Development of the lungs, thorax and respiratory diaphragm

Embryonic phase (0–7 postfertilization weeks: appearance of lung buds and main pulmonary arteries)

The endodermal epithelial lung buds grow dorsally, passing each side of the relatively smaller oesophagus, and bulge into the splanchnopleuric mesenchyme of the medial walls of the laterally situated pericardioperitoneal canals (see Figs 20.2 , 21.5 ). Proliferation of this splanchnopleuric coelomic epithelium, which is especially evident in stage 13 and decreases in stage 14, produces splanchnopleuric mesenchyme that becomes arranged in zones around the developing endoderm. This investing mesenchyme will eventually produce the supporting walls of the conducting airways, differentiating into the mucosal and submucosal laminae propriae (containing fibrocytes that synthesize the type I and II collagen, elastin, proteoglycans and glycosaminoglycans of the extracellular matrix) and smooth muscle cells.

Angiogenic mesenchyme is apparent around the primary bronchi at stage 15. It forms an extensive capillary network around each lung bud, receiving blood from the developing sixth aortic arch artery and draining it into an anastomosis connected to the dorsal surface of the left atrium in the mediastinal mesenchyme ( Ch. 13 ). The pulmonary arteries arise from the sixth aortic arch arteries and the pulmonary veins develop from a solitary channel derived from the dorsal mesocardium that establishes continuity with the vascular plexus formed in the mediastinal mesenchyme. The pulmonary veins become surrounded by myocardium to the level of the second bifurcation; the veins themselves expand and are incorporated into the roof of the left atrium, which means that cardiac muscle is found in the central branches of the pulmonary venous tree ( ).

After stage 15 the coelomic epithelium projecting into the pericardioperitoneal canals and covering the lung buds will follow a differentiation pathway to form the visceral pleura. Lobar or secondary bronchi can be seen at stage 16 and bronchopulmonary segments are present at stage 17 (see Fig. 21.3E ). Later stages of respiratory development involve the repeated division of the bronchial tree to form the subsegmental bronchi.

Pseudoglandular phase (5–17 postfertilization weeks: development of airways and blood vessels to the level of acinus)

By stage 17 the separation of the lungs from the digestive system is complete and the pseudoglandular phase of pulmonary development, including the development of the lower conducting airways and the appearance of the acinar structures, begins. During this period, virtually the complete branching structure of the future bronchial tree is laid down in 20 generations.

The growth and branching of the endoderm epithelium is controlled by the local investing splanchnopleuric mesenchyme. The airways begin to differentiate during this stage. Primitive ciliated cells appear at about postfertilization week 7, initially in the region of the membranous trachea, and in the cartilaginous region by postfertilization week 12; ciliogenesis is complete at birth in humans. The proximal airways develop basal cells from postfertilization week 11. Mucous glands develop by postfertilization week 12 and enlarge in the submucosa: secretory activity has been identified in the trachea at postfertilization week 14. The splanchnopleuric mesenchyme condenses around the epithelium, and from stages 16–17 it differentiates into connective tissue cell types and smooth muscle proximal to the tips of the developing airways. Smooth muscle cells are innervated from stage 23. Human tracheal smooth muscle cells have a fluctuating resting membrane potential associated with the spontaneous development of tone and peristalsis-like contractions of the airway of a myogenic nature, probably modified by neuro-humoral factors. Club cells develop in the peripheral airways during the pseudoglandular period and produce a surfactant apoprotein and a10-kDa protein (CC10) with immunomodulatory and anti-inflammatory activity. Cartilage also develops during this period, and is found in the airways in an adult distribution by postmenstrual week 24. By the end of this period, airway branching and pre-acinar vascular patterns are mature. A normal airway ‘template’ is essential for normal alveolar formation: a degree of pulmonary alveolar hypoplasia is inevitable in conditions characterized by an anomalous airway branching pattern (e.g. both ipsilateral and contralateral to a respiratory diaphragmatic hernia).

Further endothelial proliferation is seen in the pseudoglandular phase when capillary networks form around the developing lung buds; they will become capillary anastomoses around the future alveoli. The splanchnopleuric mesenchyme differentiates into myointimal cells and the tunica media of the developing blood vessels. Vimentin is expressed in the cells around developing vessels in the pseudoglandular stage, and is replaced by desmin in the saccular phase.

