The Embryonic Period (3–7 Weeks Postconception)

Relatively undifferentiated epithelial cells of the primitive foregut endoderm form tubules that invade the surrounding splanchnic mesoderm and undergo branching morphogenesis. This process requires highly controlled epithelial cell proliferation and migration to direct dichotomous branching of the respiratory tubules, which form the main-stem, lobar, and segmental bronchi of the primitive lung (see Table 2.1 ; Fig. 2.2 ). The respiratory epithelium remains relatively undifferentiated and is lined by columnar epithelium during this process. Proximally, the trachea and esophagus also separate into two distinct structures during this period. Experimental removal of mesenchymal tissue from the embryonic endoderm at this time arrests branching morphogenesis, demonstrating the critical role of mesenchyme in the formation of the respiratory tract. Interactions between epithelial and mesenchymal cells are mediated by a variety of signaling pathways that involve the binding of secreted peptides and extracellular matrix proteins to their respective cell surface receptors, which regulates gene transcription in differentiating lung cells. These epithelial-mesenchymal interactions involve both autocrine and paracrine signaling pathways that are critical for lung morphogenesis ( Fig. 2.3 ). Paracrine signaling pathways important for initial formation of the lung bud, as well as expansion and branching of the primitive respiratory tubules, include (1) FGF10/FGFR2, (2) sonic hedgehog (SHH/PTCH1), (3) transforming growth factor-beta (TGFβ/TGFβR2), (4) bone morphogenic protein B (BMP4/BMPR1b), (5) retinoic acid (RA/RARα, β, γ), (6) WNT (WNT2/2b, 7b, 5a and R-spondin with their receptors Frizzled and LRP5/6), and (7) the β-catenin signaling pathways. Nuclear transcription factors that are active in the primitive respiratory epithelium during this period include NKX2-1, FOXA2, GATA6, and SOX2. Likewise, transcription factors active in the mesenchyme at this time include (1) the HOX family of transcription factors (HOXA5, B3, B4); (2) the SMAD family of transcription factors (SMAD2, 3, 4), downstream transducers of the TGFβ/BMP signaling pathway; (3) the LEF/TCF family of transcription factors, downstream transducers of β-catenin, (4) the GLI-KRUPPEL family of transcription factors (GLI1, 2, 3), downstream transducers of SHH signaling; (5) the hedgehog-interacting protein, HHIP1, that binds SHH; and (6) FOXF1, another SHH target. Disruption of many of these transcription factors and signaling pathways in experimental animals causes impaired morphogenesis, resulting in laryngotracheal malformations, tracheoesophageal fistulas, esophageal and tracheal stenosis, esophageal atresia, defects in pulmonary lobe formation, pulmonary hypoplasia, or pulmonary ageneisis.

Examples of reciprocal signaling during lung morphogenesis. Examples of reciprocal signaling during lung morphogenesis. Paracrine and autocrine interactions between the respiratory epithelium and the adjacent mesenchyme are mediated by signaling peptides and their respective receptors, influencing cellular behaviors (e.g., proliferation, migration, apoptosis, extracellular matrix deposition) that are critical to lung formation. For example, fibroblast growth factor (FGF)10 is secreted by mesenchymal cells and binds to its receptor, FGFR2, on the surface of epithelial cells (paracrine signaling). Sonic hedgehog (SHH) is secreted by epithelial cells and binds to its receptor, PTCH1, on mesenchymal cells (paracrine signaling), while HHIP1 is upregulated by SHH in mesenchymal cells, secreted, and binds back to receptors on cells in the mesenchyme (autocrine signaling). Binding of SHH to mesenchymal cells activates the transcription factors, GLI1, GLI2, and GLI3, which, in turn, inhibit the expression of FGF10 (negative feedback loop). In contrast, the binding of HHIP1 to mesenchymal cells attenuates or limits the ability of SHH to inhibit FGF10 signaling. Together, these complex, interacting, signaling pathways control branching morphogenesis of the lung, differentially influencing bronchial tubule elongation (growth), arrest, and subdivision (lateral buds) into new tubules. BMP, Bone morphogenic protein; TGF-β, transforming growth factor-β; VEGF, vascular endothelial growth factor.

