Organogenesis is the development of a living organism's organs. Lung organogenesis therefore, is development of the respiratory system. Lung organogenesis is a complex process which can be classified into five separate stages. Developmental timing is fundamental in lung organogenesis, with the final stages of lung development taking place in late pregnancy. Consequently, infants that are born prematurely are often left with sever respiratory problems as a result of them being delivered before the final lung developmental stages have taken place. Respiratory Distress Syndrome for example, usually results when the lungs have not reached the stages of development that features that production of surfactant. Surfactant is a lipid-soluble compound which helps the lungs inflates with air, reduces surface tension and prevents the air-sacs collapsing. Neonates with Respiratory Distress Syndrome experience difficulty with breathing and may be confined to respiratory machinery in order to attempt to sustain life (Pickerd, 2009).
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Much of medical research concerning lung development has focussed on the genetic stimuli which control the process. However, genetic manipulation is a complex process. Attempting to rectify the genetics of development as a therapy for developmental lung diseases is a time-consuming and costly process. In recent years therefore, work has focussed on the mechanical factors that govern lung organogenesis. In theory, if the mechanical stimuli involved in organogenesis can be controlled or manipulated, then highly efficient therapeutics can be developed to remedy the devastating lung pathologies which result from incomplete lung development. This essay will discuss in the detail the roles of three dominant mechanical factors controlling lung organogenesis; lung fluid secretion, airway smooth muscle contractions and calcium.
The stages of Lung Development
Lung organogenesis can be divided into five stages: the embryonic, pseudoglandular, canicular, saccular and alveolar phases. Figure one features a simple diagrammatic representation of the five stages and where they appear in foetal development and the main features that appear in each phase. The stages are explained in further detail in table one and histological detail in figure 2.
Fig 1: Lung development can be classified into five distinct stages described in detail in table one.
Fig 2: Stages of lung development: this diagram shows the histological changes during the five stages of lung development.
Whittsett, J. Taken from:
Stage of Lung Development at x weeks gestation
Features of lung development phase
Embryonic phase (3-7 weeks)
Initial budding and branching of the lung buds from the primitive foregut. Bronchi formed.
Pseudoglandular phase (7-16 weeks)
Further branching of the duct system. Bronchioles form and branch to produce terminal bronchioles.
Canalicular phase (16-24 weeks)
The distal endoderm begins to form terminal sacs and vascularisation begins. Respiratory Bronchioles formed.
Saccular phase (24-36 weeks)
The mesenchyme thins, the number of terminal sacs increase. Endoderm differentiates into type i and type ii pneumocytes. Alveolar ducts formed.
Alveolar phase (36 weeks - term/adult)
Maturation of the lung indicated by the appearance of fully mature alveoli begins at 36 weeks, though new alveoli will continue to form for approximately three years after birth.
Table 1: Stages of Lung Development (Whitsett et al. 2004).
The mature lung develops from the primitive foregut (the anterior part of the alimentary canal). Envagination of the foregut endoderm leads to the formation of the primordial trachea which splits into two primordial lung buds. A highly patterned and tightly controlled process of budding and branching takes place throughout the initial stages of lung organogenesis. This process can be referred to as branching morphogenesis and results in the formation of the airways (Warburton, 2008). Aside from branching morphogenesis, another important feature in the lung developmental process is the production of lung liquid. The pulmonary epithelium is responsible for producing the lung fluid found inside the immature lung via chloride secretion. Lung fluid secretion is seen to be responsible for the expansion of the developing lungs. Lung morphogenesis therefore appears to be a process dependent upon a finely controlled program which strikes a balance between branching morphogenesis and fluid secretion to ultimately result in a mature pulmonary system with lungs capable of gas exchange within minutes of birth (Wilson, 2007).
Mechanical Factors involved in Lung Organogenesis
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Extensive research has been carried out to identify the genetic stimuli in the control of lung organogenesis such as the growth factor, FGF10 (fibroblast growth factor). FGF10 has been shown to induce epithelial branching in in vitro lung culture experiments. FGF10 knock-out mutants demonstrate lung agenesis (Jesudason, 2009). Aside, from these genetic factors, there is also interest in the mechanical factors involved in the process which complement the genetic stimuli and are vital to lung organogenesis. The importance of mechanical factors involved in lung organogenesis has actually been established for many years. Experiments have indicated that the developing foetus makes episodic breathing movements from as early as the first trimester. These breathing movements increase in frequency as pregnancy progresses and are believed to provide forces which contribute to the expansion of the growing lung. Experiments from the 1970s demonstrated that the foetal lung also actively secretes fluid into the lung tissue lumen which creates a transplumonary pressure in the developing airways and airspaces further contributing to lung expansion. However, it is really recent work that has demonstrated a need for another mechanical factor involved in lung organogenesis: calcium ions. Experiments involving manipulation of the calcium concentration of the extracellular environment have been shown to have marked effects on the development of the foetal respiratory system (Sanchez-Esteban, 2002).
