13. Urogenital system

### 11. Ciliary development

The cilia on the apical surface of the cells have an important role in human embryological development deciding cell specification, axis determination and pathology of human congenital disorders. A variety of congenital defects like polycystic kidney, laterality defects, nervous system defects, and retinal degeneration are associated with ciliopathies thus proving that cilia can serve as signaling factor in embryonic development. Cilia plays an important role in Hedgehog patch smoothened Gli pathway and thus it regulates the Gli protein activity. The cilia transition zone a short segment just above the basal body acts as an essential gatekeeper for movement of protein in and out of the cilia. Cilia also play an important role in movement of fluid in various confined embryonic spaces, which may act as a developmental shear force [9]. The congenital polycystic kidney disease, which results in numerous cyst formations, can be inherited as autosomal dominant or recessive. In autosomal recessive type the cysts are formed in the collecting tubules and it is a progressive disorder though less common but highly progressive and leads to kidney failure early in infancy and childhood. The autosomal dominant type is more common (1/500 to 1/1000) results in cyst formation in all segments of the nephron is less progressive and does not result in renal failure till adulthood, The above two types of conditions are related to the mutation of genes that code ciliary protein and thus fall under the group of disorders the ciliopathies which are creating a lot of interest in recent times. Bardet-Biedl syndrome and Meckel-Gruber syndrome are characterized with renal cysts and other features are also some of the ciliopathies recently identified [2].

#### 12. Reciprocal interactions

Organogenesis results due to reciprocal interaction between the differentiated epithelial cells and the underlying mesenchyme, which is brought about by signaling molecules. The transcription factor NKX2 which is a homolog of the gene tinman that regulates heart development in Drosophila, is induced in the endoderm overlying the splanchnopleuric mesoderm for human heart development. NKX2 is considered as the master gene for human heart development and its expression is brought about by BMP activity and WNT inhibition. The venous portion of the heart is specified and formed under influence of retinoic acid and cardiac looping is due to expression of PITX2 in lateral plate mesoderm.

The cephalocaudal and lateral folding of the embryo gives rise to formation of primitive gut tube which is endoderm lined yolk sac cavity incorporated into the embryo by fourth week of development. The retinoic acid (RA) concentration varies in this primitive gut and it is the highest in colon region and least in that part of this gut tube which give rise to the pharynx. This variation of RA concentrations causes expression of certain transcription factors, which leads to the specification of the gut tube to develop the parts. SOX2 is transcription factor for development of esophagus and stomach, PDX1 is for duodenum development, CDXC is for small intestine, and CDXA is for large intestine and rectum. The interaction of the epithelial and underlying mesenchyme components of the gut follows the initial patterning and brought about by sonic hedgehog expression throughout the gut. The transition of the foregut to midgut and thus hind gut derivatives and the change in the epithelial characteristics are brought about by nested expressions of HOX genes which are activated by sonic hedgehog to bring about an orchestrated development of the gut. The secretion of FGFs from cardiac mesoderm and BMPs from the septum transversum leads to the hepatic development. The FGFs inhibits the activity of inhibitors and thus specifies the development of hepatic endoderm by a negative feedback mechanism as suppressing the inhibitors brings about the induction. The sonic hedgehog expression in gut endoderm, which is destined to form dorsal pancreatic bud, is suppressed in the gut endoderm by FGF2 and activin. The ventral bud is induced by visceral mesoderm, which up regulates expression of pancreatic and duodenal homeobox gene (PDX). Two types of PAX genes specify the endocrine cells. PAX4 and PAX6 genes specify the insulin, somatostatin, and pancreatic polypeptide secreting cells whereas PAX 6 genes specify the glucagon secreting cells [5].
