Digestive and Respiratory Systems and Body Cavities

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Chapter 15

Digestive and Respiratory Systems and Body Cavities

The initial formation of the digestive system by the lateral folding of the endodermal germ layer into a tube is described in Chapter 6. From its beginnings as a simple tubular gut, development of the digestive system proceeds on several levels, including molecular patterning, elongation and morphogenesis of the digestive tube itself, inductions and tissue interactions leading to the formation of the digestive glands, and the biochemical maturation of the secretory and absorptive epithelia associated with the digestive tract. Clinical Correlations 15.1 to 15.3, later in the chapter, discuss malformations associated with the digestive system.

Formation of the respiratory system begins with a very unimposing ventral outpocketing of the foregut. Soon, however, this outpocketing embarks on a unique course of development while still following some of the basic patterns of epithelial-mesenchymal interactions characteristic of other gut-associated glands. Initially, the digestive and respiratory systems form in a common body cavity, but functional considerations later necessitate the subdivision of this primitive body cavity into thoracic and abdominal components. Later in the chapter, Clinical Correlation 15.4 presents malformations associated with the respiratory system, and Clinical Correlation 15.5 discusses malformations related to other body cavities.

Digestive System

Chapter 6 describes the formation of the primitive endodermal digestive tube, which is bounded at its cephalic end by the oropharyngeal membrane and at its caudal end by the cloacal plate (see Fig. 6.20). Because of its intimate relationship with the yolk sac through the yolk stalk, the gut can be divided into a foregut, an open-bottomed midgut, and a hindgut.

Patterning the Gut

Initial patterning of the gut endoderm begins during the period of late gastrulation, as the sheet of newly formed endoderm begins to form a gut tube. After initial broad patterning into anterior and posterior domains by nodal and fibroblast growth factor-4 (FGF-4), respectively, the overall organization of the gut gradually takes shape. Much of the patterning and of early morphogenesis of the gut occurs in response to the actions of several sets of molecular signals. As development and early organogenesis proceed, the same signaling molecules are reused. Paradoxically, the same molecule may play opposite roles in the same area, but at different times (i.e., it may first act as a stimulator, and then, within hours or days, it may function as an inhibitor).

Patterning of the broad foregut area occurs through the inhibition of Wnt signals (Fig. 15.1). The foregut domain is then marked by the expression of the transcription factors, Sox-2, Hhex, and Foxa-2. In contrast, a mix of activity by Wnts, FGFs, and bone morphogenetic proteins (BMPs), along with retinoic acid, represses foregut identity and maintains the regional identity of the hindgut. This is marked by the expression of the transcription factor Cdx-2 throughout the broad hindgut and the later expression of Pdx-1 in the midgut as this region emerges as a separate entity. Cdx-2 acts upstream of a broad range of Hox activity (Fig. 15.2) that is expressed throughout the gut. The activity of specific signaling molecules is associated with important transition points along the gut. FGF-4 is strongly expressed near the foregut-midgut boundary (around the duodenal-jejunal junction), and FGF-10 is associated with the establishment of the cecum.

Largely through the action of Cdx-2, the orderly expression of homeobox-containing genes then takes over in the regional patterning of the digestive system (see Fig. 15.2).

Mice bearing mutant copies of some of these genes develop several of the common structural malformations of the digestive tract that occur in humans. More dramatically, mice deficient in retinoic acid, a broad early patterning molecule, fail to form lungs and show severe defects of other posterior foregut derivatives, such as the stomach, duodenum, and liver. Development of the gut tube proper involves continuous elongation, herniation past the body wall, and rotation and folding for efficient packing into the body cavity, as well as histogenesis and later functional maturation.

By the end of the first month, small endodermal diverticula, which represent primordia of the major digestive glands, can be identified (Fig. 15.3). (Development of the pharynx and its glandular derivatives is discussed in Chapter 14.) The digestive glands and respiratory structures grow in complex branching patterns, resembling fractals, as the result of continuous epithelial-mesenchymal interactions. These interactions also occur in the developing digestive tube itself, with specific regional mesenchymal influences determining the character of the epithelium lining that part of the digestive tract.

Each of the glandular derivatives of the digestive tract, as well as the major regions along the gut, is the result of a specific response by a small population of founder cells for each organ to a set of environmental inductive signals. At a first level, certain regions of the gut must be prepared to be either responsive or refractive to these signals. For example, after the overall foregut is specified by the suppression of Wnt and FGF signals, transforming growth factor-β (TGF-β) signaling restricts the specification of foregut endoderm to allow the prehepatic and prepancreatic endoderm to remain receptive to inductive signals. By the same token, other influences on the dorsal side of the foregut repress the ability of these cells to become liver or pancreas.

During neurulation, as the head bends sharply to create the foregut, the ventral foregut endoderm is closely opposed to two mesodermal masses: the cardiac mesoderm and the primordium of the septum transversum (see Fig. 15.4). High levels of FGF, secreted by the cardiac mesoderm, and also retinoic acid induce the formation of the liver, lung bud, and thyroid (see Fig. 15.4). BMP-4 from the mesoderm of the septum transversum is also required for liver induction. By contrast, endodermal movements carry the preventral pancreas cells far enough from the cardiac mesoderm to expose them to a low level of FGF, thus allowing the ventral pancreas to develop. For the dorsal pancreas to develop, locally produced sonic hedgehog (shh) must be inactivated by activin and FGF emanating from the notochord. In addition, retinoic acid from the somitic mesoderm is needed for induction of the dorsal pancreas. Meanwhile, in the hindgut, the actions of Wnt and other signaling molecules repress the expression of genes, such as Hhex and Pdx1, which are essential for formation of the liver and pancreas, respectively.

Induction of these organs is marked by the activation of transcription factors specific for the organ and stage of development of that organ. Some of these factors are schematically represented in Figure 15.4B.

Formation of the Esophagus

Just caudal to the most posterior pharyngeal pouches of a 4-week-old embryo, the pharynx becomes abruptly narrowed, and a small ventral outgrowth (lung bud) appears (see Fig. 6.20). The region of foregut just caudal to the lung bud is the esophagus. This segment is initially very short, with the stomach seeming to reach almost to the pharynx. In the second month of development, during which the gut elongates considerably, the esophagus assumes nearly postnatal proportions in relation to the location of the stomach.

Although the esophagus grossly resembles a simple tube, it undergoes a series of striking differentiative changes at the tissue level. In its earliest stages, the endodermal lining epithelium of the esophagus is stratified columnar. By 8 weeks, the epithelium has partially occluded the lumen of the esophagus, and large vacuoles appear (Fig. 15.5). In succeeding weeks, the vacuoles coalesce, and the esophageal lumen recanalizes, but with multilayered ciliated epithelium. During the fourth month, this epithelium finally is replaced with the stratified squamous epithelium that characterizes the mature esophagus.

Deeper in the esophageal wall, layers of muscle also differentiate in response to inductive signals from the gut endoderm. Very early (5 weeks’ gestation), the primordium of the inner circular muscular layer of esophagus is recognizable, and by 8 weeks, the outer longitudinal layer of muscle begins to take shape. The esophageal wall contains smooth and skeletal muscle. The smooth muscle cells differentiate from the local splanchnic mesoderm associated with the gut, and the skeletal musculature is derived from paraxial mesoderm. All esophageal musculature is innervated by the vagus nerve (cranial nerve X).

The cross-sectional structure of the esophagus, similar to that of the rest of the gut, is organized into discrete layers. The innermost layer (mucosa) consists of the epithelium, derived from endoderm, and an underlying layer of connective tissue, the lamina propria (see Fig. 15.5C and D). A thick layer of loose connective tissue (submucosa) separates the mucosa from the outer layers of muscle (usually smooth muscle, with the exception of the upper esophagus). This radial organization is regulated by the epithelial expression of shh, acting through its receptor, patched, and BMP-4. Shh inhibits the formation of smooth muscle in the submucosal layer of the esophagus. Farther from the source of the endodermal shh, smooth muscle can differentiate in the outer wall of the intestine. How the developing smooth muscle layer of the mucosa (muscularis mucosae) escapes this inhibitory influence is also unclear. Intestinal mesenchyme can spontaneously differentiate into smooth muscle in the absence of an epithelium (which produces shh). Because in humans the muscularis mucosae differentiates considerably later than the outer muscular layers, it is possible that inhibitory levels of shh are reduced by that time.

Formation of the Stomach

Formation of the stomach within the foregut is first specified by the action of the transcription factors Hoxa-5 and Barx-1, which inhibits the posteriorizing effects of Wnt signaling in the region of the future stomach. A second phase of specification follows, in which a descending posterior-to-anterior gradient of FGF-10, produced in the gastric mesoderm, begins the process of regional differentiation of the glandular character of the gastric epithelium.

