Developmental pathways underlying lung development and congenital lung disorders

cells Review Developmental Pathways Underlying Lung Development and Congenital Lung Disorders Inês Caldeira 1,2,† , Hugo Fernandes-Silva 1,2,3,† , Daniela Machado-Costa 1,2, Jorge Correia-Pinto 1,2,4 and Rute Silva Moura 1,2,*   Citation: Caldeira, I.; Fernandes-Silva, H.; Machado-Costa, D.; Correia-Pinto, J.; Moura, R.S. Developmental Pathways Underlying Lung Development and Congenital Lung Disorders. Cells 2021,10, 2987. https://doi.org/ 10.3390/cells10112987 Academic Editors: Anindita Das and Arun Samidurai Received: 16 August 2021 Accepted: 29 October 2021 Published: 2 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal; [email protected] (I.C.); [email protected] (H.F.-S.); [email protected] (D.M.-C.); [email protected] (J.C.-P.) 2ICVS/3B’s—PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal 3PhDOC PhD Program, ICVS/3B’s, School of Medicine, University of Minho, 4710-057 Braga, Portugal 4Department of Pediatric Surgery, Hospital of Braga, 4710-243 Braga, Portugal *Correspondence: r[email protected]; Tel.: +35-125-360-4911 † These authors contributed equally to this work. Abstract: Lung organogenesis is a highly coordinated process governed by a network of conserved signaling pathways that ultimately control patterning, growth, and differentiation. This rigorously regulated developmental process culminates with the formation of a fully functional organ. Conversely, failure to correctly regulate this intricate series of events results in severe abnormalities that may compromise postnatal survival or affect/disrupt lung function through early life and adulthood. Conditions like congenital pulmonary airway malformation, bronchopulmonary sequestration, bronchogenic cysts, and congenital diaphragmatic hernia display unique forms of lung abnormalities. The etiology of these disorders is not yet completely understood; however, specific developmental pathways have already been reported as deregulated. In this sense, this review focuses on the molecular mechanisms that contribute to normal/abnormal lung growth and development and their impact on postnatal survival. Keywords: congenital pulmonary airway malformation (CPAM); bronchopulmonary sequestration; bronchogenic cysts; congenital diaphragmatic hernia (CDH); congenital malformations 1. Overview Lung development is a highly orchestrated and conserved multistage process sustained by molecular, cellular, and physical events, traversing all gestational ages. Proper lung formation relies upon the crosstalk between epithelial and mesenchymal compartments, which controls the temporal and spatial distribution of a multitude of factors and diffusible signals. Key signaling pathways play a role in this process, for instance, Fibroblast Growth Factor (FGF) [ 1 ], Retinoic Acid (RA) [ 2 ], Sonic Hedgehog (SHH) [ 3 ], Wingless-related Integration Site (WNT) [ 4 ], Transforming Growth Factor β (TGF β ), Bone Morphogenetic Protein (BMP) [5], Hippo [6], to name a few. Congenital lung malformations arise due to abnormal embryonic development caused by an impairment of signaling and/or genetic factors. The following sections will address the key regulatory factors involved throughout the different lung developmental stages and the molecular determinants of the most common lung congenital anomalies. 2. Lung Development Lung development is divided into five morphological stages that establish molecular and structural transitions: (1) embryonic; (2) pseudoglandular; (3) canalicular; (4) saccular; (5) alveolar. Due to the non-synchronous development of the lung, the timing of the stages overlaps. Cells 2021,10, 2987. https://doi.org/10.3390/cells10112987 https://www.mdpi.com/journal/cells Cells 2021,10, 2987 2 of 27 Primordial lung buds originate as outpunches of the primitive foregut endoderm, and the bronchial tree is generated by reinterred budding and branching of epithelial tubules [ 7 ]. The human lung epithelium derives from the endoderm, whereas the surrounding mesenchyme originates from the mesodermal germ layer [ 8 ]. Blood vessels arise from mesodermal cells surrounding the tips of branching tubules (vasculogenesis), or through migration of blood vessels from the aortic arches into the lung, by sprouting of the pulmonary artery (angiogenesis) [ 9 ]. As the lung develops, the airways and vasculature develop, fusing at the distal end of the bronchial tree to generate millions of alveolar gas exchange units. The intricate branching pattern of the airways guarantees that, during postnatal life, humidified and cleared air is uniformly distributed to alveolar units. 