Although formation of the larger, more proximal, conducting airways, including segmental and subsegmental bronchi, is completed by 6 WPC, both epithelial and mesenchymal cells of the embryonic lung remain relatively undifferentiated. At this stage, trachea and bronchial tubules lack underlying cartilage, smooth muscle, or nerves; and the pulmonary and bronchial vessels are not well developed. Vascular connections with the right and left atria are established by a common cardiopulmonary vascular progenitor at the end of this period (6–7 WPC), creating the primitive pulmonary vascular bed. Human developmental anomalies occurring during this period of morphogenesis include laryngeal, tracheal, esophageal, and bronchial atresia; tracheoesophageal and bronchoesophageal fistulas; tracheal and bronchial stenosis; bronchogenic cysts; ectopic lobes; extrapulmonary sequestration; and pulmonary agenesis. Some of these congenital anomalies are associated with documented mutations in the genes involved in early lung development, such as GLI3 (tracheoesophageal fistula found in Pallister-Hall syndrome), FGFR2 (various laryngeal, esophageal, tracheal, and pulmonary anomalies found in Pfeiffer, Apert, or Crouzon syndromes), and SOX2 (esophageal atresia and tracheoesophageal fistula found in anophthalmia-esophageal-genital, or AEG, syndrome).

The Pseudoglandular Period (6–17 Weeks Postconception)

The pseudoglandular stage is so named because of the distinct glandular appearance of the lung from 6 to 17 WPC. During this period, the lung consists primarily of epithelial tubules surrounded by a relatively thick mesenchyme. Branching of the airways continues with formation of the terminal bronchioles and primitive acinar structures, which is completed by the end of this period (see Table 2.1 ; Fig. 2.2 ). During the pseudoglandular period, epithelial cell differentiation is increasingly apparent; and deposition of cellular glycogen and expression of genes expressed selectively in the distal respiratory epithelium are initiated. The surfactant proteins, SFTPB and SFTPC, are first detected in primitive type II AECs at 12–14 weeks of gestation. Tracheobronchial glands begin to form in the proximal conducting airways; and the airway epithelium is increasingly complex, with basal, mucous, ciliated, and nonciliated secretory cells being detected. Neuroendocrine cells, often forming clusters of cells, termed neuroepithelial bodies , express a variety of neuropeptides and transmitters (e.g., bombesin, calcitonin-related peptide, serotonin, and others) and are increasingly apparent along the bronchial and bronchiolar epithelium. Smooth muscle and cartilage are now observed adjacent to the conducting airways, and the pulmonary vascular system develops in close relationship to the bronchial and bronchiolar tubules between 9 and 12 WPC when smooth muscle actin and myosin can be detected in these vascular structures.

During this period, FGF10, BMP4, TGFβ, β-catenin, and the WNT signaling pathways continue to be important for branching morphogenesis, along with several other signaling peptides and growth factors, including members of the (1) FGF family (FGF1, FGF2, FGF7, FGF9, FGF18); (2) TGFβ family, such as the SPROUTYs (SPRY2, SPRY4), which antagonizes and limits FGF10 signaling, and LEFTY/NODAL, which regulates left-right patterning; (3) epithelial growth factor (EGF) and transforming growth factor alpha (TGFα) family, which stimulate cell proliferation and cytodifferentiation; (4) insulin growth factor (IGFI, IGFII) family, which facilitates signaling of other growth factors; (5) platelet derived growth factor (PDGFA, PDGFB) family, whose members are mitogens and chemoattractants for mesenchymal cells; and (6) vascular endothelial growth factor (VEGFA, VEGFC) family, which regulates vascular and lymphatic growth and patterning. Many of the nuclear transcription factors that were active during the embryonic period of morphogenesis continue to be important for lung development. Additional transcriptional factors important for specification and differentiation of the primitive lymphatic vessels in the mesenchyme at this time include (1) SOX18, (2) the paired-related homeobox gene, PRX1, (3) the divergent homeobox gene, HEX, and (4) the homeobox gene, PROX1.

A variety of congenital defects may arise during the pseudoglandular stage of lung development, including tracheomalacia and bronchomalacia, intralobar bronchopulmonary sequestration, congenital pulmonary airway malformations (formerly cystic adenomatoid malformations), acinar aplasia or dysplasia, alveolar capillary dysplasia with or without misalignment of the pulmonary veins, congenital pulmonary lymphangiectasia, and other pulmonary vascular malformations. The pleuroperitoneal cavity also closes early in the pseudoglandular period. Failure to close the pleural cavity, often accompanied by herniation of the abdominal contents into the chest (congenital diaphragmatic hernia), may cause pulmonary hypoplasia.