Lung Fluid secretion
Arguably one of the most important mechanical factors that controls lung organogenesis in the secretion of foetal lung fluid. A deficiency of lung fluid is associated with lung hypoplasia and is presented in many clinical conditions such as congenital diaphragmatic hypoplasia. Lung fluid is seen to be present in the developing foetus from early lung development (Olver and Strang 1974). It was initially assumed that lung fluid was merely amniotic fluid that the developing foetus had inhaled. However, this view was challenged in 1941 by Potter and Bohlender who realised that the lung tissues that were distal to the congenital airway obstructions became distended with fluid. This implied that the production of the fluid must take place within the lung tissues themselves.
Mechanism of Lung fluid Secretion
Results from lung-fluid studies in rats demonstrated that Lung fluid secretion occurs due to the relatively impermeable foetal lung epithelium actively secreting chloride ions into the lung lumen which drives fluid secretion. This process is mediated via a process of secondary active transport via a sodium-potassium-2 chloride co-transporter (NKCC1) (Helve et al. 2009). Experiments using loop diuretics or NKCC1 inhibitors produced results of slow inhibition of foetal lung liquid secretion in murine models. However, Experiments involving NKCC1 knock-out mice in late gestation showed little change in lung fluid secretion compared to control mice. It was concluded therefore, that while NKCC1 does play a rate-limiting role in Cl- transport and consequently lung fluid secretion, other transporters must be present to sustain foetal lung liquid secretion in NKCC1 knock-out models (Gillie et al. 2001)
Physiological Experiments involving lung fluid secretion
Lung fluid secretion plays a vital physiological role in foetal lung developing. The fluid is secreted against the resistance provided by the larynx. Consequently, a distending pressure is established. This created intrapulmonary pressure is vital for keeping the developing pulmonary structures open (Wilson et al. 2007). In ovine foetus experiments, lung fluid was seen to be secreted at a rate of 2ml/kg/h at mid-gestation which gradually increased to 5ml/kg/h towards term. However, the rate and volume of fluid production decreased as the foetus approached term in preparation for postnatal adaptation (Joshi and Kotecha 2007).
One of the key experiments in the identification of the physiological role of lung fluid secretion was performed by Alcorn et al. 1977. The procedure involved continuous in utero tracheal ligation or drainage of lung fluid in the ovine foetuses over a period of 21-28 days. Both procedures resulted in abnormal lung growth. The lungs that were developed in the ligated animal model were hyperplastic. Correspondingly, the lungs that resulted in the drained animal model were hypoplastic. The appearance and differentiation of type ii alveolar cells (related to surfactant production) was reduced in ligated lungs but increased in drained lungs. The results from this experiment indicated in general that lung fluid secretion and therefore lung liquid volume is a mechanical factor in lung mass, formation of alveoli and alveolar cell epithelium maturation (Alcorn et al. 1977).
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Fig 3: A follow- up to Alcorns et al. 1977 experiment involving tracheal ligation and drainage showing experimental apparatus. Following ligature
of the left bronchus, the upper tracheal
catheter is used to drain foetal lung liquid in the right lung
(Moessinger et al. 1990).
Many follow up experiments similar to Alcorn et al. 1977 have been attempted. Flageole et al. 1998 describe a project involving tracheal obstruction of foetal lamb lungs by plugging. Results were consistent with Alcorn et al's suggesting a functional role for fluid secretion in producing hyperplastic lungs. However, plugged lungs also resulted in a decrease in type ii pneumocytes and consequently surfactant production. Subsequently, Flageole's study also featured unplugging of the tracheal obstruction before the delivery of the foetus to determine whether this would lead to recovery of type ii pneumocytes and hence maturity of the lung. It was determined that releasing the developing lungs from tracheal obstruction did result in recovery of the type ii pneumocytyes with the maintained advantage of increased lung distension. Flageole et al proposed that type i cells differentiate to type ii surfactant producing cells only when stimulus for lung growth and distension is removed. Tracheal obstruction therefore maintains type ii cells in their precursor state and hence, surfactant production is limited (Flageole et al. 1998).