2.1. Embryonic Stage The embryonic phase of human lung development is characterized by the formation of two primordial right and left buds that emerge from the foregut endoderm. Lung specification begins around the fourth post-conception week (pcw) in humans, at embryonic (E) day 9.5 in mice and E11 in rats, with a localized expression of NKX2.1 (also known as TTF1, thyroid transcription factor) in endodermal cells of the ventral anterior foregut [10,11]. The epithelial outgrowth of the primary buds requires complex interactions between local signals in the prespecified foregut endoderm and inductive paracrine signals from the surrounding mesoderm, both tightly regulated in dose, time, and space [ 12 ]. Defects in these molecular players impair foregut separation and cause abnormalities in epithelium and mesenchyme differentiation. WNT signaling, particularly WNT2 and WNT2B, plays an essential role in specifying NKX2.1 respiratory endoderm progenitors in the ventral anterior mesoderm surrounding the anterior foregut endoderm [ 13 , 14 ]. On the other hand, WNT2/2B expression is regulated by HOXB5 [ 15 ]. The ability of WNT/ β -catenin to induce NKX2.1 endoderm progenitor fate depends on the activation of BMP signaling [ 16 ]. BMP4 is expressed in the mesenchyme surrounding the anterior foregut and appears to have an inhibitory action on the transcription factor SOX2 (which promotes esophageal fate), allowing NKX2.1 expression [ 16 ]. SHH expression in the endoderm regulates BMP4 expression in the mesoderm through different downstream targets, such as FOXF1, GLI1, and GLI3. In its turn, SHH is regulated by mesodermal RA signaling [17]. Upon respiratory lineage specification, localized mesodermal expression of fibroblast growth factor 10 (FGF10), adjacent to the NKX2.1+ progenitors, induces FGF receptor 2b (FGFR2B) signaling resulting in primary lung bud formation due to cellular movements into the mesenchyme towards the FGF10 source. An RA-TGF-FGF10 axis triggers primary lung bud formation. Briefly, endogenous RA inhibits TGF β signaling in the lungspecified foregut to elicit FGF10 expression and, consequently, induction of primary lung buds [ 18 , 19 ]. TBX4, a member of conserved T-box-containing transcription factors, influences FGF10 expression, particularly in chicken embryos. Mesodermal tbx4 stimulates fgf10 expression, defines its anterior-posterior (AP) mesodermal expression boundaries, and induces endoderm differentiation by triggering nkx2.1 expression [ 20 ]. However, the genetic inactivation of TBX4 in mice does not prevent lung bud formation, suggesting a redundant role of these genes in foregut morphogenesis [ 21 ]. Figure 1provides a schematic representation of the key genes involved in lung specification. These early events of pulmonary development require the interplay of many other factors. For instance, the GATA family of zinc-finger transcription factors, GATA4 and GATA6, and FOXA1/2 transcription factors (Forkhead box gene superfamily) are expressed early in the endoderm and have a crucial role in survival, differentiation, and morphogenesis of the foregut [ 22 – 24 ]. Interestingly, these factors have pivotal roles in the formation of primary structures of the future lung (for instance, SOX2 and GATA6); they are also entangled in later phases of differentiation and specification of respiratory cell lineages. Once primary lung buds are formed, epithelial tubules undergo extensive branching morphogenesis that culminates in the formation of the bronchial tree and the future alveolar region. Cells 2021,10, 2987 3 of 27 Anterior Posterior SOX2 NKX2.1 WNT2/2B BMP4 SHH GLI1-3 RA TBX4 FGF10 RA SOX2 HOXB5 NOGGIN BMP4 (esophageal fate) (respiratory fate) Dorsal Ventral Figure 1. Summary of the molecular players involved in lung specification. A BMP gradient elicits dorsal SOX2 expression (esophageal progenitors) vs. ventral NKX2.1 expression (respiratory progenitors). Yellow, endoderm; blue, mesoderm. 2.2. Pseudoglandular Stage During the pseudoglandular phase (5–17 pcw human, E12–16.5 mouse, and E13–E18.5 rat), each main lung bud initiates a repetitive process of outgrowth, elongation, and bifurcation of the airway epithelium into the surrounding mesenchyme, a process called branching morphogenesis. This process is repeated over several generations; a reiterated combination of three processes, domain branching, planar bifurcation, and orthogonal bifurcation, will give rise to the respiratory bronchial tree [ 25 , 26 ]. Blood vessel development occurs concomitantly with epithelial branching, and vessels start to run along the airway, except that vessels branch more slowly [ 27 ]. Simultaneously, the proximal-distal axis of the developing lung is established. Additionally, as the airway tree is laid down, it begins to differentiate with cartilage; mucous glands and smooth muscle are already present [28]. Branching mechanisms are governed by intensive crosstalk between the epithelial and mesenchymal compartments, which in turn is regulated by a network of signaling cascades that control cellular processes, including extracellular matrix remodeling, proliferation, and differentiation in a temporal-spatial manner. Lung bud outgrowth, branching, and subsequent bud arrest result from the dynamic activity of SHH, FGF10, Sprouty 2 (SPRY2), TGFβ, and BMP4 [12,29]. FGF signaling, in particular FGF10, is essential for branching morphogenesis; in fact, fgf10 −/− mice die at birth due to complete