The Canalicular Period (16–26 Weeks Postconception)

The canalicular period is characterized by further growth and subdivision of the acinar tubules, rapid expansion of the pulmonary capillary bed, thinning of the pulmonary mesenchyme, and formation of the alveolar-capillary membrane (see Table 2.1 ; Fig. 2.2 ). By the end of this period, the terminal bronchioles have divided to form two or more respiratory bronchioles, and each of these has divided into multiple acinar tubules, forming the primitive pulmonary acini. Epithelial cell differentiation becomes increasingly complex and is especially apparent in the distal regions of the lung parenchyma. Bronchiolar cells express differentiated features, such as cilia, and secretory cells synthesize cell-specific secretory proteins, such as mucin and secretoglobin 1A1 (SCGB1A1). Cells lining the distal tubules assume cuboidal shapes and express increasing amounts of surfactant phospholipids and the associated surfactant proteins, surfactant protein A (SFTPA), SFTPB, and SFTPC. Lamellar bodies, composed of surfactant phospholipids and proteins, are seen in association with rich glycogen stores in the cuboidal pretype II AECs that line the distal acinar tubules. Some cells of the acinar tubules become squamous, acquiring features of typical type I AECs. Capillaries surround the distal acinar tubules and establish contact with the adjacent epithelium to form the primitive alveolar-capillary membrane, which ultimately forms the gas exchange region of the mature lung. By the end of the canalicular period in the human infant (26–28 WPC), gas exchange can be supported after birth, especially when surfactant is provided by administration of exogenous surfactant. Surfactant synthesis and parenchymal thinning can be accelerated by glucocorticoids at this time, which is why they are administered to mothers to prevent respiratory distress syndrome (RDS) after premature birth. Abnormalities of lung development that occur during the canalicular period include congenital alveolar dysplasia, alveolar capillary dysplasia, and pulmonary hypoplasia, and the latter is caused by (1) diaphragmatic hernia, (2) compression due to thoracic or abdominal masses, (3) prolonged rupture of membranes causing oligohydramnios, or (5) renal agenesis, in which amniotic fluid production is impaired. While postnatal gas exchange can be supported late in the canalicular stage, infants born during this period generally suffer severe complications related to decreased pulmonary surfactant, which causes RDS and bronchopulmonary dysplasia, and the latter is a complication secondary to the intensive care therapy and increasing survival of very preterm infants.

The Saccular (26–36 Weeks Postconception) and Alveolar Periods (36 Weeks Postconception Through Adolescence)

These periods of lung development are characterized by increased thinning of the respiratory epithelium and pulmonary mesenchyme, further growth and expansion of the pulmonary acini, and development of the distal alveolar capillary network (see Table 2.1 ; Fig. 2.2 ). Maturation of type II AECs occurs in association with increased synthesis of surfactant phospholipids, the surfactant proteins, SFTPA, SFTPB, and SFTPC, and the adenosine triphosphate (ATP)-binding cassette transporter, ABCA3, a phospholipid transporter important for lamellar body biogenesis. Squamous type I AECs line an ever-increasing proportion of the surface area of the distal lung. Adjacent interstitial capillaries become closely associated with the squamous type I AECs, decreasing the diffusion distance for oxygen and carbon dioxide between the alveolar space and capillary bed. Basal laminae of the epithelium and endothelium fuse to form the thin-walled alveolar-capillary membrane; and the interstitial tissue contains increasing amounts of extracellular matrix, including elastin and collagen. Secondary alveolar septa form, which partition the terminal saccules into true alveoli, greatly increasing the surface area of the lung available for gas exchange. In the human lung, the alveolar period begins near the time of birth and continues through the first decade of life, during which time, the lung grows primarily by septation, proliferation, and thinning of the alveolar walls, as well as by elongation and luminal enlargement of the conducting airways. Pulmonary arteries enlarge and elongate in close relationship to the increased growth of the lung. Pulmonary vascular resistance decreases, and considerable remodeling of the pulmonary vasculature and capillary bed continue during the postnatal period. Lung growth remains active until early adolescence, when the entire complement of approximately 300 million alveoli has been formed.

