Murine lung development is a complex process regulated by many factors guiding a carefully orchestrated series of events leading to mature lung formation. Many developmental pathways have been implicated in governing proper lung formation. Most notably, the Shh/Gli pathway shown to be crucial to the development of numerous other organ systems, is an absolute requirement for correct lung formation. Many interactions between the Shh pathway and other fundamental lung signaling molecules such as fibroblast growth factor 10 (Fgf10) have presented themselves. While the specifics of these interactions have yet to be elucidated, the consequence of their actions is paramount in guiding lung development.
Introduction and Discussion
Murine lung development initiates with the out pocketing of two endodermal lung buds from the ventral region of the primitive foregut tube around 9.5 days post coitum (dpc). The two primary lung buds then start to extend in a posterior-ventral track into the splanchnic mesenchyme, each bud representing the future left and right halves of the mature lung. Concurrently, the single foregut tube at the primary branch point begins to pinch into two distinct tubes forming the dorsal esophagus and ventrally located trachea. The right lung bud (right primary bronchus) then undergoes a secondary branching event leading to the creation of four secondary bronchi, each denoting one of the four right lung lobes (lobar bronchi). From this point, both the primary left bronchus and the four secondary bronchi of the right lung bud will continue to undergo further generations of dichotomous branching until the mature network of airways is formed. However, this branching process is not a chaotic event, but rather a carefully controlled process. A highly structured series of interactions between the developing airway epithelium and mesenchyme guides proper lung development.1,2 These interactions are directed by many tissue specific morphogenic signals.3 Much like the development of other branching organs such as the kidney, the mammalian lung requires a carefully orchestrated symphony of genes to accomplish its end goal. Several gene families have been shown to be involved in lung development, including fibroblast growth factors (Fgf ), bone morphogenic proteins (Bmp), as well as the primary focus of this chapter, the Hedgehog (Hh) family.
While many factors contribute to the formation of the mature lung, Sonic hedgehog (Shh) is absolutely required for functional lung formation. Evidence of Shh in lung development was first postulated with the observation of expression of shh transcripts throughout the epithelium of the developing mouse lung at 11.5 dpc, the highest levels occurring at the developing tips of the epithelial buds.4 Expression was also detected in the tracheal diverticulum, the esophagus, and the developing trachea.5 shh expression was originally reported to be strongly expressed until about mid gestation, after which it decreased.6 However, more recent evidence indicates shh expression increases towards birth peaking just prior to parturition.7,8 Also interesting was the detection of high levels of the transcripts for Patched (ptc) and Smoothened (smo), the downstream Shh signal relaying proteins, in the mesenchyme adjacent to the shh expressing epithelial cells in the developing buds.6 When the effect of shh over-expression in the lung epithelium using the surfactant protein (SP)-C promoter was examined, it was found that the ratio of interstitial mesenchyme to epithelial tubules had increased. More detailed analysis of these lungs revealed an abundance of mesenchyme and the absence of typical alveoli due to increased cellular proliferation in both the mesenchyme and the epithelium. Expression of ptc was also noticeably up-regulated in lungs of shh over-expressers, however no evidence of regulatory changes in other lung development related genes such as bmp4 or fgf7 was found.6 Also interesting to note is the 2.5 fold increase in gli1 expression in response to shh over-expression, while gli2 and gli3 expression levels remain unchanged.9 If we now examine the other side of the coin, a similar picture presents itself that further supports the concept of shh as a regulator of lung proliferation. A knockout of the shh gene has been created in mice in which the second exon of shh has been replaced with a PGK-neo cassette resulting in a nonfunctional truncated protein upon translation.10 While many developmental defects occur in this prenatal lethal model, we will focus on the pulmonary phenotype for the purposes of this chapter. shh null lungs have a dramatically altered phenotype; most obvious is the complete lack of asymmetry as the secondary branching in the right lung is defective (see fig. 1). The resulting single left and single right lung lobes are severely hypoplastic and fail to develop a vast network of mature air sacs. The trachea and esophagus do not divide into