Very early in the formation of the digestive tract, the stomach is recognizable as a dilated region with a shape remarkably similar to that of the adult stomach (see Fig. 15.3). The early stomach is suspended from the dorsal body wall by a portion of the dorsal mesentery called the dorsal mesogastrium. It is connected to the ventral body wall by a ventral mesentery that also encloses the developing liver (Fig. 15.6).

When the stomach first appears, its concave border faces ventrally, and its convex border faces dorsally. Two concomitant positional shifts bring the stomach to its adult configuration. The first is an approximately 90-degree rotation about its craniocaudal axis so that its originally dorsal convex border faces left, and its ventral concave border faces right. The other positional shift consists of a minor tipping of the caudal (pyloric) end of the stomach in a cranial direction so that the long axis of the stomach is positioned diagonally across the body (Fig. 15.7).

During rotation of the stomach, the dorsal mesogastrium is carried with it, thus leading to the formation of a pouchlike structure called the omental bursa (bursa is a Latin word meaning “sac” or “pouch”). Both the spleen and the tail of the pancreas are embedded in the dorsal mesogastrium (see Fig. 15.6). Another viewpoint suggests that the right pneumatoenteric recess, a projection from the pleural cavity into the dorsal mesogastrium, persists as the omental bursa.

As the stomach rotates, the dorsal mesogastrium and the omental bursa that it encloses enlarge dramatically. Soon, part of the dorsal mesogastrium, which becomes the greater omentum, overhangs the transverse colon and portions of the small intestines as a large, double flap of fatty tissue (Fig. 15.8). The two sides of the greater omentum ultimately fuse and obliterate the omental bursa within the greater omentum. The rapidly enlarging liver occupies an increasingly large portion of the ventral mesentery.

At the histological level, the gastric mucosa begins to take shape late in the second month with the appearance of folds (rugae) and the first gastric pits. During the early fetal period, the individual cell types that characterize the gastric mucosa begin to differentiate. Biochemical and cytochemical studies have shown the gradual functional differentiation of specific cell types during the late fetal period. In most mammals, including humans, cells of the gastric mucosa begin to secrete hydrochloric acid shortly before birth.

The caudal end of the stomach is physiologically separated from the small intestine by the muscular pyloric sphincter. Formation of the pyloric sphincter is directed by the transcription factors Sox-9 and Nkx 2.5, whose expression in the area of pyloric mesoderm is stimulated by BMP-4 signals. In addition, several Hox genes are needed to make each of the three major sphincters (pyloric, ileocecal, and anal) in the digestive tract.

Clinical Correlation 15.1 presents malformations of the esophagus and stomach.

Clinical Correlation 15.1   Malformations of the Esophagus and Stomach

Esophagus

The most common anomalies of the esophagus are associated with abnormalities of the developing respiratory tract (see p. 364). Other rare anomalies are stenosis and atresia of the esophagus. Stenosis is usually attributed to abnormal recanalization of the esophagus after epithelial occlusion of its lumen. Experimental evidence suggests that abnormal separation of the early notochord from the dorsal foregut endoderm is often associated with esophageal atresia, possibly through the incorporation of some of the dorsal foregut cells into an abnormal notochord. Atresia of the esophagus is most commonly associated with abnormal development of the respiratory tract. In both these conditions, impaired swallowing by the fetus can lead to an excessive accumulation of amniotic fluid (polyhydramnios). Just after birth, a newborn with these anomalies commonly has difficulty swallowing milk, and regurgitation and choking while drinking are indications for examination of the patency of the esophagus.

Stomach

Pyloric Stenosis

Pyloric stenosis, which seems to be more physiological than anatomical, consists of hypertrophy of the circular layer of smooth muscle that surrounds the pyloric (outlet) end of the stomach. The hypertrophy causes a narrowing (stenosis) of the pyloric opening and impedes the passage of food. Several hours after a meal, the infant violently vomits (projectile vomiting) the contents of the meal. The enlarged pyloric end of the stomach can often be palpated on physical examination. Although pyloric stenosis is commonly treated by a simple surgical incision through the layer of circular smooth muscle of the pylorus, the hypertrophy sometimes diminishes without treatment by several weeks after birth. The pathogenesis of this defect remains unknown, but it seems to have a genetic basis. Pyloric stenosis is more common in male than in female infants, and the incidence has been reported as 1 in 200 to 1 in 1000 infants.

Heterotopic Gastric Mucosa

Heterotopic gastric mucosa has been found in a variety of otherwise normal organs (Fig. 15.10). This condition is often clinically significant because if the heterotopic mucosa secretes hydrochloric acid, ulcers can form in unexpected locations. Many cases of heterotopic tissue within the gastrointestinal tract are now thought to be caused by the inappropriate expression of genes that are characteristic of other regions of the gut, but the question remains: What is the basis of the inappropriate gene expression, and why does it often occur within a very restricted area? Given the recognition of increasingly complex networks of genetic control in the gut, it is not difficult to imagine that on occasion normal developmental controls go awry, but understanding the genetic basis of specific instances of ectopic mucosa still remains elusive.

Development of the Spleen

Development of the spleen is not well understood. Initially, two bilaterally symmetrical organ fields are reduced by regression to one on the left. The spleen is first recognizable as a mesenchymal condensation in the dorsal mesogastrium at 4 weeks and is initially closely associated with the developing dorsal pancreas. Initiation of splenic development requires the cooperative action of a basic helix-loop-helix protein (Pod-1) and a homeobox-containing protein (Bapx-1), acting through another transcription factor, Pbx-1. Such a combination is emerging as a common theme in the initiation of development of several organs. These substances act on the downstream molecules Nkx 2.5 and the oncogene Hox-11 (T-cell leukemia homeobox-1) in early splenic development (Fig. 15.9).

The splenic primordium consists of a condensation of mesenchyme covered by the overlying mesothelium of the dorsal mesogastrium, both of which contribute to the stroma of the spleen. Its normal left-sided location is determined early by the mechanisms that dictate heart asymmetry; Nkx 2.5, an important determinant of early heart development, is also expressed in the splenic primordium. Hematopoietic cells move into the spleen in the late embryonic period, and from the third to fifth months, the spleen and the liver serve as major sites of hematopoiesis during the first trimester of pregnancy. Later, the splenic primordium becomes infiltrated by lymphoid cells, and by the fourth month, the complex vascular structure of the red pulp begins to take shape.

Formation of the Intestines

The intestines are formed from the posterior part of the foregut, the midgut, and the hindgut (Table 15.1). Table 15.2 summarizes the chronology of major stages in the development of the digestive tract. Two points of reference are useful in understanding the gross transformation of the primitive gut tube from a relatively straight cylinder to the complex folded arrangement characteristic of the adult intestinal tract. The first is the yolk stalk, which extends from the floor of the midgut to the yolk sac. In the adult, the site of attachment of the yolk stalk is on the small intestine about 2 feet cranial to the junction between small and large intestine (ileocecal junction). On the dorsal side of the primitive gut, an unpaired ventral branch of the aorta, the superior mesenteric artery, and its branches feed the midgut (see Fig. 15.7). The superior mesenteric artery itself serves as a pivot point about which later rotation of the gut occurs.

Table 15.1

Derivatives of Regions of the Primitive Gut

Blood Supply Adult Derivatives
Foregut  
Celiac artery (lower esophagus to duodenum) Pharynx
Esophagus
Stomach
Upper duodenum
Glands of pharyngeal pouches, respiratory tract, liver, gallbladder, pancreas
Midgut  
Superior mesenteric artery Lower duodenum
Jejunum and ileum
Cecum and vermiform appendix
Ascending colon
Cranial half of transverse colon
Hindgut  
Inferior mesenteric artery Caudal half of transverse colon
Descending colon
Rectum
Superior part of anal canal

image

Table 15.2

Timelines in Development of the Digestive System

Normal Time (wk) Developmental Events
3 Tubular gut beginning to form; early induction of major digestive glands
4 Most of gut tubular; primordia of liver, dorsal and ventral pancreas, and trachea visible; rupture of oropharyngeal membrane
5 Expansion and early rotation of stomach; intestinal loop beginning to form; cecum and bile duct evident
6 Rotation of stomach completed, prominent intestinal loop; appearance of allantois and appendix; urorectal septum beginning to subdivide cloaca into rectum and urogenital sinus
7 Herniation of intestinal loop; rapid growth of liver; fusion of dorsal and ventral pancreas; cloacal septation complete
8 Counterclockwise rotation of herniated intestinal loop; recanalization of intestine; early penetration of parasympathetic neuronal precursors from cranial neural crest into gut
9 Return of herniated gut into body cavity; differentiation of epithelial types in intestinal lining
11 Villi appearing in small intestine; differentiation of goblet cells
16 Villi lining entire intestine (including colon)
20 Peyer’s patches seen in small intestine

By 5 weeks, rapid growth of the gut tube causes it to buckle out in a hairpinlike loop. The growth in length results in large part from the effect of FGF-9, which is produced by the epithelium and stimulates proliferation of the fibroblasts in the intestinal walls. The major change that causes the intestines to assume their adult positions is a counterclockwise rotation of the caudal limb of the intestinal loop (with the yolk stalk attachment and superior mesenteric artery as reference points) around the cephalic limb from its ventral aspect. The main consequence of this rotation is to bring the future colon across the small intestine so that it can readily assume its C-shaped position along the ventral abdominal wall (see Fig. 15.7). Behind the colon, the small intestine undergoes great elongation and becomes packed in its characteristic position in the abdominal cavity.