Canalicular phase (17–27 postmenstrual weeks: formation of respiratory airways and blood–gas barrier thins)

Two to three generations of branching occur during the canalicular phase, after which the amount of mesenchyme around the branching tips of the dividing respiratory tree decreases and the distal airspaces widen. At 23 postmenstrual weeks, longitudinal histological sections of the future distal regions show a sawtooth margin, which may indicate the site of further acini. Peripheral growth is accompanied by an increase in the capillary network around the distal airspace: in many places, the capillaries are in close contact with the respiratory cuboidal epithelium. The primitive endodermal cuboidal epithelial cells, previously the predominant cell in this part of the lung, differentiate into type I epithelial cells (pneumocytes) and type II epithelial cells (pneumocytes) containing lamellar bodies that form the intracellular storage bodies of surfactant. Type II pneumocytes are believed to be the stem cells of the alveolar epithelium. Apposition of the capillary networks to the thin type I pneumocytes and reduction of the interstitial tissue of the lung are prerequisites for future effective gas exchange. By postmenstrual week 24, the histological characteristics of the airways, including cartilage distribution, are the same as in the adult.

Saccular/alveolar phase (28 postmenstrual weeks–term: first appearance of alveoli in humans)

Thin-walled terminal saccules are apparent at the saccular stage and will become alveolar ducts as development proceeds. The expansion of the prospective respiratory airspaces that occurs during this period is accompanied by a further decrease in the amount of mesenchymal interstitial tissue, and the capillary networks become ever more closely opposed to the pneumocyte epithelium. Secondary crests develop from the invaginations of the saccule walls, although this may be an apparent movement when the saccule walls expand each side of a subjacent blood vessel. As an alveolar crest protrudes into a saccule, part of the capillary network becomes drawn into it. After the later expansion of the saccules on each side of the crest, a double capillary layer becomes annexed between what are now alveolar walls. During the saccular stage, elastin is deposited beneath the epithelium (an important step for future alveolar formation), and surfactant production, from type II pneumocytes, matures (essential for the survival of a preterm neonate).

Alveolar phase

An epithelial layer is usually supported by an underlying lamina propria, as in the conducting airways. The mutual support of two epithelial layers, a specialized epithelium and an endothelium, where both contribute to the generation of mutual basal laminal proteins, is very unusual and seen elsewhere only in the kidney glomerular basement membrane and in the brain (see Fig. 11.2 ). This close relationship develops during the alveolar phase between type I pneumocytes and pulmonary endothelium. A number of genes and growth factors are expressed during the formation and maturation of the type I pneumocyte:endothelium interface (alveolar–capillary membrane; blood–gas barrier) and in the final fibrocyte deposition of matrix molecules between developing alveoli. Retinoic acid regulates the expression of a number of growth signalling molecules, homeobox genes and lung epithelial genes. TGF-β, platelet- derived growth factor (PDGF) and fibroblast growth factor are involved in the final stages of conducting airway branching. TGFβ is also involved in septal formation and the induction of myofibroblasts which co-localize at ridges signalling the start of alveolar crest development. Myofibroblasts are found later at the tips of alveoli and play a particular role in production of elastin: their failure to produce elastin leads to a lack of alveoli. PDGF also stimulates myofibroblasts: PDGF-null mice fail to form secondary septa. Mechanical stretching of developing lung tissue stimulates human smooth muscle cell differentiation and increases tropoelastin content. Elastin gene expression, possibly modulated through retinoic acid, appears to play a pivotal role. (For further reading about factors controlling alveogenesis, see , , .)

Exactly when the saccular structure of the lung can be termed alveolar is not yet clear: estimates range from postmenstrual weeks 28 to 32. The formation of millions of alveoli is accomplished by a complex process of folding and division of type I pneumocytes. Mechanical stretch drives type II to type I cell differentiation. The existing walls of the alveoli become thinner as type I pneumocyte cells flatten, forming primitive alveoli. Septa (ridges) initially form where the type I pneumocytes meet subjacent capillaries and myofibroblasts. Subsequent areal expansion of type I pneumocytes, of more than ten times, and increased capillary development, combine to form new alveoli. Mouse embryo studies indicate that each type I pneumocyte may form the walls of multiple adjacent alveoli ( ). Vessels remain within the developing secondary septa whereas the myofibroblasts are temporary. The process of remodelling of the final capillary network between adjacent alveoli is not yet elucidated, however, it has been noted that type I pneumocytes secrete VEGF ( ).

The distal airspaces expand during late gestation and continue to do so after birth. The number of alveoli present at birth (variously estimated as between none to 5 x10 ⁷ ) is controversial; the number probably increases particularly rapidly over postmenstrual weeks 41–64 (postnatal months 0–6). This process is accompanied by fusion of adjacent capillary networks, so that shortly after birth there is an extensive double capillary net. Fusion of these layers is apparent at postnatal day 28, extensive at 1.5 years and probably complete by 5 years. The alveolar stage is now considered to continue beyond infancy and throughout childhood (whereas heterochronic differences in rat and mouse development show that alveolar formation is exclusively post-natal in these animals).

