0031-3998/05/5705-0026R PEDIATRIC RESEARCH Vol. 57, No. 5, Pt 2, 2005 Copyright © 2005 International Pediatric Research Foundation, Inc. Printed in U.S.A. Molecular Mechanisms of Early Lung Specification and Branching Morphogenesis DAVID WARBURTON, SAVERIO BELLUSCI, STIJN DE LANGHE, PIERRE-MARIE DEL MORAL, VINCENT FLEURY, ARNAUD MAILLEUX, DENISE TEFFT, MATHIEU UNBEKANDT, KASPER WANG, AND WEI SHI Developmental Biology Program [D.W., S.B., S.D.L., P.-M.D.M., D.T., K.W., W.S.], The Saban Research Institute of Childrens Hospital Los Angeles and the Center for Craniofacial Molecular Biology, Keck School of Medicine and School of Dentistry, University of Southern California, Los Angeles, California, 90027, Department of Cell Biology [A.M.], Harvard Medical School, Boston, Massachusetts, 02115, Laboratoire de Physique de la Matiere Condensee [V.F., M.U.], Ecole Polytechnique, 91128 Palaiseau Cedex, France ABSTRACT The “hard wiring” encoded within the genome that determines Abbreviations the emergence of the laryngotracheal groove and subsequently early BMP, bone morphogenetic protein lung branching morphogenesis is mediated by finely regulated, DKK, Dickkopf interactive growth factor signaling mechanisms that determine the EGF (R), epidermal growth factor (receptor) automaticity of branching, interbranch length, stereotypy of branch- ERK, extracellular regulated kinase ing, left-right asymmetry, and finally gas diffusion surface area. The FGF (R), fibroblast growth factor (receptor) extracellular matrix is an important regulator as well as a target for FN, fibronectin growth factor signaling in lung branching morphogenesis and al- LRP, lipoprotein receptor-related proteins veolarization. Coordination not only of epithelial but also endothe- MAP, membrane-associated protein lial branching morphogenesis determines bronchial branching and PDGF, platelet-derived growth factor the eventual alveolar-capillary interface. Improved prospects for RAR, retinoic acid receptor lung protection, repair, regeneration, and engineering will depend on sFRP, secreted Frizzled-related protein more detailed understanding of these processes. Herein, we con- SHH, Sonic hedgehog cisely review the functionally integrated morphogenetic signaling Sp-C, network comprising the critical bone morphogenetic protein, fibro- surfactant protein C ␣ ␤ blast growth factor, Sonic hedgehog, transforming growth factor-␤, TGF- ( ), transforming growth factor alpha (beta) vascular endothelial growth factor, and Wnt signaling pathways that VEGF (R), vascular endothelial growth factor (receptor) specify and drive early embryonic lung morphogenesis. (Pediatr Res 57: 26R–37R, 2005) THE 70 M2 ALVEOLAR SURFACE ORIGINATES which in turn give rise to the left and right lobar branches of the FROM THE LARYNGOTRACHEAL GROOVE bronchial tree. In mice, four lobes form in the right lung, whereas the left lung forms a single lobe. In humans, the left Lung development begins with the appearance of the laryn- gotracheal groove, which is a small diverticulum that arises lung is trilobed, whereas the right lung is bilobed. Lobation of from the floor of the primitive pharynx at E9 in mouse and 4 the lungs, as well as the first 16 of 23 airway generations in wk in human. The proximal portion of the laryngotracheal humans is stereotypic, which implies the presence of a hard- groove gives rise to the larynx and trachea, whereas the distal wired, genetic program that controls early embryonic lung portion gives rise to the left and right mainstem bronchial buds, branching morphogenesis. The latter seven generations of lower airway branching and onward into the folding and expansion phase of the alveolar surface is nonstereotypic, but Received December 15, 2004; accepted January 21, 2005. nevertheless follows a recognizable, proximal-distal fractal Correspondence: David Warburton, D.Sc., M.D., F.R.C.P., Developmental Biology Program, MS #35, The Saban Research Institute of Childrens Hospital Los Angeles, 4650 pattern that is repeated automatically at least 50 million times. Sunset Blvd., Los Angeles, CA 90027; e-mail: [email protected] The lung morphogenetic program thus drives the formation of Supported by grants HL44060, HL44977, HL60231, HL75773, HL73014 to D.W.; ␮ 2 grants ALA 9PB-0082, HL074832, HL74862, HL74862 to S.B.; CNRS to V.F.; and an alveolar gas diffusion surface 0.1 m thick by 70 m in grants HL68597 and HL61286 to W.S. surface area that is perfectly matched to the alveolar capillary 26R MECHANISMS OF EARLY LUNG MORPHOGENESIS 27R and lymphatic vasculature. This huge gas exchange surface LEFTY-1, LEFTY-2, AND NODAL ARE IMPLICATED area is packed into the chest in a highly spatially efficient IN DETERMINATION OF LEFT-RIGHT manner (1,2). LATERALITY OF THE LUNG Lefty-1 null mutant