While the successful results of experimental studies using tracheal ligation as a method of increasing lung fluid secretion and stimulating distension of the lungs have been established for many years, trials on human foetuses have only recently taken place. Harrison et al. 1997 describes a prospective trial of surgical correction of congenital diaphragmatic hernia to reverse pulmonary hypoplasia. Four foetuses with diagnosed CDH underwent open foetal surgery for repair. Unfortunately, there was no significant difference in the survival between foetuses that had been treated and control foetuses. Results indicated that foetuses that had undergone surgery were more likely to be born prematurely. From the results of this trial, it could be concluded that while open-foetal surgery is a feasible option, in the case of CDH, it does not improve survival rates compared with standard post-natal treatment (Harrison et al.1997).
Lung Fluid removal
At birth, the newborn must immediately commence breathing. Pre-term infants often possess retention of lung fluid which leads to a variety of lung pathologies. This is because In order for this pulmonary switch to occur, lung fluid must be removed. This theory has been proved by early animal experiments which show a significant reduction in lung fluid content after birth. Foetal breathing movements (mentioned above) are seen to be vital in clearing some of this fluid, but most of the liquid is cleared by another mechanism. An experiment involving the addition of amiloride to the trachea of guinea pigs lead to results of respiratory distress in the animal indicated the importance of amiloride-sensitive sodium channels in lung-fluid clearance. Consequently, electrogenic sodium ion transport must be responsible for the fluid clearance. The sodium channel ENaC has been found to be rate-limiting for the process of lung-fluid absorption across the epithelium. This theory was further supported after experiments using alpha-ENaC knock-out mice which resulted in lethal phenotype production where the mice were unable to clear their lungs of prenatal fluid and died (Helve et al. 2007).
In the airway epithelium, the ENaC consists of three subunits: alpha, beta, gamma. Western blot studies have confirmed the presence of all three subunits of ENaC in rat and foetal fat lung. ENaC expression appears to occur late in gestation and seems to be complimentary to production of vasopressin and adrenaline. It can thus be concluded that these hormones influence ENaC expression. Therefore, there is potential for therapeutic intervention for the removal if lung-liquid fluid that has been retained by the pre-term infant hence enhancing the chances of survival (Jain and Eaton 2006).
Airway Smooth Muscle: Appendix or Architect of the developing Lung?
There has been much debate about whether airway smooth muscle could contribute to mechanical stimuli in lung development. From a functional perspective, there seems no need for airway smooth muscle. Seow and Fredberg commented that there is no known disease associated with the loss of airway smooth muscle (Seow, 2001) However, recent work has indicated a mechanical role for airway smooth muscle in pulmonary development and many interesting connections have been made between airway smooth muscle peristalsis and some of the other mechanical factors in lung development (Mitzner 2004).
From the earliest stages of lung development, experiments have demonstrated that the developing foetal lung is vigorously active. This has been observed in airway explants from avian, rodent and humans. Peristaltic waves of airway smooth muscle (ASM) are seen to propagate rhythmically throughout the developing airway tree. This results in the propulsion of lung fluid. The growing end buds of the lungs (where ASM is at its minimum) experience distension and relaxation. It has been hypothesized that rhythmic stretch-relaxation cycles modulate gene expression and therefore the growth of certain cell types, such as the pulmonary epithelium. Jesudason et al. suggested that ASM peristalsis could be an important bridge between biomechanical and biochemical or genetic stimuli to lung development and could therefore provide promise for strategies to treat developmental lung diseases which result in lung hypoplasia. The data showed that embryonic rodent explants equivalent for 5 weeks human gestation demonstrate powerful ASM peristalsis. It appeared that ASM contraction arises throughout the proximal airways and shows a preference for the right lung. To ascertain the stimulus for the peristaltic activity, calcium imaging and c-kit immunoreactivity techniques were undertaken. The results indicated that ASM peristalsis was underpinned by spontaneous propagating calcium ion waves. The purpose of ASM contractility was then explored using an technique that alternated inhibition of either peristalsis or lung growth itself. The results from these experiments suggested that growth and peristalsis are coupled. The emergence of growth factor FGF10 was followed throughout the experiment. FGF10 was seen to emergence when a threshold frequency of ASM peristalsis was reached. The experiments indicated therefore that ASM regulates FGF10 or vice versa and that both these mechanisms could be calcium-regulated (Jesudason, 2009).