Development of the intrapulmonary vasculature and lymphatics

Two vascular circulations arise in the developing lungs, bronchial and pulmonary. Two or three bronchial arteries grow from the descending aorta from stages 21–22. They enter the lung at the hilum and branch, the airways acting as a template for their development. The vessels extend adjacent to the developing airways and eventually form sub-epithelial and adventitial plexuses that reach the distal portions of the bronchioles by birth. Bronchial arteries also supply the pleura near the hilum and form vasa vasorum in the adventitia of the large arteries and veins. The bronchial veins from the periphery of the lungs drain into the pulmonary veins. Postnatally, inflammatory lung conditions such as asthma, cystic fibrosis and bronchiectasis cause hypertrophy of the bronchial circulation and these vessels may bleed, sometimes giving rise to massive haemoptysis ( ). Some congenital thoracic anomalies have a systemic arterial blood supply.

The earliest pulmonary vessels form in the mesenchyme by vasculogenesis: the capillaries coalesce to form small blood vessels alongside the branching airways. Pulmonary arteries form adjacent to the conducting airways, joining the angiogenic mesenchyme of the sixth aortic arch arteries. Pulmonary veins develop from mediastinal mesenchyme in the dorsal mesocardium and become separated from the airways by the growth of alveoli and lymphatic vessels. By stage 15 a circulation is present from the aortic sac through a branching network of pulmonary arteries into capillary plexuses around the two lung buds, and returning via pulmonary veins to the developing left atrium. As each new distal airway forms in the mesenchyme, a new angiogenic plexus forms as a halo around it, and coalesces with the pulmonary vessel already alongside the proximal airway. In this way, addition of the newly formed tubules to the existing pulmonary vessels is sustained. Vessel formation occurs at least until the end of the pseudoglandular stage and has been shown to be controlled by production of VEGF from type I pneumocytes ( ). As the arteries increase in size they acquire a muscular wall, initially from the bronchial smooth muscle of the adjacent airway and then from the splanchnopleuric mesenchyme. The muscle layer is thick relative to the lumen, and this in part increases resistance to blood flow. Although only 10% of blood flow goes through the pulmonary circulation during intrauterine life, it is important for normal lung development. By postmenstrual week 20, the structure of the pulmonary vessels is the same as it is in the adult. Vascular innervation follows muscularization; the vasoactive peptides contained in these nerves are predominantly vasoconstrictor.

As the pulmonary veins enlarge, they become separated from the airways by the lymphatic vessels that lie within the mesenchyme. At around postmenstrual week 12, the peripheral veins develop a single layer of smooth muscle cells in their walls. This layer is derived exclusively from the splanchnopleuric mesenchyme and not from bronchial smooth muscle (as occurs in the arteries). The lumen of each vein is relatively large and the wall is relatively thin at all levels. Arteries and veins continue to develop in the canalicular phase, probably by angiogenesis (dividing cells are seen in the peripheral capillaries). The epithelium of the most peripheral conducting airways flattens when the developing capillaries come to lie immediately subjacent to it. (For a review of the development of the pulmonary circulation see ).

Lymphatic capillaries and vessels run within bronchovascular bundles. Lymphatic channels develop from outgrowths of vascular endothelial cells: the molecular trigger for lymphatic sprouting is VEGF-C and D, acting through the VEGF receptor 3 ( ). See for a review of lymphatic development.

Intrauterine maturation of the lungs

More than 30 different cell types found within the adult lung mature during development. Only a few important changes relevant to human disease will be highlighted here.

Pulmonary surfactant is produced by type II alveolar epithelial cells, and stored in lamellar bodies; surfactant production is under complex molecular control ( ). Surfactant proteins (Sp) B and C are surface active, whereas A and D are part of the collectin family of pattern recognition receptors. Surfactant is crucial for maintaining the functional integrity of alveoli: the main morbidity of extreme prematurity reflects surfactant deficiency causing neonatal respiratory distress syndrome (treatment has been transformed by the availability of exogenous surfactant). Mutations in the genes encoding SpB and SpC , ABCA3 (responsible for surfactant processing) and the transcription factor NKX2.1 (controlling SpB , SpC and ABCA3 expression) can all present in the neonatal period in full-term babies (notionally 40 postmenstrual weeks) with relentlessly progressive respiratory distress and diffuse ground glass shadowing on imaging studies ( ). Fetal breathing movements appear to be important in surfactant synthesis: the functional maturation of the surfactant system can be accelerated therapeutically by the administration of steroids to the mother with important benefits to the preterm baby ( ).