The rotation and other positional changes of the gut occur partly because the length of the gut increases more than the length of the embryo. From almost the first stages, the volume of the expanded gut tract is greater than the body cavity can accommodate. Consequently, the developing intestines herniate into the body stalk (the umbilical cord after further development) (Fig. 15.11). Intestinal herniation begins by 6 or 7 weeks of embryogenesis. By 9 weeks, the abdominal cavity has enlarged sufficiently to accommodate the intestinal tract, and the herniated intestinal loops begin to move through the intestinal ring back into the abdominal cavity. Coils of small intestine return first. As they do, they force the distal part of the colon, which was never herniated, to the left side of the peritoneal cavity, thus establishing the definitive position of the descending colon. After the small intestine has assumed its intra-abdominal position, the herniated proximal part of the colon also returns, with its cecal end swinging to the right and downward (see Fig. 15.7).

During these coilings, herniations, and return movements, the intestines are suspended from the dorsal body wall by a mesentery (Fig. 15.12). Experimentation has shown that looping of the intestine is caused principally by tension-compression relationships between the intestine and its dorsal mesentery. When the intestine is separated from the mesentery, the normal looping does not occur. As the intestines assume their definitive positions within the body cavity, their mesenteries follow. Parts of the mesentery associated with the duodenum and colon (mesoduodenum and mesocolon) fuse with the peritoneal lining of the dorsal body wall.

Starting in the sixth week, the primordium of the cecum becomes apparent as a swelling in the caudal limb of the midgut (see Fig. 15.7). In succeeding weeks, the cecal enlargement becomes so prominent that the distal small intestine enters the colon at a right angle. The sphincterlike boundary at the cecum between the small and large intestines, similar to that in other regions of the gut, is regulated by a high concentration of Cdx-2 and a sequence of Hox gene expression. In mice, deletion of Hoxd4, Hoxd8 to Hoxd11, and Hoxd13 results in the absence of this region. When the overall pattern has been set by combinations of Hox genes, cecal development depends on an interaction between FGF-9 produced by the cecal epithelium and FGF-10 produced by the overlying mesoderm.

The tip of the cecum elongates, but its diameter does not increase in proportion to the rest of the cecum. This wormlike appendage is aptly called the vermiform appendix.

Partitioning of the Cloaca

In the early embryo, the caudal end of the hindgut terminates in the endodermally lined cloaca, which, in lower vertebrates, serves as a common termination for the digestive and urogenital systems. The cloaca also includes the base of the allantois, which later expands as a common urogenital sinus (see Chapter 16). A cloacal (proctodeal) membrane consisting of apposed layers of ectoderm and endoderm acts as a barrier between the cloaca and an ectodermal depression known as the proctodeum (Fig. 15.13). A shelf of mesodermal tissue called the urorectal septum is situated between the hindgut and the base of the allantois. During weeks 6 and 7, the urorectal septum advances toward the cloacal membrane. At the same time, lateral mesodermal ridges extend into the cloaca.

The combined ingrowth of the lateral ridges and growth of the urorectal septum toward the cloacal membrane divide the cloaca into the rectum and urogenital sinus (see Fig. 15.13B). Double mutants of Hoxa13 and Hoxd13 result in the absence of cloacal partitioning, along with hypodevelopment of the phallus (genital tubercle). In addition, they lead to the absence of the smooth muscle component of the anal sphincter. According to classic embryology, the urorectal septum fuses with the cloacal membrane, thus dividing it into an anal membrane and a urogenital membrane before these membranes break down (see Fig. 15.13C). Other research suggests that the cloacal membrane undergoes apoptosis and breaks down without its fusion with the urorectal septum. The area where the urorectal septum and lateral mesodermal folds fuse with the cloacal membrane becomes the perineal body, which represents the partition between the digestive and urogenital systems.

The actual anal canal consists of a craniocaudal transition from columnar colonic (rectal) epithelium to a transitional region of cloacally derived endodermal epithelium leading into a zone of squamous epithelium that merges with the external perianal skin. These zones are surrounded by the smooth muscle internal anal sphincter.

Histogenesis of the Intestinal Tract

Shortly after its initial formation, the intestinal tract consists of a simple layer of columnar endodermal epithelium surrounded by a layer of splanchnopleural mesoderm. Three major phases are involved in the histogenesis of the intestinal epithelium: (1) an early phase of epithelial proliferation and morphogenesis, (2) an intermediate period of cellular differentiation in which the distinctive cell types characteristic of the intestinal epithelium appear, and (3) a final phase of biochemical and functional maturation of the different types of epithelial cells. The mesenchymal wall of the intestine also differentiates into several layers of highly innervated smooth muscle and connective tissue. An overall craniocaudal gradient of differentiation is present within the developing intestine.

The endoderm of the early foregut is capable of producing cell types other than those of the gut tube itself, such as liver cells. A two-phase series of inhibitory influences from the gut mesoderm restricts the overlying endoderm to forming only the appropriate epithelial cell types through the activity of the transcription factors Foxa-2 (formerly called hepatic nuclear factor-3) and GATA-4, which are essential for formation of anterior regions of endoderm.

Early in the second month, the epithelium of the small intestine begins a phase of rapid proliferation that causes the epithelium temporarily to occlude the lumen by 6 to 7 weeks’ gestation. Within a couple of weeks, recanalization of the intestinal lumen has occurred. At about this time, small, cracklike secondary lumina appear beneath the surface of the multilayered epithelium, and aggregates of mesoderm push into the epithelium. A combination of coalescence of the secondary lumina with continued mesenchymal upgrowth beneath the epithelium results in the formation of numerous fingerlike intestinal villi, which greatly increase the absorptive surface of the intestinal surface. By this time, the epithelium has transformed from a stratified into a simple columnar type.

With the formation of villi, pitlike intestinal crypts form at the bases of the villi. Toward the bottom of the crypts are epithelial intestinal stem cells, which, in response to Wnt signaling, have a high rate of mitosis and serve as the source of epithelial cells for the entire intestinal surface (Fig. 15.14). Despite the presence of four to six stem cells per crypt, it has been shown that each crypt is monoclonal (i.e., all the existing cells are descendants of a single stem cell from earlier in development). Toward the top of a crypt, shh and Indian hedgehog (Ihh) signaling stimulates the activity of BMP. This BMP has two main functions. It counteracts the effects of Wnt and thus keeps proliferation deep in the crypt, and it also facilitates cellular differentiation. Aided in part by an ephrin-eph gradient, progeny of the stem cells make their way up the wall of the crypt as multipotential transit amplifying cells, which, under the influence of the Delta-Notch system, begin to differentiate into the four main mature cell types of the intestinal epithelium (see Fig. 15.14). These cells then both differentiate and migrate toward the tip of the villus, until after about 4 days they die and are shed into the intestinal lumen as they are replaced below by new epithelial cells derived from the crypts. Human intestinal epithelial cells develop the intrinsic capacity for apoptosis by 18 to 20 weeks of gestation.

By the end of the second trimester of pregnancy, all cell types found in the adult intestinal lining have differentiated, but many of these cells do not possess adult functional patterns. Several specific biochemical patterns of differentiation are present by 12 weeks’ gestation and mature during the fetal period. For example, lactase, an enzyme that breaks down the disaccharide lactose (milk sugar), is one of the digestive enzymes synthesized in the fetal period in anticipation of the early postnatal period during which the newborn subsists principally on the mother’s milk. Further biochemical differentiation of the intestine occurs after birth, often in response to specific dietary patterns.

Histodifferentiation of the intestinal tract is not an isolated property of the individual tissue components of the intestinal wall. During the early embryonic period and sometimes into postnatal life, the epithelial and mesodermal components of the intestinal wall communicate by inductive interactions. In region-specific manners, these interactions involve hedgehog signaling (shh for the foregut and midgut and Ihh for the hindgut) from the endodermal epithelium. BMP signaling from the mesoderm is involved in positioning the crypts and villi in the small intestine and the glands of the colon. Interspecies recombination experiments show that the gut mesoderm exerts a regional influence on intestinal epithelial differentiation (e.g., whether the epithelium differentiates into a duodenal or colonic phenotype). When regional determination is set, however, the controls for biochemical differentiation of the epithelium are inherent. This pattern of inductive influence and the epithelial reaction are similar to those outlined earlier for dermal-epidermal interactions in the developing skin (see Chapter 9).