Airway Smooth Muscle peristalsis. The figure showing rapid sequence photography of the whole lung aims to demonstrate the contractions of ASM. On the left hand panels: the fluid flux within the epithelial lumen (dark) of the cultured lung is visualized due to the peristaltic propulsion of debris (arrowed).
The right hand panels show the airway contractions (boxed) during a wave of airway peristalsis.
Intracellular calcium and ASM peristalsis
Jesudason undertook further experiments in order to underpin the role of ASM pulmonary development. The investigation featured the L-type calcium antagonist nifedipine. Nifedipine follows a mechanism of action which therefore reduces intracellular calcium ion concentration. Results from these experiments on lung explants showed that nifedipine addition halts airway peristalsis and causes a decrease in lung size. Branching morphogenesis however, was unaffected. Alternate experiments using cyclopiazonic acid; a compound which blocks sarcoplasmic calcium reuptake showed halted airway contractility and reduced branching morphogenesis. Jesudason et al. therefore hypothesized that cyclopiazonic acid may act on ASM progenitors to halt smooth muscle actin expression and consequently the expression of FGF10, the branching morphogenesis marker. It was also theorized that FGF10 and ASM coupling may operate as a feedback mechanism to maintain the intraluminal pressure in the developing lung. There is extensive evidence that loss of this pressure is associated with lung hypoplasia. As the lung lumen increases in volume, intraluminal pressure maintenance would require increased lung liquid production as well as further wall tension. In theory, both these stimuli could be supplied by increasing ASM contractility. Early studies suggest that ASM peristalsis could also be associated with chloride efflux from the muscle itself. The chloride ions may be taken up at the adjacent basolateral epithelial surface to drive fluid production (described above). Therefore, ASM peristalsis may not only influence the pressure that propels the lung liquid and causes inflation of the lungs, but also modulate fluid production which further contributes to lung distension. On the other hand, ASM peristalsis does not influence lung branching morphogenesis.
Fig. 5. Proposed regulation of lung growth by ASM activity via feedback regulation of intraluminal pressure in the expanding lung. (Jesudason 2006).
Extracellular Calcium ions: mechanical regulators of the developing lung?
Intracellular calcium has long been known to have a fundamental role in processes such as muscle contraction, but extracellular calcium has emerged as an important component of many signalling mechanisms. Recently, research has identified that extracellular calcium may provide a mechanical role in lung organogenesis with the foetal lung responding to different concentrations of extracellular calcium.
A key discovery in medical research was that the concentrations of extracellular calcium in the developing foetus and the newborn differ. Calcium ion concentration in the foetus is around 1.7mM, a higher value than the concentration of calcium ions in the newborn which is around 1.0-1.3mM. (Finney et al. 2008). Lung development therefore, must take place in a hypercalcaemic environment. It has recently become clear that mammalian cells can respond to changes in the extracellular environment, such as variations in the concentration levels of ions. A specialised G-protein Coupled Receptor (GCPR); the calcium-sensing receptor was discovered to sense and respond to extracellular calcium concentrations (Kovacs et al. 1998). The calcium-sensing receptor (CaR) appears to be the master regulator of the adult serum calcium homeostatic system. Work by Brown and Macleod demonstrated that this system is activated via a phospholipase-c signalling pathway. CaR cloning studies have demonstrated that the receptor is highly expressed in organs involved in extracellular free ionized calcium homeostasis. However, until recently, previous work was unable to detect CaR expression in the adult lung. Doctor Riccardi and Professor Kemp at Cardiff University therefore began work to attempt to detect CaR expression prenatally in order to test the involvement of the CaR in lung organogenesis (Finney et al. 2008).
Methods and Results
Finney et al. isolated and purified RNA from mouse lung samples aged between E11.5-E18.5 and postnatal day 10. Polymerase Chain Reaction was then carried out using specific primers for CaR sequences. The results demonstrated the changing expression of the CaR over the embryonic period. CaR expression was first detected in the mouse lung epithelium at 10.5E using immunohistochemistry technique. Expression of the receptor remained exclusively in the epithelium until a peak at day 12.5. Epithelial expression of CaR then decreased until E18.5 where no expression of the receptor was recorded. As development continued, CaR expression began to appear in the mesenchyme. In neonate and adult lung tissue, CaR expression was totally absent. As expression of CaR was at its highest at E12.5, E12.5 samples were incubated in the presence of varying calcium concentrations (as shown in fig.2.). Branching morphogenesis (quantified using time-lapse images from a dissecting microscope linked to a digital camera) was maximal at the physiological adult calcium concentration of 1.05mM. In contrast, at calcium concentrations of 1.7mM (similar to those of the foetus) branching morphogenesis was decreased. This research was further supported by the fact that the presence of a CaR positive modulator produced suppressive effects on branching morphogenesis. Inhibitors of ip3 and phospholipase C (signalling molecules produced after the activation of the G protein) reversed this suppressive effect.