Signaling pathways that are critical for growth, differentiation, and maturation of the alveolar epithelium and capillary bed during these periods include the FGF, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VGEF), RA, BMP, WNT, β-catenin, and NOTCH signaling pathways. For example, targeted deletion of the FGF receptors, Fgfr3 and Fgfr4, blocks alveologenesis in mice; likewise, targeted deletion of Pdgfa interferes with myofibroblast proliferation and migration, resulting in failure of alveologenesis and postnatal alveolar simplification in mice. Notch2, expressed by type II AECs in the developing lung, induces PDGFA expression, activating platelet-derived growth factor receptor (PDGFR) signaling in alveolar myofibroblast progenitors, which is required for myofibroblast differentiation and normal alveologenesis. Recently, a WNT responsive epithelial cell has been identified that serves as a progenitor for both type I and type II AECs during perinatal alveologenesis.

Nuclear transcription factors found earlier in lung development, that is, FOXA2, NKX2-1, GATA6 and SOX2, continue to be important for maturation of the lung, influencing sacculation, alveolarization, vascularization, and cytodifferentiation of the peripheral lung. Transcription factors associated with cytodifferentiation during these periods include (1) FOXJ1 (ciliated cells), (2) MASH1 (or HASH1) and HES1 (neuroendocrine cells), (3) FOXA3 and SPDEF (mucus cells), (4) ETV5/ERM (type II AECs), and (5) HOPX (type I AECs). Morphogenesis and cytodifferentiation are further influenced by additional transcription factors expressed in the developing respiratory epithelium at this time, including (1) several ETS factors (ETV5/ERM, SPDEF, ELF3/5); (2) SOX genes (SOX-9, SOX11, SOX17); (3) nuclear factor of activated T cells/calcineurin-dependent signaling (NFATC); (4) nuclear factor-1 (NF-1); (5) CCAAT/enhancer binding protein alpha (CEBPα); (6) Krüppel-like factor 5 (KLF5); (7) transcription factors, GLI2/GLI3, SMAD3, FOXF1, POD1, and HOX (HOXA5, HOXB2-B5); all of which are expressed in the mesenchyme.

Transcription Factors and Gene Regulatory Networks

Transcription factors, which are nuclear proteins that bind to specific DNA nucleotide sequences (motifs) in regulatory regions throughout the genome, represent one major mode of transcriptional regulation. The binding of transcriptional factors to the cis-acting elements influences the activity of RNA polymerase II, which binds, in turn, to sequences near the transcription start site of target genes, initiating mRNA synthesis. Transcription factors may recruit additional proteins to the site of binding, modifying various chemical signatures on the DNA or chromatin (e.g., methylation, acetylation) that can continue to influence gene transcription long after the transcription factor itself leaves the region. Numerous families of transcription factors have been identified, and their activities are regulated by a variety of mechanisms, including posttranslational modification and interactions with other proteins or DNA, as well as by their ability to translocate or remain in the nucleus. Due to the large number of individual transcription factors and transcription-binding sites across the genome, these proteins interact combinatorally to form complex gene regulatory networks that direct patterns of gene transcription throughout development.

Although our knowledge of the various factors regulating gene transcription during lung development is still in its infancy, a number of transcription factors and signaling networks that play critical roles in lung morphogenesis have been identified. A general schematic of important transcriptional regulators of lung epithelial morphogenesis is seen in Figs. 2.5 and 2.6 . Lung morphogenesis depends on formation of definitive endoderm, which, in turn, signals from the splanchnic mesenchyme to initiate organogenesis along the foregut, forming thyroid, liver, pancreas, lung, and portions of the gastrointestinal tract. FOXA2, a member of the winged helix family of transcription factors, is a “pioneering” transcription factor that is known to play a critical role in committing progenitor cells of the endoderm to form the primitive foregut. Global loss of Foxa2 in mice results in catastrophic failure of development at the time of gastrulation. Loss of Foxa2 at later developmental timepoints, alone or in combination with the related transcription factor Foxa1, has revealed its essential role in specification of the pancreas and liver and formation of the lung epithelium, and it influences the expression of specific genes in the respiratory epithelium later in development. Conditional deletion of Foxa2 in the lung epithelium of mice highlights its ongoing role in both the proximal and distal lung epithelium. Conditional loss of Foxa2 prior to birth resulted in impaired maturation of type II cells, altered regulation of surfactant protein and phospholipid production, and death from RDS after birth. Loss of Foxa2 after birth caused goblet cell metaplasia, airspace enlargement, and inflammation during the postnatal period. Thus, FOXA2 plays a critical role in specification of foregut endoderm in the early embryo, and it is used again in the perinatal and postnatal period to direct surfactant production, alveolarization, postnatal lung function, and homeostasis (see Fig. 2.5 ).