abrogation of pulmonary branching morphogenesis [ 30 ]. FGF10 is expressed in the distal lung mesenchyme, specifically at sites of branch point formation and outgrowth; it acts in a paracrine fashion on the adjacent epithelium, where its cognate receptor FGFR2 is expressed [ 31 , 32 ], prompting a signaling cascade that culminates in the expression of SPRY2, SHH, and BMP4, which subsequently regulate the response and outgrowth of the lung bud [ 33 , 34 ]. This interaction is repeated each time a new series of bud initiation and outgrowth is initiated—the buds that branch from the preexisting ones grow towards the regions of high FGF10 expression [ 35 ]. Recently, there has been emerging evidence for a critical role of mammalian target of rapamycin (mTOR) complexes in coordinating branching morphogenesis and defining organ structural complexity [ 36 ]. FGF10/ FGFR2b downstream signaling through ERK1/2 (extracellular regulated kinase 1/2) and mTORC1, which is active in the epithelial progenitor cells at the tip of the airway tube, induce outward growth towards FGF10 induction signaling, controlling the duration of this event (a determinant of branch length and patterning of the airway tree) [ 37 ]. Furthermore, though the mechanisms that link vascular growth to airway Cells 2021,10, 2987 4 of 27 branching remain unknown, it is recognized that mTORC1 directly links the primary cues for airway (FGF10) and vascular (VEGF-A; vascular endothelial growth factor A) growth in the pulmonary branching morphogenesis program [ 38 ]. The secretion of VEGF from the branching epithelium initiates the differentiation of mesenchyme progenitor cells into vascular tissue and is regulated by hypoxia-inducible transcription factors (HIFs). From the three HIFα isoforms expressed in the developing lung, HIF-1 α , present in the branching epithelium, plays a major role in early pulmonary vasculogenesis [ 39 ]. Land et al. demonstrated that FGF10 induces mTORC1 activity via SPRY2 in fetal airway epithelium, and that amplifies HIF-1 α vasculogenic activity to drive VEGF-A expression and secretion from the airway endoderm [ 37 ]. The RA pathway has also been recognized as a crucial regulator for correct lung formation in mammalian [ 40 , 41 ] and chicken models [ 42 ]. Impairment of the RA signaling (by interfering with RDH10 or RALDH2) undermines branching morphogenesis and, thus, lung formation [ 43 , 44 ]. Likewise, WNT signaling is also a key regulator of lung branching morphogenesis [ 45 , 46 ]; targeted deletion of β -catenin [ 47 ] or the expression of the Dickkopf-1 (DKK1) WNT antagonist hamstrings normal lung branching [ 48 ]. Ligands such as WNT5A, WNT2A, and WNT7B play crucial roles in lung epithelial and mesenchymal growth/differentiation through a WNT-FGF crosstalk [ 49 – 52 ]. Branching morphogenesis also depends on the timely expression of numerous transcription factors. Among the subgroup of homeobox-containing genes (HOX family), HOXB5 is essential for establishing AP airway patterning. hoxb5 is highly expressed during the pseudoglandular period, in the mesenchymal compartment, surrounding active branching sites [ 42 , 53 ]. As branching morphogenesis is completed and lung development progresses, hoxb5 expression diminishes. Additionally, sox2 and sox9, belonging to the SRY-related HMG-box family of transcription factors, regulate cell specification and differentiation. In the mouse, the proximal-distal patterning of the respiratory tree is defined by a distinct expression of SOX9 (and ID2) in the distal epithelium and SOX2 in the proximal airway epithelium. SOX2 is restricted to the pulmonary epithelium and is undetectable at the tips of the emerging secondary bronchi, whereas SOX9 expression delimits branching epithelium and originates distal pulmonary cell lineages [ 54 ]. In fact, it has been shown that loss of sox2 expression at branching sites is required for branching morphogenesis to occur [ 55 ]. SOX2-SOX9 spatial distribution along the lung epithelium is regulated by FGF10 signaling; FGF10 induces sox9 expression in the distal epithelium thus maintaining SOX9+ multipotent progenitor cells in an undifferentiated and stimulating their self-renewal; conversely, FGF10 inhibits sox2 expression, and proximal cellular fate, in the distal lung [ 56 ]. Furthermore, loss of SOX2 signaling impairs the differentiation of secretory and multiciliated cells that line the proximal airway epithelium in the subsequent lung stage [57]. More recently, the Hippo effectors YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) have been described as having important growth-modulator functions in branching morphogenesis and epithelial cell differentiation. YAP functions at the transition zone between the airway and the distal lung compartments and controls sox2 expression, thus promoting proximal airway differentiation [ 58 , 59 ]. In the absence of YAP, epithelial progenitors are unable to respond to local cues and, consequently, control SOX2 levels and spatial distribution and properly form airways [ 59 ]. Furthermore, Volckaert and colleagues proposed that cytoplasmatic YAP activity in the proximal epithelium promotes epithelial lineage commitment by inhibiting the β -catenin signaling pathway and FGF10 [60]. MicroRNAs (miRNAs/miR) also contribute to the lung developmental program. For instance, in this stage, miR-326 negatively regulates SHH signaling by targeting GLI2 and SMO (Smoothened) [ 61 ]. miR-142-3p regulates the proliferation and differentiation of mesenchymal progenitors by controlling the level of WNT signaling [ 62 ]. Mouse miR-17 (and its paralogs miR-20a and miR-106b) is expressed in the epithelium and controls branching morphogenesis through an FGF10-mediated molecular mechanism targeting MAPK14 and STAT3 [ 63 ]. Overexpression or deletion of the miR17–92 cluster (belonging to the miR-17 Cells 2021,10, 2987 5 of 27 family) leads to increased proliferation/decreased differentiation or hypoplastic lungs, respectively, pointing to a crucial role in lung branching morphogenesis [ 64 , 65 ]. Moreover, miR-200b −/− mice display disturbed airway distal lung branching and impairment in lung parenchyma [ 66 ]. miR-449a is crucial in the pseudoglandular to canalicular transition, and its inhibition results in increased mycn and sox9 mRNA and Ki-67 and SOX9 protein levels [ 67 ]. Furthermore, overexpression of rat miR-127 results in decreased terminal number of buds and increased terminal and internal bud sizes, suggesting an important role during lung morphogenesis [ 68 ]. In chicken, the expression pattern of miRNA processing machinery drosha,dgcr8,exportin-5, and dicer1 was described, thus supporting the importance of these regulatory elements during the early stages of branching morphogenesis [ 69 ]. Branching morphogenesis is also highly influenced by biomechanical forces, such as the transmural pressure in the chest cavity and smooth muscle contractions that influence the synchronization of the branching events [ 70 ]. HIPPO/YAP signaling regulates myosin light chain kinase activity, creating mechanical forces that influence cell shape required for branching [ 71 ]. In addition, recent data highlighted the importance of metabolic regulation during the early stages of lung branching morphogenesis [ 72 ]. The key signaling pathways underlying branching morphogenesis are shown in Figure 2. SOX2 SOX9 FGF10 FGF10 RA Proximal Distal WNT mTOR HIPPO WNT SPRY2 BMP4 SHH miR-142-3p miR-326 GLI2/3 miR-17 miR-20a miR-106b MAPK14 STAT3 SOX2 Figure 2. Signaling pathways that mediate epithelial–mesenchymal interactions during pseudoglandular stage, specifically in the distal epithelial tip. SOX2+ cells define proximal epithelial cell lineages whereas SOX9+ cells define distal epithelial cell lineages. Yellow, epithelium; blue, mesenchyme. Furthermore, the extracellular matrix (ECM) undergoes a series of remodeling events for the branching process to occur. ECM composition/structure plays a decisive role in lung development and organ architecture; it directly affects the availability and activity of soluble factors (as, for instance, FGF) by influencing their diffusion rates and the accessibility of their receptors [ 73 ]. ECM components, particularly fibronectin, laminin, and collagen, actively participate in the epithelium and mesenchyme interactions by accumulating within the clefts that mark the branch points. Fibronectin deposition possibly regulates cellular migration by fixing some cells at the cleft, while the un-fixed ones can migrate and proliferate distally [ 74 ]. By the end of the pseudoglandular phase, the complete human airway structure has been established, and airway epithelial differentiation is proceeding. 2.3. Canalicular Stage The canalicular stage spans pcw 16–26 in human (E16.5–E17.5 in mouse and around E18.5–E20 in rat) [ 25 ]. During this period, the existing epithelial airways continue to increase in size, and the epithelial terminal buds project into the distal airspaces as their surrounding mesenchyme thins, giving rise to the primitive pulmonary acini (terminal sacs), the primitive alveoli. This phase is also characterized by alveolar cellular differentiation. Distal epithelial cells differentiate into alveolar epithelial cells type 1 (AEC1) and Cells 2021,10, 2987 6 of 27 type 2 (AEC2), also known as type 1 and type 2 pneumocytes. AEC2 are responsible for surfactant production and serve as AEC1 progenitor cells, whereas AEC1 are responsible for gas exchange. One of the key signaling pathways for the determination of cellular fate is NOTCH signaling. NOTCH transmembrane receptors mediate communication between neighboring cells and have a central role in balancing differentiation of multiciliated vs. secretory lineages in the proximal airway epithelium; loss of NOTCH signaling leads to the absence of secretory cells and airways populated by multiciliated cells [ 75 ]. HIPPO signaling ceases branching morphogenesis and promotes alveolar differentiation through degradation of β -catenin in the epithelium, disrupting the WNT-FGF feedback loop and directing bud tip epithelial