mice show a variety of left-right isom- erisms in visceral organs, but the most common feature is SOLUBLE FACTORS ARISING FROM thoracic left isomerism. The lack of Lefty-1 expression results THE MESENCHYME DETERMINE LUNG in abnormal bilateral expression of Nodal, Lefty-2, and Pitx2 (a EPITHELIAL MORPHOGENESIS homeobox gene normally expressed on the left side). This suggests that Lefty-1 normally restricts Lefty-2 and Nodal Soluble factors arising from the mesenchyme have long been expression to the left side, and that Lefty-2, Pitx2, and/or Nodal implicated in lung branching morphogenesis distal to the tra- encode a signal for “leftness” in the lung (26,27). We speculate chea, since it was discovered that transposition of early stage that proximal-distal, anteroposterior, and left-right stereotypy distal lung mesenchyme onto the primitve tracheal epithelium must be superimposed on the automatic morphogenetic branch- can induce supernumary branching (3–7) as well as distal ing mechanisms proposed below. epithelial marker gene expression (8–10). Null mutation in mice of Fgf10, or its cognate receptor Fgfr2, as well as TRACHEOESOPHAGEAL SEPTATION misexpression of dominant negative kinase dead FGFR, or of soluble FGFR, all display a spectrum of closely related tra- Septation of the trachea from the esophagus is normal in the cheobronchial truncation phenotypes, in which epithelial, en- Fgf10 null mutation, indicating that this process can also be dothelial, and lymphatic branching of the bronchi distal to the distinguished genetically from peripheral lung branching. trachea is abrogated (11–15). This strongly suggests that the However, null mutations of the transcriptional factor Nkx2.1 genetic controls of laryngotracheal groove formation are sep- and of Shh display severe anomalies of larngyo-tracheal- arate from those that induce the peripheral lung. Herein, we esophageal septation, together with abrogation of distal lung review the little that is known about the molecular signals that morphogenesis (28–31). Haploinsufficency of Foxf1, which is induce the formation of the laryngotracheal groove. Then, we itself a SHH target, causes esophageal atresia and tracheo- consider the rather more extensive information on some of the esophageal fistula (32). SHH also plays a key role in determin- key molecular signaling mechanisms that mediate branching of ing correct patterning of the tracheal cartilages, inasmuch as the airways during early lung development. peripheral expression of Shh under the control of the Sp-C promoter fails to rescue tracheal cartilage morphogenesis on the Shh null background (33). SHH also regulates Gli3 gene expression (34). Laryngeal clefts, tracheal atresias, and esoph- THE LARYNX AND TRACHEA ARE GENETICALLY ageal atresias, with or without tracheoesophageal fistula, occur DISTINGUISHABLE FROM THE DISTAL AIRWAYS quite frequently in humans. Clinical associations with multiple other anomalies, including the VACTERL association, duode- ␤ Null mutation of Hnf3 results in failure of closure of the nal atresia and pancreatic malformations, have been noted. In primitive foregut, thus no laryngotracheal bud can form (16). the Pallister-Hall syndrome, among other anomalies, bifid epi- Hoxa3 and 5 also play key roles in specifying the larynx and glottis and cleft larynx have been described as an effect of Gli3 trachea (17–20). Retinoic acid deficiency in mice has long been mutation (34). Defects in the Shh-Gli pathway as well as the associated with total agenesis of the lungs, in association with FGF pathway have also been noted in the adriamycin-mediated other complex anomalies (21,22). Certain RAR null mutations teratogenic model of tracheoesophageal anomalies (35–38). are also among the few known causes of complete laryngo- Hox family genes are also likely to play a key role in tracheo- tracheo-pulmonary agenesis in mice (22). The other known bronchial differentiation, since in Hoxa-5-/- mice, tracheal oc- null mutations associated with complete absence of the larynx, clusion and respiratory distress are associated with marked trachea, and lung being the double Gli2/3 null mutation (23). decrease in surfactant protein production, together with altered Recently, it has been shown that retinoic acid induces Fgf10 gene expression in the pulmonary epithelium (19). Because expression within the mesoderm subjacent to the site of origin Hoxa-5 expression is restricted to the lung mesenchyme, the of the laryngotracheal groove (24). Blockade of RAR signaling null mutant phenotype strongly supports the inference that in E9 foregut cultures not only blocks the local expression of Hoxa-5 expression is necessary for induction of epithelial gene Fgf10 at this restricted site but also disrupts expression of expression by