A blueprint for lung epithelial cell development. Cytodifferentiation of the respiratory epithelium is controlled by transcriptional networks of genes that are expressed throughout lung development, in conjunction with autocrine and paracrine signaling pathways that control structural morphogenesis of the lung. Additional transcription factors are induced or repressed later in development and in the adult organ, to influence the differentiation of specific cell types. BMP, Bone morphogenic protein; FGF, fibroblast growth factor; SHH, sonic hedgehog.

Morphogenesis of the conducting and peripheral airway epithelium. The lung bud, visualized by expression of NKX2-1 (green), forms from ventral foregut endoderm on embryonic day 9 in the mouse. Endoderm is dependent upon the transcription factors FOXA2 and SOX17. Initiation of lung buds from the foregut endoderm is dependent upon Wnt2/Wnt2b signaling. At E12, bronchial tubes undergo branching morphogenesis marked by NKX2-1 (green), while endomucin staining (red) identifies endothelial cells. Cells destined to line the conducting airways express SOX2. In contrast, epithelial cells located at the tips of the branches expressing SOX9 and elevated levels of NKX2-1 are progenitors and cells that form the alveoli in the perinatal period. In the human lung, trachea, bronchi, and bronchioles (shown at 20 months of age) are lined primarily by ciliated, basal (stained by TP63), goblet (MUC5B), and other secretory cells, including neuroendocrine cells. In the normal postnatal human lung (shown at 3 years of age), alveoli are lined by squamous, Alveolar Type I that express AGER (green). In contrast, cuboidal Alveolar Type II cells express NKX2-1 (white). Endothelial cells in alveolar capillaries express PECAM (red). The interface between endothelial and alveolar type I cells creates the efficient gas exchange region that facilitates transport of oxygen and carbon dioxide.

The transcription factor NXK2-1 is the earliest known marker of lung formation. This 38-kd nuclear protein contains a homeodomain DNA-binding motif, and it is critical for formation of the lung and for regulation of a number of highly specific gene products produced selectively in the respiratory epithelium. NKX2-1 is also expressed in the thyroid and in specific regions of the developing central nervous system. Ablation of Nkx2-1 in mice impairs lung morphogenesis, resulting in hypoplastic lungs lined by a poorly differentiated respiratory epithelium and lacking gas exchange areas. Substitution of a mutant Nkx2-1 gene that lacks phosphorylation sites substantially rescues lung function in the Nkx2-1 knockout mouse. Nkx2-1 directly regulates the transcription of numerous downstream targets, including genes involved in surfactant homeostasis, fluid and electrolyte transport, host defense, and vasculogenesis. Just as important, NKX2-1 functions in concert with other transcription factors, including GATA6, the NF-1 family of transcription factors, STAT3, NFAT, FOXA1/FOXA2, and RARs to form a tightly integrated gene regulatory network to direct lung epithelial development and differentiation. For example, NKX2-1 gene transcription itself is modulated by the activity of FOXA2, which binds to the promoter enhancer region of the NKX2-1 gene, thus creating a transcriptional network. The stoichiometry, timing, and distinct combinations of transcription factors, as well as posttranscriptional modification of these transcription factors, are all critical factors in determining the precise transcriptional output at each stage of development (see Fig. 2.5 ), and our comprehensive understanding of how these signals are integrated on the systems-level is just beginning to emerge.