progenitors to differentiate [ 76 ]. Finally, both human and mouse miR-449a are upregulated in the distal lung, throughout the canalicular stage, to promote distal epithelium differentiation by regulating N-MYC and SOX9 but has no effect in SOX2 expression [67]. Figure 3illustrates the signaling events governing the canalicular stage. NOTCH signaling PD cellular fate SOX2 SOX9 miR-449a WNT signaling Figure 3. Simplified representation of the signaling pathways implicated in the canalicular stage. Proximal SOX2+ cells generate conducting airway cells (neuroendocrine, secretory, multiciliated and basal). Distal SOX9+ cells generate alveolar epithelial cells. Lastly, vascularization begins during this phase. The course of capillaries leaning against the distal epithelium airspaces and the continued angiogenic process contribute to the formation of the first thinned air-blood barrier in the future alveolar ducts and saccules [77]. 2.4. Saccular Stage The saccular phase, occurring between pcw 24 and 38 in humans (between E17.5–Postnatal day (P) 4 in mouse and E21–P4 in rat), is an intermediate phase in which branching morphogenesis ceases, and alveolarization is yet to commence. This intermediate stage is necessary since branching morphogenesis and alveolarization do not occur simultaneously [ 12 ]. Furthermore, this period is characterized by the widening and further division of the distal airspaces into numerous thin-walled terminal saccules, the alveoli precursors. The expansion of the future gas exchange region causes the condensation of the mesenchyme in-between airspaces, originating a thick immature primary septum at locations where two airspaces meet. [ 25 ]. Capillary networks remain very close to septal surfaces and are separated by a dense central layer of mesenchyme that forms a core of connective tissue [ 25 ]. During this period, the exceptional expansion of the prospective respiratory airspaces leads to a decrease in the interstitial tissue, which greatly impacts capillary arrangement. As each saccule further increases in size, blood vessels become closely associated and form a capillary bilayer wrapping each saccule. This process is crucial for alveoli formation and subsequent gas exchange [ 8 ]. Compared to the mature lung, the capillaries are embedded in a broad interstitial layer that is very poor in extracellular fibers but is very cellular [ 8 ]. Nonetheless, soon after, elastin starts to be deposited under the epithelium, which prepares the lungs for further alveolar formation. Cells 2021,10, 2987 7 of 27 The primary septa’s surface is mainly covered by AEC1 and AEC2, which continue to differentiate and populate the distal tubules. The surfactant system of AEC2 maturates and lamellar bodies appear, and surfactant secretion is detected [ 25 ]. With the development and maturation of the surfactant system during the saccular phase, the chances of survival to early premature infants’ increase. [78]. RA signaling pathway seems to play an important role in preparing the lungs for sacculation and, later, in preparing for alveolar formation. Throughout the saccular stage, expression levels of the RA receptor-alpha (RAR α ) transcript decrease, which seems to be essential for sacculation and differentiation of mature AEC1. RA receptor-beta (RAR β ) expression increases significantly in the saccular phase, matching AEC1 and AEC2 induction, pointing to a plausible role in preparing the lungs for alveolarization [ 79 ]. Similarly, loss of WNT signaling has been proven to be required to facilitate the transition from canalicular to saccular phase and allow air sac formation in the canalicular-saccular stages [ 80 ]. Interestingly, activation of WNT signaling results in the expansion of AEC2, whereas its inhibition constrains AEC2 development and shunts alveolar epithelial development toward the AEC1 cell lineage. These findings revealed that a wave of WNT-dependent AEC2 expansion is required for lung alveologenesis and maturation [ 81 ]. Moreover, studies from YAP/TAZ loss-of-function mouse mutants have shown that HIPPO signaling pathway plays a key role in promoting AEC1 fate [ 82 ]. miR-26a-1/miR-26a-2 knockout mice exhibit alterations in the morphology of distal epithelial cells, and display an increase in AEC2 cell number in the saccular stage with a concomitant increase in surfactant production; this study points to a role of miR-26a in lung maturation [ 83 ]. miR-127 expression levels are highest during the saccular/alveolar stage, and its spatial distribution shifts from mesenchymal to epithelial cells, suggesting a role in the cellular reorganization and differentiation of alveolar epithelial cells [ 68 ]. During sacculation, miR-17-92 should be repressed by HDAC3 to allow proper TGFβ signaling crucial for AEC1 remodeling [ 84 ]. Figure 4displays the main signaling events in this phase. (miR-26a-1/2) WNT signaling RARβ miR-17-92 TGFβ RARα WNT signaling AEC2AEC1 Figure 4. Simplified scheme of the signaling pathways involved in alveolar epithelial cell type 1 (AEC1) and type 2 (AEC2) differentiation during the saccular stage. Blue label: AEC1 differentiation; red label: AEC2 differentiation. (miR), present in the lung compartment. 