Final enzymatic differentiation of intestinal absorptive cells is strongly influenced by glucocorticoids, and the underlying mesoderm seems to mediate this hormonal effect. In a converse inductive influence, the intestinal endoderm, through the action of shh signaling, induces the differentiation of smooth muscle from mesenchymal cells in the wall of the intestine.

Although the intestine develops many functional capabilities during the fetal period, no major digestive function occurs until feeding begins after birth. The intestines of the fetus contain a greenish material called meconium (see Fig. 18.9), which is a mixture of lanugo hairs and vernix caseosa sloughed from the skin, desquamated cells from the gut, bile secretion, and other materials swallowed with the amniotic fluid.

Formation of Enteric Ganglia

As outlined in Chapter 12, the enteric ganglia of the gut are derived from neural crest. Pax-3–expressing cells from the vagal neural crest migrate into the foregut and spread in a wavelike fashion throughout the entire length of the gut. Slightly later, cells from the sacral crest enter the hindgut and intermingle with cells derived from the vagal neural crest. The migratory properties of vagal crest cells are much more pronounced than are those of cells from the sacral crest. Initially, the vagal crest cells migrate throughout the mesenchyme of the gut, but as the smooth muscle of the intestines begins to differentiate, the migrating vagal crest cells become preferentially distributed between the smooth muscle and the serosa, where the myenteric plexuses form. They are absent from the connective tissue of the submucosa because of the inhibiting effects of shh, secreted by the epithelial cells. During migration through the gut, the population of neural crest cells undergoes a massive expansion until the number of enteric neurons ultimately exceeds the number of neurons present in the spinal cord. Glial cells also differentiate from neural crest precursors in the gut, but the environmental factors that contribute to the differentiation of neural crest cells in the gut wall remain poorly understood.

Clinical Correlation 15.2 presents malformations of the intestinal tract.

Clinical Correlation 15.2   Malformations of the Intestinal Tract

Vitelline Duct Remnants

The most common family of anomalies of the intestinal tract is some form of persistence of the vitelline (yolk) duct. The most common member of this family is Meckel’s diverticulum, which is present in 2% to 4% of the population. Typical Meckel’s diverticulum is a blind pouch a few centimeters long located on the antimesenteric border of the ileum about 50 cm cranial from the ileocecal junction (Fig. 15.15A and E). This structure represents the persistent proximal portion of the yolk stalk. Simple Meckel’s diverticula are often asymptomatic, but they occasionally become inflamed or contain ectopic tissue (e.g., gastric, pancreatic, or even endometrial tissue), which can cause ulceration. It has been suggested that in the absence of intestinal mesodermal restriction (the wall of Meckel’s diverticulum is derived from the yolk duct) of the endoderm lining Meckel’s diverticulum, the endoderm retains the developmental capability to form various types of cellular phenotypes derived from endoderm.

In some cases, a ligament connects Meckel’s diverticulum to the umbilicus (see Fig. 15.15B), or a simple vitelline ligament that may have an associated persisting vitelline artery can connect the intestine to the umbilicus. Occasionally, the intestine rotates about such a ligament and causes a condition known as volvulus (see Fig. 15.15D). This disorder can lead to strangulation of the bowel.

A persistent vitelline duct can take the form of a vitelline fistula (see Fig. 15.15C), which constitutes a direct connection between the intestinal lumen and the outside of the body via the umbilicus. Rarely, a vitelline duct cyst is present along the length of a vitelline ligament.

Omphalocele

Omphalocele represents the failure of return of the intestinal loops into the body cavity during the tenth week. The primary defect in omphalocele is most likely a reduced prominence of the lateral body wall that does not provide sufficient space for the complete return of the intestines to the body cavity. After birth, the intestinal loops can be easily seen within an almost transparent sac consisting of amnion on the outside and peritoneal membrane on the inside (Fig. 15.16). The incidence of omphalocele is approximately 1 in 3500 births, but half of the infants with this condition are stillborn.

Abnormal Rotation of the Gut

Sometimes the intestines undergo no or abnormal rotation as they return to the abdominal cavity; this can result in a wide spectrum of anatomical anomalies (Fig. 15.17). In most cases, these anomalies are asymptomatic, but occasionally, they can lead to volvulus or another form of strangulation of the gut. The major rotation of the gut occurs after smooth muscle has formed in the intestinal walls. Mice mutant for both sonic hedgehog (shh) and Indian hedgehog have greatly reduced smooth muscle in the gut wall and frequently exhibit malrotation of the intestines. Research has placed much more emphasis on mechanical relationships between the dorsal mesentery and the intestine for patterns of gut rotation.

Aganglionic Megacolon (Hirschsprung’s Disease)

The basis of aganglionic megacolon, which is manifested by great dilation of certain segments of the colon, is the absence of parasympathetic ganglia in the affected walls of the colon. Hirschsprung’s disease seems to be truly multifactorial, with dominant and recessive mutations resulting in the condition. Many patients with Hirschsprung’s disease do not express the c-RET oncogene. C-RET, along with a coreceptor, Gfra-1, is a receptor for glia-derived neurotrophic factor (GDNF). This gene is activated by the combination of Pax-3 with SOX-10, both of which are necessary for the formation of enteric ganglia. Similarly, mutations of SOX10, which probably interferes with Pax-3 function, can result in a combined Waardenburg-Hirschsprung syndrome. Mutants of Ret, Gfra1, and GDNF interfere with the migration of vagal neural crest cells into the gut.

Known mutations currently account for only about half of the cases of Hirschsprung’s disease. Other mutations underlying aganglionic megacolon could involve defects in the migration or proliferation of neural crest precursor cells. Death of precursor cells before or after they reach their final destination in the hindgut could reduce the number of enteric ganglia. Alternatively, the local environment could prevent the successful migration of neural crest cells into the colon. Evidence from mutant mice developing aganglionic segments of the bowel strongly suggests that the environment of the gut wall inhibits the migration of neural crest cells into the affected segment of gut. This was shown by experiments in which crest cells from mutant mice were capable of colonizing normal gut, but normal crest cells could not migrate into gut segments of mutant mice. The accumulation of laminin in the gut wall as the result of overproduction of endothelin-3 serves as a stop signal for neural crest migration.

The distal colon is the most commonly affected region for aganglionosis, but in a few cases, aganglionic segments extend as far cranially as the ascending colon. Estimates of the frequency of megacolon vary widely, from 1 in 1000 to 1 in 30,000 births.

More recent experiments have shown that cells derived from the vagal neural crest exhibit much stronger migratory properties in the hindgut than do cells from the sacral crest. This finding has led to the suggestion that vagal crest cells be transplanted into aganglionic segments of the colon in an attempt to correct the deficit of enteric neurons in this area.

Imperforate Anus

Imperforate anus, which occurs in 1 of every 4000 to 5000 births, includes a spectrum of anal defects that can range from a simple membrane covering the anal opening (persistence of the cloacal membrane) to atresia of various lengths of the anal canal, rectum, or both. Grossly, all defects are characterized by the absence of an anal opening (Fig. 15.19). Deletions of Hoxa13 and Hoxd13 in mice result in defects in morphogenesis of the anal sphincter, and mutants of shh and its downstream molecules Gli2 and Gli3 cause the colon to end in a blind sac, with no anus forming. Any examination of a newborn must include a determination of the presence of an anal opening. The extent of the atretic segment is important when considering the surgical treatment of imperforate anus. Treatment of a persistent anal membrane can be trivial, whereas more extensive defects, especially defects involving the anal musculature, constitute very challenging surgical problems.

Glands of the Digestive System

The glands of the digestive system arise through inductive processes between the early gut epithelium and the surrounding mesenchyme. The various glandular epithelia have considerably different requirements in the types of mesenchyme that can support their development. In tissue recombination experiments, pancreatic epithelium undergoes typical development when it is juxtaposed with mesenchyme from almost any source. The development of salivary gland epithelium is supported by mesenchyme from lung or accessory sexual glands, but not by many other types of mesenchyme. Inductive support of hepatic (liver) epithelium follows a distinctive pattern. Normal epithelial development is supported by mesenchyme derived from lateral plate or intermediate mesoderm, but axial mesenchyme (either somitic or neural crest) fails to support hepatic differentiation. The inductive properties of certain glandular mesenchymes may be correlated with different modes of vascularization of these mesenchymes (see p. 414).

Formation of the Liver

After initial induction by the cardiac mesoderm and the septum transversum (see Fig. 15.4), the gut-derived hepatic endoderm thickens to form a pseudostratified epithelium (Fig. 15.21). The nuclear dynamics within the pseudostratified epithelium parallel those in the early neural tube (see Fig. 11.4). The nuclei undergo DNA synthesis (S phase of the cell cycle) in the basal position near the basal lamina surrounding the liver bud. Then the nuclei migrate to the apical (luminal) position, where they undergo mitotic division. The pathways and fates of the daughter cells have not yet been clearly determined. The transition to the pseudostratified stage requires the activity of the homeobox gene Hhex, without which the liver does not form (see Fig. 15.21).