Normal lung development requires sufficient intrathoracic space, normal fetal breathing movements and sufficient amniotic fluid. There is evidence that perturbation of fetal lung blood flow also affects lung growth, even though most of the right ventricular output of the heart is shunted away from the lungs through the ductus arteriosus: pulmonary valve stenosis is associated with pulmonary hypoplasia. Although many fetal organs are able to grow to normal proportions even if they are in abnormal locations, this is not the case for the lungs. Lung growth becomes impaired by restricted expansion. Absence or impairment of fetal breathing movements, and defects affecting respiratory diaphragmatic activity, are all associated with pulmonary hypoplasia. Distension of the developing lung may provide a major stimulus to growth during normal development. It is believed that normal fetal breathing movements increase the lung volume and stimulate growth of the distal airspaces. Fetal breathing movements involve rhythmic activation of the respiratory diaphragm and the muscles of the upper respiratory tract. Even though such movements are necessarily very small compared with those seen after birth, because the fetal airways are filled with lung fluid, there is evidence that these phasic movements are important in the release of growth factors. Fetal breathing becomes more prominent in the second and third trimesters, by which time the fetus spends nearly one-third of the time breathing ( ). One effect of the absence of fetal breathing movements, for example in severe neuromuscular diseases of antenatal origin such as severe (type I) spinal muscular atrophy and spinal muscular atrophy with respiratory distress, is pulmonary hypoplasia ( ).

During development, the mucous glands of the trachea and bronchi secrete a chloride-rich fluid that usually passes up the respiratory tract to mix with the amniotic fluid secreted by the fetal kidneys. Amniotic fluid volume, pressure and, possibly, content significantly influence lung development by maintaining appropriate lung distension. Renal agenesis, severe congenital urinary obstruction and oligohydramnios (Potter’s syndrome; Potter’s sequence), leads to pulmonary hypoplasia at birth. In renal agenesis, reduced bronchial branching occurs as early as postmenstrual weeks 12–14 (i.e. at a time before amniotic fluid is produced by the metanephric kidneys), which suggests that a direct renal factor supports lung development. Later, the presence of amniotic fluid is necessary for normal fetal lung development. The fetal lung is a net fluid secretor, the output of fluid reaching as high as 5 ml/kg/hour shortly before birth. Most of the fluid produced within the lungs remains there because of the mechanical effect exerted by amniotic fluid pressure, and normally only a small amount of this fluid contributes to the amniotic fluid. The normal functioning of the kidneys regulates the volume and pressure of the lung airway fluid and may in turn provide the pressure needed for expansion and enlargement of the bronchial and pulmonary systems. Interestingly, obstruction to the fetal airway causes accelerated maturation of alveoli, a finding that has been used therapeutically in congenital diaphragmatic hernia, based on studies showing that intermittent inflation and deflation of an intra-tracheal balloon in a lamb model resulted in better lung growth and maturation ( ). This treatment is the subject of interventional trials in human fetuses ( https://www.totaltrial.eu/; ).

Two other cell types that participate in normal lung development, neuroendocrine cells and pulmonary lipofibroblasts, have been implicated in postnatal paediatric interstitial lung disease with early onset of respiratory distress ( ).

Neuroendocrine cells are found in the normal developing airway: neuroendocrine cell hyperplasia of infancy (NEHI) is characterized by persistence of bombesin-positive neuroendocrine cells. Abnormal glycogen-containing cells derived from lipofibroblasts ( ) result in pulmonary interstitial glycogenosis (PIG), which is not related to any of the systemic glycogen storage diseases. Both conditions tend to remit over time, and in neither case is it known whether the abnormal cells are of pathophysiological significance or merely markers of another process. These likely represent part of a spectrum of pulmonary dysmaturation syndromes, including alveolar capillary dysplasia-congenital alveolar dysplasia, see and .

Antenatal influences on intrauterine lung development

There is increasing evidence from epidemiological studies that antenatal factors have long-lasting effects on lung development. The earliest major effect is congenital respiratory diaphragmatic hernia (see below): this leads to both ipsilateral and contralateral abnormalities in branching patterns in the first 16 postmenstrual weeks that cannot be corrected subsequently, even by intrauterine tracheal balloon occlusion. Inevitably abnormal branching leads to abnormal alveolarization. The most important influence on later development is maternal smoking; there is evidence that maternal exposure to pollution is also an important cause of subsequent airflow obstruction ( ). Animal studies have confirmed that antenatal nicotine exposure leads to structural changes in the fetal lung ( ), and it should be remembered that e-cigarettes contain nicotine. Other factors that may be important include maternal hypertension in pregnancy and maternal antibiotic and paracetamol usage ( ).

Congenital anomalies of the trachea, bronchi and lungs

The concept of the lung as six ‘trees’ has been mentioned earlier; all except the systemic venous tree may contribute to a congenital thoracic malformation ( ). Congenital anomalies of the heart and great vessels, and of the chest and abdominal walls, may adversely impact lung development. It is somewhat artificial to describe airway malformations in isolation. The possibility of associated vascular abnormalities must always be considered and descriptions of what is seen clinically should be kept separate from speculations about the putative developmental origins of an anomaly.