2.5. Alveolar Stage The final alveolar stage refers to the process of alveoli formation (alveologenesis, also known as alveolization) that gives rise to the functional units for gas exchange. The timing of alveolar development varies among species. In humans, some alveoli are already formed before birth, and this process continues postnatally until young adulthood, whereas in mice, it is mainly a postnatal process. Alveologenesis can be divided into classical (or bulk) Cells 2021,10, 2987 8 of 27 alveolarization (week 36 of gestation—~3 years), continued alveolarization (birth-young adulthood), and microvascular maturation (week 36 of gestation—young adulthood) [ 25 ]. During classical alveolization, new secondary septa, risen from the pre-existing septa, grow from the saccular walls to subdivide the distal saccules into smaller units, the alveoli, broadening the surface area for gas exchange [ 28 ]. Like the primary septa, the secondary septa initially display a double capillary network [ 85 ]. Endothelial cells, myofibroblasts progenitors, fibroblasts, and lipofibroblasts cover the secondary septa, and matrix elastic fibers are deposited at the tip of the crests [85,86]. Classic alveolarization starts rather rapidly, but as it switches to continued alveolarization, the rate of increase in the number of alveoli declines [ 87 , 88 ]. Moreover, throughout postnatal lung development, the size and surface area of alveoli remain singularly stable in mice and humans. Simultaneously, microvascular maturation occurs, and the doublelayered capillary network observed in the immature primary and secondary septa fuse into a more efficient single-layered one in a thin septum [ 25 ]. This process involves multi-focal fusing of capillary segments and preferential growth of the mature single-layer capillary network [ 85 ]. Since it is now widely recognized that alveolarization continues until young adulthood, the timing of microvascular maturation was re-examined. Based on stereological estimations performed during rat lung development, microvascular maturation occurs in parallel to alveolarization and continues as long as new septa or alveoli are formed [ 25 ]. Once the alveolus has matured, every AEC1 has become in close contact with the endothelium. Reciprocal crosstalk between the airway epithelium and the vascular endothelium has been investigated in mice and found to be crucial for the prosperous development of one another. In pericytes, which are mesenchymal cells that strongly interact with endothelial and epithelial cells, mechanosensitive YAP1/TAZ signaling is stimulated to release hepatocyte growth factor (HGF), which is important for effective secondary septation [ 89 ]. In addition, the respiratory epithelium is a source of alveolar epithelium-derived VEGF-A that promotes vascularization [90]. Alveologenesis involves extensive cellular and tissue remodeling that culminates in the establishment of a large gas exchange surface area. Remodeling processes include the final specification and maturation of AEC2, surfactant synthesis, AEC1 flattening, and mesenchymal differentiation. Several signaling events are associated with the processes of secondary septation and alveolarization. For instance, platelet-derived growth factor A chain (PDGF-A) and its receptor (PDGFR α / β ) have been shown to play key roles in myofibroblast differentiation and production of elastin [ 91 ]. In PDGF-A deficient mice, alveolarization failed to occur and exhibited reduced elastic fibers deposition, showing that the appearance of elastin and alveolar crests are closely linked [ 92 ]. Bundles of elastin laid down by myofibroblasts restrain the differentiating alveolar cells that are expanding into sacculi as development proceeds. This remodeling process is driven by mesenchymal cells, such as the alveolar myofibroblasts (MYF), which are stimulated by PDGF-A and SHH. Furthermore, the contractibility of the MYFs physically shapes the alveolus. Additionally, WNT-responsive AEC2 receives WNT ligands from the mesenchymal cells and proliferate during this time, increasing surfactant production, which is crucial for the shift to air breathing [81]. Ephrin-B2 has been shown to play an important role in endothelial cells. Loss of ephrin-B2 signaling impairs the normal development of secondary septa and disrupts the deposition of several matrix proteins [ 93 ]. Additionally, the TGF superfamily of transforming growth factors has received great attention as a mediator of normal and aberrant lung alveolarization [ 94 , 95 ]. BMP, the alternative branch of the TGFβ superfamily, is also implicated in postnatal lung maturation, particularly surfactant production in neonates during respiratory adaptation to the extrauterine environment [ 96 ]. Furthermore, evidence from fgfr3/fgfr4 double null mice points to a critical role for FGF signaling in controlling the alveolarization