Fig 6. Effect of 1.05 mm (upper panel) or 1.7 mm (lower panel) [Ca2+] o on branching by E12.5 at t=0, 24 and 48 hours.
Significant increase in branching shown at 24 and 48 hours for 1.05mM [Ca2+] o compared to 1.7mM [Ca2+] o
It could therefore be concluded, that a threshold extracellular calcium ion concentration (similar to foetal E12.5 calcium concentration) must be established to activate or agonise the calcium-sensing receptor. Once activated, the receptor induces a signalling cascade which eventually results in the cessation of lung branching morphogenesis.
Embryonic lung fluid secretion is driven by secondary active chloride transport and therefore results in a negative transepithelial potential difference (Wilson, 2007) the activation of the calcium-sensing receptor with a calcium agonist appeared to promote the generation of a more negative epithelial potential difference. Therefore, it could perhaps be concluded that CaR activation stimulates transepithelial electrogenic transport and hence fluid secretion in the developing lungs despite decreasing branching morphogenesis. The reduction of lung branching morphogenesis could be a consequence of either decreased proliferation or increased apoptosis which accompanies a high level of expansion and increase in luminal volume in the developing lung. Consequently, Finney et al.'s results suggest that CaR activation could be involved in both the regulation of peripheral tubule formation and airway expansion.
The work provides a potential for the synthesis of pharmacological manipulators of the CaR which could rescue hypoplastic/hyperplastic lung conditions in premature newborns. Calcium-modulating drugs already exist, so one of these could be adapted and fast-track the drug development process to produce a compound to target CaR in the lungs to develop a newborn's lungs after birth (Finney, 2008)
Linking lung pathologies to mechanical stimuli
Many developmental lung diseases can be classed into two categories: hypoplastic lungs (smaller than normal lungs) or hyperplastic lungs (enlarged lungs). Hypoplastic lung conditions such as congential diaphragmatic hernia appear to result from a lack of lung liquid secretion, or propulsion. Therefore, in hypoplastic lung disease, it is hypothesised that ASM peristalsis is deficient or lung-fluid production itself is deficient. Hypoplastic lungs often have normal branching morphogenesis, suggesting that the branching of the lung bud is not related to these mechanical stimuli (Wright 2006). Pulmonary hyperplasia on the other hand is a rarer but critical condition which leads to severe pulmonary hypertension. Malfunctions that cause upper airway obstruction such as laryngeal atresia cause an increase in lung liquid secretion resulting in lungs which demonstrate normal histology but are vastly increased in weight and volume compared to regular post-natal lungs (Silver, 1988)
Lung organogenesis is a complex process that can be separated into five phases. Each stage is controlled molecularly by a series of transcription factors, growth factors and polypeptides. However, it has been suggested that mechanical or physical factors are also fundamental in organogenesis and complement the genetic control of the process. The most dominant physical factor in lung development is the chloride-driven secretion of the lung fluid. The lung liquid provides forces which lead to a positive intraluminal pressure which leads to distension and inflation of the growing lung. Propulsion of lung liquid leading to this pressure appears to be influenced by foetal breathing movements and airway smooth muscle contractions which appear to be regulated by intracellular calcium. However, when airway smooth muscle contractions are at their most frequent, branching morphogenesis of the developing lung appears to be suppressed. Results of experiments with varying extracellular calcium concentrations demonstrate this antagonistic relationship between lung fluid secretion or propulsion and branching morphogenesis. Research has isolated a calcium-sensing receptor that is expressed towards the beginning of gestation which once activated suppresses branching morphogenesis but appears to positively modulate fluid secretion. Expression of this receptor decreases towards the end of gestation and branching morphogenesis is restored. Consequently, lung development appears to be a balance between two processes; branching morphogenesis and fluid secretion. These events can be controlled by the mechanical factors described above. Infants that are born prematurely have often not completed one of these two processes, resulting in lung pathology. An increase in fluid secretion may lead to hyperplastic lungs with a lack of branching. An increase in branching morphogenesis could result in hypoplastic yet mature lungs. Consequently, with knowledge of the mechanical factors which influence lung development, there is promise for manipulators of these physical stimuli to be developed which could rescue the hypoplastic or hyperplastic lungs of a premature newborn.