Early in the third week, through the actions of Hhex and other transcription factors, cells within the hepatic epithelium lose their epithelial characteristics by the downregulation of E-cadherin and migrate through the underlying basal lamina, which has been degraded by matrix metalloproteinases (MMPs). These migrating cells make their way into the underlying mesenchyme of the septum transversum and form hepatic cords. Early in the formation of the liver, the future hepatic cells already express the albumin gene, one of the major characteristics of mature hepatocytes.

The original hepatic diverticulum branches into many hepatic cords, which are closely associated with splanchnic mesoderm of the septum transversum. The mesoderm supports continued growth and proliferation of the hepatic endoderm. This occurs partly through the actions of hepatic growth factor (HGF), which is bound by the receptor molecule, c-met, located on the surface of the endodermal hepatocytes. Experimental studies have shown that mesoderm from either the splanchnopleural or the somatopleural components of the lateral plate mesoderm can support further hepatic growth and differentiation, whereas paraxial mesoderm has only a limited capacity to support hepatic development.

The cells of the hepatic cords (hepatoblasts) are bipotential: they can form either hepatic parenchymal cells (hepatocytes) or intrahepatic bile duct cells (cholangiocytes). Guided by the transcription factors hepatic nuclear factor-4 (HNF-4) and FoxA, some hepatoblasts differentiate into hepatocytes, which begin to express molecules (e.g., albumin and α-fetoprotein) characteristic of mature hepatic parenchymal cells. Other hepatoblasts, under the influence of TGF-β and Notch, gather as a single layer around branches of the portal vein as a ductal plate (Fig. 15.22A). Through mechanisms still not well understood, two ductlike structures (future bile ducts) begin to form around each vein. Initially, the cells comprising the walls of the ducts are hybrid in character (Fig. 15.22B). Cells closest to the vein have the characteristics of cholangiocytes, whereas those on the opposite face more closely resemble hepatocytes. Eventually, all the cells lining the bile duct become full-fledged cholangiocytes. These bile ducts branch to form networks leading toward the edges of the hepatic lobes and constitute the intrahepatic component of the bile duct system.

The other major component of the bile duct system, consisting of the larger hepatic ducts, the cystic duct, the gallbladder, and the common bile duct, arises outside the main body of the liver and is called the extrahepatic biliary tree. Its precursor cells arise as a component of a common pancreatobiliary precursor (see Fig. 15.21), which is located caudal to the prehepatic endoderm. Expressing Sox-17 and Pdx-1, these cells are bipotential, like those of the hepatic diverticulum. Some of these cells cease expressing Sox-17, but continue to express Pdx-1 and go on to form the ventral pancreas. Others, which lose Pdx-1 expression but continue to express Sox-17, become precursors of the extrahepatic biliary tree. They extend to form the cystic duct, and a dilatation from that foreshadows the further development of the gallbladder (Fig. 15.23). How the intrahepatic and extrahepatic bile ducts become connected remains unclear.

Within the substance of the liver, the hepatic cords form a series of loosely packed and highly irregular sheets that alternate with mesodermally lined sinusoids, through which blood circulates and exchanges nutrients with the hepatocytes. The sinusoids are the first vessels to form in the liver, and they arise from the mesenchyme of the proepicardium and the septum transversum. This same source also gives rise to stellate cells, cells residing in the space (of Dissé) between the hepatocytes and the sinusoidal endothelium. These cells store vitamin A and can also modulate the sinusoidal circulation, but when chronically activated postnatally, they form the basis for fibrosis of the liver.

The entire liver soon becomes too large to be contained in the septum transversum, and it protrudes into the ventral mesentery within the abdominal cavity. As it continues to expand, the rapidly growing liver remains covered by a glistening, translucent layer of mesenteric tissue that now serves as the connective tissue capsule of the liver. Between the liver and the ventral body wall is a thin, sickle-shaped piece of ventral mesentery: the falciform ligament. The ventral mesentery between the liver and the stomach is the lesser omentum (see Fig. 15.6).

Development of Hepatic Function

Development of the liver is not only a matter of increasing its mass and structural complexity. As the liver develops, its cells gradually acquire the capacity to perform the many biochemical functions characterizing the mature, functioning liver. One characteristic major function of the liver is to produce the plasma protein serum albumin. The mRNA for albumin has been detected in mammalian hepatocytes during the earliest stages of their ingrowth into the hepatic mesoderm, and it seems to depend on the earlier expression of the transcription factor HNF-3 (Foxa-3).

Major functions of the adult liver include the synthesis and storage of glycogen, which serves as a carbohydrate reserve. As the fetal period progresses, the liver actively stores glycogen. This function is strongly stimulated by adrenocortical hormones and is indirectly stimulated by the anterior pituitary. Similarly, the fetal period includes the functional development of the system of enzymes involved in the synthesis of urea from nitrogenous metabolites. By birth, these enzymes have attained full functional capacity.

A major function of the embryonic liver is the production of blood cells. After yolk sac hematopoiesis, the liver is one of the chief sites of intraembryonic blood formation (see Fig. 17.2). Hematopoietic cells, seeding the liver from origins in other sites, appear in small clusters among the hepatic parenchymal cells. As the liver matures, the intrahepatic environment no longer supports blood cell development, and hematopoiesis migrates to other sites in the fetus.

At approximately 12 weeks’ gestation, the hepatocytes begin to produce bile, largely through the breakdown of hemoglobin. The bile drains down the newly formed bile duct system and is stored in the gallbladder. As bile is released into the intestines, it stains the other intestinal contents a dark green, which is one of the characteristics of meconium.

Formation of the Pancreas

The pancreas begins as separate dorsal and ventral primordia within the duodenal endoderm (see Fig. 6.20D). Early development of each of these two primordia is under different molecular controls. As discussed previously, ventral endoderm of the hepatic diverticulum is patterned to differentiate into ventral pancreatic tissue by a default mechanism in areas where liver induction does not occur. In primitive vertebrates, pancreatic function is distributed in cells within the foregut, rather than in a discrete gland, and some investigators have speculated that the ventral pancreatic bud represents the extension of this system, whereas the discrete dorsal pancreas is an evolutionary newcomer.

The dorsal pancreas is induced from the dorsal gut endoderm by activin and FGF signals emanating from the notochord, which early in development is directly opposed to the endoderm (Fig. 15.24). Shh activity in the dorsal endoderm must be repressed, or pancreatic differentiation does not occur. Initiation of development of the ventral pancreas is under a different set of developmental controls and heavily depends on the activity of the transcription factor Ptf-1a. During the earliest stages of dorsal pancreatic bud formation, the pancreatic progenitor cells express the transcription factors Pdx-1 and Hoxb-9. If Pdx-1 expression is eliminated by targeted mutagenesis, development of the pancreatic bud ceases. From this point, different environmental signals and intracellular responses result in the differentiation of two lineages of cells: the pancreatic exocrine cells and the pancreatic endocrine cells (see Fig. 15.24).

During the phase of early growth, the dorsal pancreas becomes considerably larger than the ventral pancreas. At about the same time, the duodenum rotates to the right and forms a C-shaped loop that carries the ventral pancreas and common bile duct behind it and into the dorsal mesentery. The ventral pancreas soon makes contact and fuses with the dorsal pancreas.

Both the dorsal pancreas and the ventral pancreas possess a large duct. After fusion of the two pancreatic primordia, the main duct of the ventral pancreas makes an anastomotic connection with the duct of the dorsal pancreas. The portion of the dorsal pancreatic duct between the anastomotic connection and the duodenum normally regresses, leaving the main duct of the ventral pancreas (duct of Wirsung) the definitive outlet from the pancreas into the duodenum (see Fig. 15.23).

The pancreas is a dual organ with endocrine and exocrine functions. The exocrine portion consists of large numbers of acini, which are connected to a secretory duct system. The endocrine component consists of roughly 1 million richly vascularized islets of Langerhans, which are scattered among the acini.

In certain pancreatic progenitor cells, the action of the signaling molecules, follistatin, and several FGFs emanating from the surrounding mesoderm, in combination with the activation of the Notch receptor system (see p. 69), results in the differentiation of many of the pancreatic cells along the exocrine pathway. These cells, which secrete digestive hormones, such as amylase and carboxypeptidase, are ultimately responsible for the gross morphogenesis of the pancreas. During outgrowth of the pancreatic primordia, the exocrine cells assume the form of sequentially budding cords. From these cellular cords, the acini and their ducts differentiate. Tissue recombination experiments, conducted in vitro and in vivo, have shown that the presence of mesenchyme is necessary for the formation of acini, but that ducts can form in the absence of mesenchyme if the endodermal precursor cells are exposed to a gel rich in basement membrane material.

Although the presence of mesenchyme is required for the differentiation of acini, the mesenchyme need not be of pancreatic origin. In vitro, pancreatic endoderm combined with salivary gland mesoderm differentiates even better than that exposed to pancreatic mesenchyme. This finding shows that in the case of the pancreas, the inductive influence of the mesenchyme is permissive, rather than instructive.