Cis-Regulatory Networks Controlling Gene Expression

Since the launch of the ENCODE (Encyclopedia of DNA elements) project, it has become increasingly clear that a significant fraction of the genome outside of protein-coding genes plays a key role in regulating gene expression. Cis-regulatory elements are a key class of regulatory noncoding DNA sequences, which act to regulate the transcription of a neighboring gene (although the distance of the regulated gene may be many megabases away). Promoters and enhancers are examples of cis-regulatory elements and characteristically contain many transcription factor binding motifs, integrating signals derived from the intricate gene regulatory networks described above. Promoter elements are essential for proper assembly of the key machinery necessary to initiate and establish transcription. Enhancers are “canonically defined as short (~100–1000 bp) noncoding DNA sequences that act to drive transcription independent of their relative distance, location, or orientation to their cognate promoter.” Consisting of clusters of transcription factor motifs, combinations of transcription factors interact with enhancer elements, recruit additional cofactor protein complexes, and act combinatorally to interact with the promoter of their regulated gene to modulate gene expression. Genome-wide association studies (GWAS) of many common disorders, including lung diseases such as interstitial pulmonary fibrosis, chronic obstructive pulmonary disease, and asthma, demonstrate that many of the genetic variants identified to date, which have been implicated in susceptibility to these diseases, fall within these noncoding regions of the genome. Thus, even though our understanding of the location and function of all cis-regulatory elements (referred to as the “cistrome”) critical for proper pulmonary development and function remains limited, a deeper understanding of this area of gene regulation will be essential to make progress in understanding the pathogenesis of a wide variety of common pulmonary disorders.

Epigenetic Mechanisms: DNA Methylation and Chromatin State

Genomic DNA does not exist in isolation within the cell, but genomic DNA instead is wrapped around proteins called histones in a very orderly fashion that consists of multiple levels of coiling and packing. Both the efficiency or “tightness” with which chromatin is compacted, as well as numerous associated chemical modifications to either the histone proteins or the DNA itself, are emerging as critical mechanisms by which gene expression is regulated during lung development. Methylation of cytosine at position 5 of the pyrimidine ring (largely on cytosines found in the CpG context) is the best studied of these “epigenetic” modifications, with roles in a large variety of biological processes, including X-chromosome inactivation, genomic imprinting, repression of transposable elements, aging, and cancer. In the context of regulating gene expression, methylation of a promoter region generally acts to repress expression of that gene. The promoters of active genes are typically unmethylated regions, or UMRs, whereas distal regulatory elements such as enhancers often have low levels of methylation, or LMRs. These methylation states have been observed to change dynamically during embryonic stem cell models of development and in organ systems such as the brain. Moreover, widespread differences in methylation are observed in a variety of lung diseases such as interstitial pulmonary fibrosis. The precise mechanisms, however, by which DNA methylation directs normal development and subsequent homeostasis of the adult lung remain poorly understood. Over 40 posttranslational modifications to the peptide tails of histones have been described, and they are associated with at least a dozen different chromatin “states.” For example, tri-methylation of lysine 4 on histone H3 (notated as H3K4me ) is found on active promoter regions, whereas colocalization of H3K4me and H3K27ac mark active enhancers. Other marks, such as H3K27me , are found on regions of silenced heterochromatin. Large-scale efforts such as the ENCODE Project and International Human Epigenome Consortium (IHEC) have generated thousands of datasets to create maps of the cis-regulatory landscape in dozens of tissue- and cell-types. It is thought that these various chemical marks act to integrate and retain the directions provided by the binding of transcription factors to chromatin throughout development, helping to “lock-in” specific cell fate decisions and provide cellular memory for these transient, but powerful, developmental cues long after they have passed. Dysregulation of chromatin state has recently been recognized to contribute to a wide variety of human disorders from congenital heart disease to lung adenocarcinoma. However, whether these posttranslational modifications of histones are merely associated with various regulatory states or play direct and causal roles in regulation of gene expression is just beginning to be explored, particularly in the context of lung development.

Epigenetic Mechanisms: Chromatin Topology/3D Structure

It has long been recognized that many cis-regulatory elements such as enhancers act at great distances, often thousands or even millions of bases-pairs, to regulate the expression of their target genes. Three-dimensional folding of chromatin is essential to mediate these interactions, bringing distant enhancer elements with bound transcription factors and coactivator protein complexes into physical proximity to their target gene promoters. Chromatin conformation capture (3C)-based technologies have revealed that the genome is organized into discrete physical domains, called topological-associated domains, or TADs, often forming loops of coregulated genes. Other regions of chromatin have been observed to associate with the nuclear membrane (laminin-associated domains or LADs), which typically consist of genes that are silenced or only expressed at low levels, that are thought to help further partition chromatin within the nucleus. Examples of local or global dysregulation of chromatin topology have been reported in human diseases, ranging from blood diseases to Down syndrome, and forced chromatin looping has even been used as a treatment strategy for hemoglobinopathies. Future research in this area is likely to have significant implications for our understanding of lung development and pulmonary diseases.