process since these animals fail to undergo secondary septation [ 97 ]. Moreover, these animals did not present the typical downregulation of elastin production that occurs at the end of this process. Finally, the retinoids have been recognized Cells 2021,10, 2987 9 of 27 as alveolar morphogens, especially RA, which is crucial for alveolar formation [ 85 , 86 ]. Both increased and decreased RA signaling impair alveolar development. Several studies have demonstrated an association between vitamin A deficiency and major histological alterations, including thinner alveolar walls, airspace enlargement, and an increase in alveolar breaks [ 98 ]. On the other hand, increased epithelial RA signaling by transgenic expression of a dominant active RAR α resulted in lung immaturity and a blockage in distal epithelial maturation, preventing the appearance of AEC1 cells [ 79 ]. Despite all evidence, the precise mechanism by which RA functions to regulate alveolarization is not entirely understood. Notwithstanding, the paracrine regulation of lung myofibroblast proliferation and elastin synthesis by RA is dependent on the interaction with FGF signaling, specifically FGF18 [ 99 ]. RA also acts in an autocrine manner to regulate proliferation and tube formation in endothelial cells [ 100 ]. The main interactions eliciting alveologenesis are displayed in Figure 5. Secondary septa formation Elastic fibers Alveolar macrophage Endothelium Myofibroblast AEC2AEC1 Lipofibroblast Fibroblast HGF VEGF-A YAP1/TAZ FGF18 Pericyte Elastin RA PDGFA, SHH Figure 5. Schematic representation of the signaling events occurring during alveologenesis, particularly secondary septa formation. Left image: alveolar niche. Right image: magnification of the secondary septa. Finally, it has been demonstrated that targeting miR-34a partially improves alveologenesis in the hyperoxia-induced alveolar impairment mice model [ 101 ]. miR-29b supplementation improves alveolarization in mice that were exposed to neonatal hyperoxia and maternal inflammation [ 102 ]. miR-876-3p gain of function improved alveolar structures in the bronchopulmonary dysplasia mice model [ 103 ]. Mice miR-421 inhibition improves bronchopulmonary dysplasia condition by targeting fgf10 [104]. 3. Congenital Lung Malformations Congenital lung malformations arise during development and include numerous anatomical anomalies of the lung and respiratory tree. They are usually detected prenatally by ultrasonography and comprise congenital pulmonary airway malformation (CPAM), bronchopulmonary sequestration (BPS), bronchogenic cysts (BC), and more rarely bronchial atresia, congenital lobar emphysema (CLE), and congenital tracheal obstruction. This section focuses on the molecular and genetic determinants of the most frequent anomalies: CPAM, BPS, and BC. Congenital diaphragmatic hernia (CDH) is not usually included in this group; however, since the lung is also highly affected in this condition, we have also incorporated evidence related to lung hypoplasia. Cells 2021,10, 2987 16 of 27 Table 1. Summary of the molecular players and corresponding major events underlying normal lung development. Stage Major Events Molecular Players Expression Site Function Embryonic Lung bud develops from the foregut; Tracheoesophageal septation NKX2.1 (or TTF1) Endoderm Specification of respiratory progenitors Epithelial marker TBX4 Mesoderm Induction of endodermal differentiation (nkx2.1-dependnent) & budding (fgf10-dependent) WNT2/2B Mesoderm Specification of NKX2.1 respiratory progenitors GATA4/6 Endoderm Formation of primary lung structures Differentiation of visceral endoderm SHH/ GLI1-3 Endoderm/ Mesoderm Embryonic foregut development FOXF1 Mesoderm Lung & gastrointestinal morphogenesis Regulation of mesenchymal-epithelial interactions FOXA1/A2 Endoderm Specification of foregut endoderm Branching morphogenesis Pseudoglandular Formation of bronchial buds: Branching morphogenesis. Formation of bronchial tree and blood vessels; Cellular differentiation (cartilage, smooth muscle); FGF10/ FGFR2 Mesenchyme/ Epithelium Lung bud outgrowth SPRY2 Distal epithelium Negatively regulates FGF10 signaling Inhibits lung budding TGFβMesenchyme/ Epithelium Lung branching BMP4 Epithelium of distal tips Negatively regulates FGF10 signaling Inhibits lung budding SHH Epithelium Branching morphogenesis RDH10 Lung buds Lung bud outgrowth RALDH2 Mesothelial region Lung growth and branching WNT7B Distal epithelium Branching morphogenesis WNT2A Distal lung mesothelium Mesenchymal cell proliferation WNT5A Mesenchyme/ Epithelium Tracheal development/ Distal lung morphogenesis β-Catenin Airway epithelium Branching morphogenesis Epithelial differentiation and proliferation Cells 2021,10, 2987 17 of 27 Table 1. Cont. Stage Major Events Molecular Players Expression Site Function HOXB5 Mesenchyme Anterior-posterior patterning Directing epithelial morphogenesis SOX2 Proximal epithelium Proximal-distal patterning SOX9 Distal epithelium Proximal-distal patterning miR-17 miR-20a miR-106b Epithelium FGF10-mediated epithelial