Differentiation of the acini is divided into three phases (Fig. 15.25). The first, called the predifferentiated state, occurs while the pancreatic primordia are first taking shape. A population of pancreatic progenitor cells that exhibits virtually undetectable levels of digestive enzyme activity is established. As the pancreatic buds begin to grow outward, the epithelium undergoes a transition into a second, protodifferentiated state. During this phase, the exocrine cells synthesize low levels of many hydrolytic enzymes that they will ultimately produce. After the main period of outgrowth, the pancreatic acinar cells pass through another transition before attaining a third, differentiated state. By this time, these cells have acquired an elaborate protein-synthesizing apparatus, and the inactive forms of the polypeptide digestive enzymes are stored in the cytoplasm as zymogen granules. Glucocorticoid hormones from the fetal adrenal cortex stimulate increased production of certain digestive enzymes.

Development of the islets of Langerhans follows a different course from that of the acini. The islets of Langerhans are formed from groups of epithelial cells that break away from the acinar epithelial cells during the second (protodifferentiated) phase of acinar cell development. In a pathway not involving activation of the Notch system, but requiring signals from the local vasculature, a bipotential precursor cell has the potential to become either an endocrine cell or a ductal cell (see Fig. 15.24). Those cells entering the endocrine lineage as endocrine progenitor cells express the transcription factors neurogenin-3 and Isl-1. The endocrine progenitor cells give rise to two types of progenies (committed precursor cells), each of which is characterized by the expression of a different Pax gene. One type, which differentiates at 8 to 9 weeks, gives rise to α and γ cells, which produce glucagon and pancreatic polypeptide. The other type, which differentiates later, gives rise to β and δ cells, which produce insulin and somatostatin. During the second phase of pancreatic differentiation (protodifferentiated state), the levels of glucagon synthesis considerably exceed the levels of insulin. By the third phase of pancreatic development, secretory granules are evident in the cytoplasm of most islet cells. Insulin and glucagon are present in the fetal circulation by the end of the fifth month of gestation.

Clinical Correlation 15.3 presents anomalies of the liver and pancreas.

Clinical Correlation 15.3   Anomalies of the Liver and Pancreas

Many minor variations in the shape of the liver or bile ducts occur, but these variations normally have no functional significance. One of the most serious malformations involving the liver is biliary atresia. This malformation can involve any level ranging from the tiny bile canaliculi to the major bile-carrying ducts. Alagille’s syndrome, which is characterized by biliary atresia and cardiac defects, is caused by mutations in Jagged-1, a ligand for the Notch receptor. Newborns with this condition typically develop severe jaundice shortly after birth. Some patients can be treated surgically; for others, a liver transplant is necessary.

Rarely, a ring of pancreatic tissue completely encircles the duodenum and forms an annular pancreas (Fig. 15.26). This anomaly can sometimes cause obstruction of the duodenum after birth. The cause of annular pancreas is not established, but a commonly accepted explanation is that outgrowths from a bifid ventral pancreas may encircle the duodenum from both sides. Studies in mice suggest that locally reduced sonic hedgehog (shh) signaling may permit overgrowth of the ventral pancreatic bud tissue.

Heterotopic pancreatic tissue can occasionally be found along the digestive tract, and it occurs most frequently in the duodenum or mucosa of the stomach (Fig. 15.27). About 6% of Meckel’s diverticula contain heterotopic pancreatic tissue.

A serious genetic condition affecting both the liver and the pancreas, as well as the kidneys and other organs, is polycystic disease. This condition results from malfunction of primary cilia, and it can be caused by the defective formation of any of several proteins (e.g., Polycystin-1 or -2 in autosomal primary polycystic kidney disease) involved in the function of the primary cilia. Mutations of the gene coding for the transmembrane protein polyductin in cholangiocytes are responsible for some cases of polycystic disease in the liver. Symptoms of polycystic liver disease include swelling of the liver and abdominal discomfort, especially after eating.

Respiratory System

The location of the future respiratory system in the ventral foregut is indicated by a zone of expression of the transcription factor Nkx 2.1, which also marks the site of formation of the thyroid gland. The dorsal wall of the foregut in this area is characterized by Sox-2 expression. Specification (induction) of the respiratory region is mediated by Wnt and FGF signals from the adjacent mesoderm. Late in the fourth week, paired lung buds begin to protrude from the posterior part of the respiratory endoderm (Fig. 15.28A). Dorsal to the lung buds, a pair of lateral mesodermal ridges begins to grow inward. Under the influence of Wnt signaling, these ridges fuse in a posterior-to-anterior direction. As they do so, they create a septum that separates the newly forming trachea from the esophagus. Through a series of interactions with the surrounding mesoderm, the early respiratory diverticulum (trachea plus lung buds) elongates, and the lung buds begin a set of 23 bifurcations that continue into postnatal life.

Formation of the Larynx

During weeks 4 and 5 of gestation, a rapid proliferation of the fourth and sixth pharyngeal arch mesenchyme around the site of origin of the respiratory bud converts the opening slit from the esophagus into a T-shaped glottis bounded by two lateral arytenoid swellings and a cranial epiglottis. The mesenchyme surrounding the laryngeal orifice ultimately differentiates into the thyroid, cricoid, and arytenoid cartilages, which form the skeletal supports of the larynx. Similar to the esophagus, the lumen of the larynx undergoes a temporary epithelial occlusion. In the process of recanalization during weeks 9 and 10, a pair of lateral folds and recesses forms the structural basis for the vocal cords and adjacent laryngeal ventricles. The somitomere-derived musculature of the larynx is innervated by branches of the vagus nerve (cranial nerve X), the musculature associated with the fourth arch is innervated by the superior laryngeal nerve, and the musculature of the sixth arch is innervated by the recurrent laryngeal nerve.

Formation of the Trachea and Bronchial Tree

At the initial appearance of the respiratory diverticulum, a pair of bronchial buds appears at its end (Fig. 15.28B). It now seems that the precursors of the trachea and lung buds are derived from separate sources of cells, and that the lung buds give rise to the bronchi and distal respiratory tree. The straight portion of the respiratory diverticulum is the primordium of the trachea. The bronchial buds, which ultimately become the primary bronchi, give rise to additional buds—three on the right and two on the left. These buds become the secondary, or stem, bronchi, and their numbers presage the formation of the three lobes of the right lung and the two lobes of the left (see Fig. 15.28). From this point, each secondary bronchial bud undergoes a long series of branchings throughout embryonic and fetal life.

Morphogenesis of the lung continues after birth. Stabilization of the morphological pattern of the lungs does not occur until about 8 years of age. An array of Hox genes (Hoxa3 to Hoxa5 and Hoxb3 to Hoxb6) is expressed early in the developing respiratory tract. Combinatorial patterns of expression of Hox genes are involved in regional specification of the respiratory tract.

The mesoderm surrounding the endoderm controls the extent of branching within the respiratory tract. Numerous tissue recombination experiments have shown that the mesoderm surrounding the trachea inhibits branching, whereas the mesoderm surrounding the bronchial buds promotes branching. If tracheal endoderm is combined with bronchial mesoderm, abnormal budding is induced. Conversely, tracheal mesoderm placed around bronchial endoderm inhibits bronchial budding. Mesoderm of certain other organs, such as salivary glands, can promote budding of the bronchial endoderm, but a pattern of branching characteristic of the mesoderm is induced. A mesoderm capable of promoting or sustaining budding must maintain a high rate of proliferation of the epithelial cells. Generally, the pattern of the epithelial organ is largely determined by the mesoderm. Structural and functional differentiation of the epithelium is a specific property of the epithelial cells, but the epithelial phenotype corresponds to the region dictated by the mesoderm.

The basic principles underlying pulmonary branching are similar to those operating in the development of the salivary glands and pancreas. At points of branching, epithelial cell proliferation is reduced, and the deposition of types I, III, and IV collagen, fibronectin, and proteoglycans stabilizes the morphology of the branching point and more proximal ductal regions. Heightened epithelial cell proliferation characterizes the rapidly expanding portions of the epithelial buds (Fig. 15.29).

The activities of many molecules contribute to lung morphogenesis. More than 50 genes are involved in morphogenesis of the tracheal system in Drosophila, which shows remarkable parallels to the mammalian respiratory system. A prime mover in generating branching is FGF-10, which, in response to the action of retinoic acid* and Tbx-4 and Tbx-5, is produced by the mesenchyme off the tip of a growing bud in the respiratory system. In FGF-10 knockout mice, budding of the developing lungs fails to occur. FGF-10 acts as a signaling center, by stimulating cell proliferation in the epithelium at the tip of the bud and causing the epithelium to grow out toward the source of the FGF-10 (see Fig. 15.29A). Apical epithelial proliferation is also promoted by the expression of the transcription factor Nkx 2.1 in these cells.