Noncoding RNA

With the advent of widespread availability of next-generation sequencing-based analysis of RNA, it has become clear that many regions of the genome (as much as 60%–80%) are transcribed into RNA, even if many of these RNAs do not progress to make a protein. The transcripts are called “non-coding RNAs” (ncRNA) and are typically divided into two broad categories based on their size: transcripts greater than 200 bp are referred to as “long non-coding RNAs” (lncRNAs), and their smaller counterparts (<200 bp) are referred to as small ncRNAs. A wide variety of small ncRNAs have been described, including microRNAs (miRNAs), small nuclear RNAs (snRNAs), and Piwi-interacting RNAs (piRNAs). A number of miRNAs have been specifically implicated in lung development, including miR-17-92, miR20a, miR-302-367, miR106a-106b, and miR-34/449. For example, loss of the miR-17-92 cluster leads to pulmonary hypoplasia due to decreased cellular proliferation, while overexpression enhances proliferation and differentiation of the lung epithelium. More broadly, loss of the Dicer complex, a key component of the machinery necessary to process longer, immature RNA transcripts (pri- or pre-miRNAs) into their mature miRNA form, results in severe defects in branching morphogenesis, and germline mutations in the DICER gene have been identified in familial cases of the rare pediatric lung malignancy pleuropulmonary blastoma. Long noncoding RNAs also exhibit dynamic expression patterns throughout development, and new studies have started to unravel important contributions to lung organogenesis. An important subset of lncRNAs appears to be genomically positioned near critical transcription factors, including Nkx2-1, Foxa2, and Foxf1. Loss of the lncRNA, Nanci, adjacent to the transcription factor, Nkx2-1, results in a reduction in NKX2-1 expression and recapitulates aspects of the Nkx2-1 haploinsufficiency phenotype, while loss of the lncRNA, Fendrr, adjacent to Foxf1 results in a phenotype similar to the human lung developmental disorder alveolar capillary dysplasia. Other lncRNAs, such as MALAT1, have been implicated in the pathogenesis of multiple types of malignancy, including lung cancer, but global loss of MALAT1 demonstrated that this lncRNA is not necessary for normal pulmonary development. Although individual elements may be dispensable for normal development, it is likely that lncRNAs, similar to miRNAs and many of the other gene regulatory mechanisms discussed earlier, act in highly redundant, coordinated systems to fine-tune gene expression throughout life and will play key roles in response to physiologic stress and disease.

Receptor-Mediated Signal Transduction

Receptor-mediated signaling is well recognized as a fundamental mechanism for transducing extracellular information. Such signals are initiated by the occupancy of membrane-associated receptors capable of transducing additional signals (known as secondary messengers), such as cyclic adenosine monophosphate, calcium, and inositide phosphates, which influence the activity and function of intracellular proteins (e.g., kinases, phosphatases, and proteases). These proteins, in turn, may alter the abundance of transcription factors, the activity of ion channels, or changes in membrane permeability, which subsequently modify cellular behaviors. Receptor-mediated signal transduction, induced by ligand-receptor binding, mediates endocrine, paracrine, and autocrine interactions on which cell differentiation and organogenesis depend. For example, signaling peptides and their receptors, such as FGF, SHH, WNT, BMP, VEGF, PDGF, and NOTCH have been implicated in organogenesis of many organs, including the lung.

Gradients of Signaling Molecules and Localization of Receptor Molecules

Chemical gradients within tissues, and their interactions with membrane receptors located at distinct sites within the organ, can provide critical information during organogenesis. Polarized cells have basal, lateral, and apical surfaces with distinct subsets of signaling molecules (receptors) that allow the cell to respond in unique ways to focal concentrations of regulatory molecules. Secreted ligands (e.g., FGFs, TGFβ/BMPs, WNTs, SHH, and HHIP1) are further influenced by binding of the ligand to basement membranes or to proteoglycans in the extracellular matrix. Spatial information is established by gradients of these signaling molecules and by the presence and abundance of receptors at specific cellular sites. Such systems provide positional information to the cell, which influences its behavior (e.g., shape, movement, proliferation, differentiation, and polarized transport).