branching morphogenesis miR-200b Epithelium & Mesenchyme Distal airway branching miR-449a Distal epithelium Regulation of differentiation & proliferation miR-326 Mesenchyme SHH signaling modulator miR-142-3p Mesenchyme Proliferation & differentiation of mesenchymal progenitors Control of WNT signaling Canalicular Formation of distal most airways; Alveolar cellular differentiation; Appearance of the first air-blood barrier; Surfactant production initiation NOTCH Determination of cellular fate Development of microvasculature network miR-449a Distal epithelium Regulation of differentiation & proliferation Saccular Formation of thin-walled terminal saccules (alveoli precursors); Maturation of vasculature and surfactant system RARαEpithelium Sacculation & differentiation of mature AEC1 RARβEpithelium AEC1 & AEC2 induction miR-26a-1/ miR-26a-2 Alveolar epithelial cells Pulmonary surfactant synthesis miR-17-92 Epithelium AEC1 remodeling Alveolar Alveologenesis: establishment of secondary septa and alveoli formation; Microvascular maturation: single-layered capillary network formation. VEGF-A Epithelium Vascular development PDGF-A/ PDGFRα/β Epithelium/ Mesenchyme Myofibroblast differentiation Elastin production Secondary septation & alveolarization Ephrin-B2 Microvasculature Secondary septation & alveolarization miR-34a Impairs alveolarization miR-29b Promotes alveolarization miR-876-3p Promotes alveolarization miR-421 Alveolar epithelial cells Disrupts alveolarization Cells 2021,10, 2987 18 of 27 A deeper understanding of the mechanisms controlling lung morphogenesis is crucial for managing congenital/neonatal respiratory diseases. Furthermore, it is highly relevant for developing new strategies to improve the regenerative response of the lung to injury. With this review, we aimed to describe the key signaling pathways underlying normal lung development. Additionally, we reviewed the current knowledge regarding the signaling events impaired in congenital lung lesions. Congenital lung lesions present several alterations in both the epithelial and mesenchymal compartments that, altogether, contribute to the abnormal lung phenotype. Table 2summarizes the molecular players known to be altered in CPAM and CDH. Nonetheless, more studies are in demand to determine the etiology of these congenital conditions. Table 2. Summary of the molecular players impaired in congenital lung diseases. Congenital Lung Disease Altered Epithelial Signaling/Expression Altered Mesenchymal Signaling/Expression References CPAM Integrin Integrin [117] E-cadherin - [117] - PDGF-BB [118] GDNF - [119] - FABP-7 [120] CC10 - [115] KRAS; PI3K-AKT-mTOR - [121] VEGFR2 - [122] - HOXB5 [123,125] TTF1/Nkx2.1 - [124,125,140] fgf9 - [125,129] -fgf7 [125,127,130] -fgf10 [126,128,131,132,134,135] fgfr2 - [135] DICER - [131] yy1 - [132,133] shh - [24,132,134] cdc42 - [134] -ptc1 [134] foxa1;foxa2 - [24] fzd2 - [135] RhoA - [135] hdac1;hdac2 - [136] bmpr1a - [137] Mycn - [138] Yap - [59] SOX2 - [55,139,140] SOX9 - [54] Notch - [141] - Elastin [142] CDH - RALDH2 [187] RAR RAR [186] - STRA6 [174,187] Midkine Midkine [186] FGF signaling - - - FGF10 [189] FGF2 FGF2 [186] - FGF7 [189,190] FGF9 - [186] - FGF18 [191] FGFR2 FGFR2 [186] FGFR3 FGFR3 [186] BMP signaling - - BMP4 - [186] BMP7 - [186] BMPR2 - [186] WNT signaling - - - WNT2 [186] WNT5A WNT5A [186] WNT7B - [186] - GATA6 [198] Cells 2021,10, 2987 19 of 27 Table 2. Cont. Congenital Lung Disease Altered Epithelial Signaling/Expression Altered Mesenchymal Signaling/Expression References - GATA4 [192,193] - NR2F2 [194] ZFPM2 - [195] - WT1 [196] - MYRF [199] - GLI [200] - KIF7 [201] - PBX1 [202] - SLIT3 [174,179,203] ROBO1 ROBO1 [174,179] NDST1 - [174,179] FREM1 - [174,179] - FRAS1 [174,179] - FREM2 [174] miR-200b miR-200b [207] Author Contributions: Conceptualization, J.C.-P. and R.S.M.; writing—original draft preparation, I.C., H.F.-S., D.M.-C., and R.S.M.; writing—review and editing, I.C., H.F.-S., D.M.-C., J.C.-P., and R.S.M.; supervision, J.C.-P. and R.S.M. All authors have read and agreed to the published version of the manuscript. Funding: This work has been funded by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020. Hugo Fernandes-Silva was supported by a doctoral fellowship (PD/BD/137655/2018) from FCT, as part of the Inter-University Doctoral Programme in Ageing and Chronic Disease (PhDOC). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. References 1. Danopoulos, S.; Shiosaki, J.; Al Alam, D. FGF Signaling in Lung Development and Disease: Human Versus Mouse. Front. Genet. 2019,10, 170. [CrossRef] 2. Fernandes-Silva, H.; Araújo-Silva, H.; Correia-Pinto, J.; Moura, R. Retinoic Acid: A Key Regulator of Lung Development. Biomolecules 2020,10, 152. [CrossRef] 3. Fernandes-Silva, H.; Correia-Pinto, J.; Moura, R.S. Canonical Sonic Hedgehog Signaling in Early Lung Development. J. Dev. Biol. 2017,5, 3. [CrossRef] 4. Pongracz, J.E.; Stockley, R.A. Wnt signalling in lung development and diseases. Respir. Res. 2006,7, 15. [CrossRef] 5. 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