Branching is initiated with the stimulation of secretion of BMP-4 in the apical epithelial cells; this inhibits their proliferation. Simultaneously, shh, which is also produced by the epithelium, stimulates proliferation of the mesenchymal cells off the tip and inhibits the formation of FGF-10 (see Fig. 15.29B). These mesenchymal cells begin to produce TGF-β1, which, in addition to inhibiting FGF-10 production along with shh, promotes the synthesis of extracellular matrix molecules just distal to the apical epithelial cells. These molecules, including fibronectin and collagen types I, III, and IV, stabilize the formerly growing epithelial tip.

While the cell proliferation in the epithelial tip is reduced and the cells become bound by newly secreted extracellular matrix molecules, FGF-10 is secreted by the mesenchyme lateral to the old apex, where the concentrations of shh and TGF-β1 are reduced to less than the inhibitory level (see Fig. 15.29C). This activity sets up two new signaling centers on either side of the original one, and the cycle of apical epithelial proliferation begins anew. As the new apical growth centers mature, FGF-10 signaling is again inhibited, and each of the two existing tips begins its own branching cycle. The concurrent presence of the epithelial cell–associated proteoglycan syndecan is important for maintaining the stability of epithelial sheets along the ducts. Interacting with the extracellular matrix protein tenascin, syndecan is found along already formed ducts, but not in areas where branching is occurring in terminal saccular regions of the developing airway (see Fig. 15.29).

As with branching morphogenesis, the formation and maintenance of epithelially lined ducts involve special sets of molecular components. Hoxb-5 is expressed during the early development of smaller bronchioles (e.g., terminal bronchioles), but not in the components of the lung that are involved in actual respiratory exchange (i.e., respiratory bronchioles, alveolar ducts, alveoli). The protein epimorphin is important in the later formation of epithelial tubes. Epimorphin is located in the mesenchyme and seems to provide a signal that allows overlying epithelial cells to establish proper polarity or cell arrangements. In the embryonic lung, the developing epithelial ducts become disorganized and do not form lumina if epimorphin is blocked by specific antibodies.

Smooth muscle formation in the mesenchyme alongside the respiratory tract depends on shh and BMP-4 signals coming from the distal epithelial buds. In addition, FGF-9 secreted by the surrounding pleura helps to control the proliferation and differentiation of the smooth muscle cell precursors.

Stages in Lung Development

Embryonic Stage (Weeks 4 to 7)

The embryonic stage includes the initial formation of the respiratory diverticulum up to the formation of all major bronchopulmonary segments. During this period, the developing lungs grow into and begin to fill the bilateral pleural cavities. These structures represent the major components of the thoracic body cavity above the pericardium (Fig. 15.30).

Canalicular Stage (Weeks 17 to 26)

The canalicular stage is characterized by the formation of respiratory bronchioles as the result of budding of the terminal components of the system of bronchioles that formed during the pseudoglandular stage. An array of many different cell types forms along the developing respiratory tree. A gradient of BMP-4 and Wnt signaling, which is highest at the distal tips of the branches, prevents the distal cells from forming phenotypes more characteristic of the larger branches of the bronchial tree. The other major events during this stage are the intense ingrowth of blood vessels into the developing lungs and the close association of capillaries with the walls of the respiratory bronchioles (see Fig. 15.31). Occasionally, a fetus born toward the end of this period can survive with intensive care, but respiratory immaturity is the principal reason for poor viability.

Terminal Sac Stage (Weeks 26 to Birth)

During the terminal sac stage, the terminal air sacs (alveoli) bud off the respiratory bronchioles that largely formed during the canalicular stage. The epithelium lining the alveoli differentiates into two types of cells: type I alveolar cells (pneumocytes), across which gas exchange occurs after birth; and type II alveolar (secretory epithelial) cells. Type II alveolar cells form pulmonary surfactant, the material that spreads over the surface of the alveoli to reduce surface tension and facilitate expansion of the alveoli during breathing. Research involving specific markers of the epithelial cells has shown that type II cells form first in the alveolar lining. After proliferation, some type II cells flatten, lose their characteristic secretory function, and undergo terminal differentiation into type I pneumocytes. Other type I cells may differentiate directly from a pool of epithelial precursor cells in the early alveolar lining. With increasing amounts of pulmonary surfactant being formed, the fetus has a correspondingly greater chance of survival if born prematurely. In the fetus, the respiratory passageways in the lungs are filled with fluid (see Chapter 18). During the last 4 weeks of pregnancy, greatly increased formation of alveoli results in an exponential increase in the respiratory surface area of the lung. These weeks are sometimes referred to as the alveolar period of lung development.

Postnatal Stage

At birth, the mammalian lung is far from mature. An estimated 90% or more of the roughly 300 million alveoli found in the mature human lung are formed after birth. The major mechanism for this increase is the formation of secondary connective tissue septa that divide existing alveolar sacs. When they first appear, the secondary septa are relatively thick. In time, they transform into thinner mature septa capable of full respiratory exchange function.

Clinical Correlation 15.4 presents malformations of the respiratory system.

Clinical Correlation 15.4   Malformations of the Respiratory System

Tracheoesophageal Fistulas

The most common family of malformations of the respiratory tract is related to abnormal separation of the tracheal bud from the esophagus during early development of the respiratory system. Many common anatomical varieties of tracheoesophageal fistulas exist (Fig. 15.32), but virtually all involve the stenosis or atresia of a segment of trachea or esophagus and an abnormal connection between them. These are manifested early after birth by the newborn’s choking or regurgitation of milk when feeding.

Expression of certain genes is important in the normal formation of a mesenchymal partition between the esophagus and the developing trachea. Nkx 2.1 and bone morphogenetic protein-4 (BMP-4) are expressed in the ventral foregut mesoderm in the area where the trachea forms. Mutants of these genes are characterized by a high incidence of tracheoesophageal fistulas. The loss of Wnt signaling, leading to the downregulation of Nkx 2.1 ventrally, and reduced Sox-2 activity in the dorsal foregut have both been linked to tracheoesophageal fistulas.

Respiratory Distress Syndrome (Hyaline Membrane Disease)

Respiratory distress syndrome is often manifested in infants born prematurely and is characterized by labored breathing. In infants who die of this condition, the lungs are underinflated, and the alveoli are partially filled with a proteinaceous fluid that forms a membrane over the respiratory surfaces (Fig. 15.33). This syndrome is related to insufficiencies in the formation of surfactant by the type II alveolar cells.

Body Cavities

Formation of the Common Coelom and Mesentery

As the lateral plate mesoderm of the early embryo splits and then folds laterally, the space between the somatic and splanchnic layers of mesoderm becomes the common intraembryonic coelom (Fig. 15.34). The same folding process that results in the completion of the ventral body wall and the separation of the intraembryonic from the extraembryonic coelom also brings the two layers of splanchnic mesoderm around the newly formed gut as the primary (common) mesentery. The primary mesentery suspends the gut from the dorsal body wall as the dorsal mesentery and attaches it to the ventral body wall as the ventral mesentery. This placement effectively divides the coelom into right and left components. Soon, however, most of the ventral mesentery breaks down and causes a confluence of the right and left halves of the coelom. In the region of the developing stomach and liver, the ventral mesentery persists, thus forming the ventral mesogastrium and the falciform ligament of the liver (see Fig. 15.6). Further cranially, the tubular primordium of the heart is similarly supported by a dorsal mesocardium and briefly by a ventral mesocardium, which soon breaks down.

Formation of the Septum Transversum and Pleural Canals

A major factor in division of the common coelom into thoracic and abdominal components is the septum transversum. This septum grows from the ventral body wall as a semicircular shelf, which separates the heart from the developing liver (Fig. 15.35). During its early development, a major portion of the liver is embedded in the septum transversum. Ultimately, the septum transversum constitutes a significant component of the diaphragm (see p. 367).

The expanding septum transversum serves as a partial partition between the pericardial and the peritoneal portions of the coelom. By the time the expanding edge of the septum transversum reaches the floor of the foregut, it has almost cut the common coelom into two parts. Two short channels located on either side of the foregut connect the two major parts (Fig. 15.36). Initially known as the pleural (pericardioperitoneal) canals, these channels represent the spaces into which the developing lungs grow. The pleural canals enlarge greatly as the lungs increase in size and ultimately form the pleural cavities.

The pleural canals are partially delimited by two paired folds of tissue: the pleuropericardial and pleuroperitoneal folds. The pleuropericardial folds (see Fig. 15.30) are ridges of tissue associated with the common cardinal veins, which bulge into the dorsolateral wall of the coelom as they arch toward the midline of the thoracic portion of the coelom and enter the sinus venosus of the heart (Fig. 15.37). Initially, the pleuropericardial folds are not large and cause only a narrowing at the junction of the pericardial cavity and pleural canals. As the lungs expand, however, the folds form prominent shelves that meet at the midline and form the fibrous (parietal) layer of the pericardium.

The paired phrenic nerves are associated with the pleuropericardial folds. These nerves arise from joined branches of cervical roots 3, 4, and 5 and supply the muscle fibers of the diaphragm. With the shifts in positions of various components of the body during growth, the diaphragm ultimately descends to the level of the lower thoracic vertebrae. As it does, it carries the phrenic nerves with it. Even in adults, the pathway of the phrenic nerves through the fibrous pericardium is a reminder of their early association with the pleuropericardial folds.

At the caudal ends of the pleural canals, another pair of folds, the pleuroperitoneal folds, becomes prominent as the expanding lungs push into the mesoderm of the body wall. The pleuroperitoneal folds occupy successively greater portions of the pleural canal until they fuse with the septum transversum and the mesentery of the esophagus, thereby effectively obliterating the pleural canal (Fig. 15.38). Cells from the pleuroperitoneal folds continue into the abdominal cavity and contribute to the connective tissue that connects the liver and the right adrenal gland. All connections between the abdominal cavity and the thoracic cavity are eliminated.

Formation of the Diaphragm

The diaphragm, which separates the thoracic from the abdominal cavity in adults, is a composite structure derived from several embryonic components (see Fig. 15.38). The large ventral component of the diaphragm arises from the septum transversum, which fuses with the ventral part of the esophageal mesentery. Converging on the esophageal mesentery from the dorsolateral sides are the pleuroperitoneal folds. These components form the bulk of the diaphragm. As the lungs continue to grow, their caudal tips excavate additional space in the body wall. The body wall mesenchyme separated from the body wall proper becomes a third component of the diaphragm by forming a thin rim of tissue along its dorsolateral borders. In keeping with their motor innervation by the phrenic nerve, arising from the third to fifth cervical roots, the cellular precursors of the diaphragmatic musculature shift caudally into the body cavity from their site of origin in the cervical somites.

Clinical Correlation 15.5 presents malformations of the body cavities, diaphragm, and body wall.

Clinical Correlation 15.5   Malformations of the Body Cavities, Diaphragm, and Body Wall

Ventral Body Wall Defects, Ectopia Cordis, Gastroschisis, and Omphalocele

The opposing sides of the body wall occasionally fail to fuse as the embryo assumes its cylindrical shape late in the first month. Several defective mechanisms, such as hypoplasia of the tissues, can account for these defects. A quantitatively minor defect in closure of the thoracic wall is manifested as failure of sternal fusion (Fig. 15.39). If growth of the two sides of the thoracic wall is severely defective, the heart can form outside the thoracic cavity, thus resulting in ectopia cordis (Fig. 15.40).

Closure defects of the ventral abdominal wall can lead to similar gross malformations. In many cases of omphalocele (see Fig. 15.16), hypoplasia of the abdominal wall itself and deficiencies of abdominal musculature are evident. More serious cases involve evisceration of the abdominal contents through a fissure between the umbilicus and sternum (gastroschisis) (Fig. 15.41). Caudal to the umbilicus, an associated closure defect of the urinary bladder (exstrophy of the bladder [see Fig. 16.20]) is common.

Diaphragmatic Hernias

Incomplete fusion or hypoplasia of one or more of the components of the diaphragm can lead to an open connection between the abdominal and thoracic cavities. If the defect is large enough, various structures in the abdominal cavity (usually part of the stomach or intestines) can herniate into the thoracic cavity, or, more rarely, thoracic structures can penetrate into the abdominal cavity. Minor cases of herniation can cause digestive symptoms. In the case of major defects, herniation of massive portions of the intestines can press against the heart or lungs and interfere with their function. Some common sites of defects in the diaphragm are shown in Figure 15.42.

More recent laboratory studies on rodents have pointed to a relationship between a deficiency in vitamin A (retinoic acid) and diaphragmatic hernia. The laboratory evidence suggests that the primary defect may arise at a time earlier than what would be predicted by the commonly held belief that most diaphragmatic hernias are caused by failure of closure of the pleuroperitoneal canals. Whether the laboratory findings can be directly extrapolated to the human condition remains to be seen.

Summary

image The digestive system arises from the primitive endodermally lined gut tube, which is bounded cranially by the oropharyngeal membrane and caudally by the cloacal membrane. The gut is divided into foregut, midgut, and hindgut segments, with the midgut opening into the yolk sac. Specification of the various regions of the gut tract depends on a pattern set up by tightly regulated combinations of Hox genes. Development of virtually all parts of the gut depends on epithelial-mesenchymal interactions. Responding to such interactions, primordia of the respiratory system, the liver, the pancreas, and other digestive glands bud out from the original gut tube.

image The esophagus takes shape as a simple tubular structure between the pharynx and stomach. At one stage, the epithelium occludes the lumen of the esophagus; the lumen later recanalizes. The developing stomach is suspended from a dorsal and ventral mesogastrium. Through two types of rotation, the stomach attains its adult position. Common malformations of the stomach include the following: pyloric stenosis, which interferes with emptying of the stomach; and ectopic gastric mucosa, which can produce ulcers in unexpected locations.

image As they grow, the intestines form a hairpinlike loop that herniates into the body stalk. Further growth of the small intestine causes small intestinal loops to accumulate in the body stalk. As the intestines retract into the body cavity, they rotate around the superior mesenteric artery. This rotation results in the characteristic positioning of the colon around the small intestine in the abdominal cavity. During these changes in position, parts of the dorsal mesentery fuse with the peritoneal lining of the dorsal body wall. In the posterior part of the gut, the urorectal septum partitions the cloaca into the rectum and urogenital sinus.

image During its differentiation, the lining of the intestinal tract passes through phases of (1) epithelial proliferation, (2) cellular differentiation, and (3) biochemical and functional maturation. Similar to the esophagus, the small intestine goes through a period of occlusion of the lumen by the epithelium. At later stages, intestinal crypts located at the base of villi contain epithelial stem cells, which supply the entire intestinal epithelial surface with various epithelial cells.

image The intestinal tract is subject to a variety of malformations, including local stenosis, atresia, duplications, diverticula, and abnormal rotation. Incomplete resorption of the vitelline duct can give rise to Meckel’s diverticulum, vitelline duct ligaments, cysts, or fistulas. Omphalocele is the failure of the intestines to return to the body cavity from the body stalk. Aganglionic megacolon is caused by the failure of parasympathetic neurons to populate the distal part of the colon. Failure of the anal membrane to break down (imperforate anus) may be associated with fistulas connecting the digestive tract to various regions of the urogenital system.

image Digestive glands arise as epithelial diverticula from the gut. Their formation and further outgrowth are based on inductive interactions with the surrounding mesenchyme. The primordium of the liver arises in the septum transversum, but as it expands, it protrudes into the ventral mesentery. As it develops, the liver acquires the capacity to synthesize and secrete serum albumin and to store glycogen, among other biochemical functions. The pancreas grows out as dorsal and ventral pancreatic buds, which ultimately fuse to form a single pancreas. Within the pancreas, the epithelium forms exocrine components, which secrete digestive enzymes, and endocrine components (islets of Langerhans), which secrete insulin and glucagon.

image The respiratory system arises as a ventral outgrowth from the gut just caudal to the pharynx. Through epithelial-mesenchymal interactions, the tip of the respiratory diverticulum undergoes up to 23 sets of dichotomous branchings. Other interactions with the surrounding mesenchyme stabilize the tubular parts of the respiratory tract by inhibiting branching. Lung development goes through several stages: (1) the embryonic stage, (2) the pseudoglandular stage, (3) the canalicular stage, (4) the terminal sac stage, and (5) the postnatal stage.

image Important malformations of the respiratory tract include tracheoesophageal fistulas, which result in abnormal connections between the trachea and esophagus. Atresia of components of the respiratory system is rare, but anatomical variations in the morphology of the lungs are common. Respiratory distress syndrome, commonly seen in premature infants, is related to insufficiencies in the formation of pulmonary surfactant by type II alveolar cells.

image In its most basic condition, the intraembryonic coelom is separated into right and left components by the dorsal and ventral mesenteries, which suspend the gut. Except for the region of the stomach and liver, the ventral mesentery disappears. In the region of the heart, the dorsal mesocardium persists, and the ventral mesocardium disappears.

image The septum transversum divides the coelom into thoracic and abdominal regions, which are connected by pleural canals. The developing lungs grow into the pleural canals, which are partially delimited by paired pleuropericardial and pleuroperitoneal folds. The definitive diaphragm is formed from (1) the septum transversum, (2) the pleuroperitoneal folds, and (3) ingrowths from body wall mesenchyme.

image Quantitative deficiencies in ventral body wall tissue can result in abnormalities ranging from failure of sternal fusion to ectopia cordis in the thorax and omphalocele to gastroschisis or exstrophy of the bladder or both in the abdomen. Defects in the diaphragm are diaphragmatic hernias and can result in herniation of the intestines into the thoracic cavity.