separate entities. Ultimately the lack of shh results in severely reduced mesenchymal proliferation and an extensive reduction in epithelial branching. When effects on gene regulation were examined, it was shown that ptc, gli1 and gli3 were all down-regulated in the lung mesenchyme.11 Like the shh over-expression model, proximal-distal differentiation of the lung was unaffected while prominent proliferative defects were evident. However, shh over-expression with the SP-C promoter in the lungs of shh-/- mice showed a significant improvement in growth, branching morphogenesis and vascularization.12 But, the peripheral over-expression failed to correct lobulation as well as cartilage defects in the trachea and bronchi seen in the shh-/- mice, signifying the importance for shh expression in areas to secondary branching and cartilage formation. More recently, a lung specific knockout of shh has been achieved in mice, which can also be temporally controlled through administration of doxycycline.12,13 shh expression appears to primarily be required for lung development prior to 13.5 dpc, after this point in development only mild defects in peripheral lung structure are observed when shh function is removed. However, removal of shh prior to 13.5 dpc resulted in severe malformations similar to the shh-/- null mouse, with defects in the trachea, bronchi and peripheral lungs, as well as many changes in gene expression levels as evident from microarray data. This study also demonstrated the localized spatial requirement for shh in proper cartilage formation in the conducting airways of the developing lung.12
Ptc is a twelve pass transmembrane receptor protein that is a fundamental component of the Shh signaling pathway. While Ptc itself is not a transcriptional regulator, nor a diffusible morphogen, its presence and function is absolutely critical for normal lung development. Ptc is expressed in high concentrations in the mesenchyme near the epithelial border of the developing tips neighboring to shh expression, as well as at lower concentrations in the distal epithelium.6 A ptc null mutant has been created, however this embryonic lethal genetic defect offered no clues to the role of ptc in lung development as these mice die around 9.0 dpc to 10.5 dpc, at the start of lung formation.14 While a mouse with lung specific over-expression of ptc has not been created, other experiments show that increased expression of ptc results in a reduction of Shh signaling, consequentially down-regulating expression of Shh responsive genes such as gli1 and ptc itself.14,15 This would suggest that over expression of ptc in the lung near the mesenchymal border would attenuate the Shh epithelial to mesenchymal signaling, resulting in somewhat of a less severe shh null phenotype.16 Most likely proliferative and branching defects would present themselves, however depending on the onset of over-expression, early lung development may proceed to further stages than the shh null phenotype.
Smoothened (Smo), another trans-membrane protein essential to the Hh signaling pathway, has been targeted for gene deletion in mice. The resulting phenotype is very severe, and offers little insight into effects on lung develop with embryos dying prior to 9.5 dpc.17 To speculate on possible effects of a smo null mutation on lung development, it must be taken into consideration that there is only one mammalian homologue of smo, and that it has been suggested that all three Hh signaling pathways would use smo.17 Therefore one would expect a lung specific smo null mutant to have a phenotype at least as severe to that of the shh null lung. Further more, since the transcripts for Indian Hedgehog (Ihh) have more recently been found to be expressed in the lung as well, the resulting phenotype could be even more detrimental to lung formation as the effects of Ihh on lung development are not known.18
Another protein important in the regulation of Shh through a negative feedback loop is Hedgehog-interacting protein 1 (Hip1). Hip1 is induced in Shh responsive cells upon Shh signaling and encodes a membrane-bound protein capable of directly binding to Shh, Ihh and Desert Hedgehog (Dhh).19 Hip1 expression has been found in the lung epithelium, as well as the underlying mesenchyme. Closer inspection shows hip1 is transcribed in cells near sources of Hh signaling, in a domain that overlaps with ptc expression.20 An increase in hip1 expression is observed in shh over-expression models, and conversely hip1 is decreased in shh null mutants. Experiments in which hip1 was ectopically expressed in the developing endochondral skeleton where Ihh is accountable for Hh signaling, it was found to attenuate Hh signaling showing a similar phenotype to the Ihh null mutant.20 More recently in continuing their investigation into Hip1 function, Chuang and coworkers (2003) have demonstrated that the hip1 loss-of-function mutant mouse has increased Hh signaling, disrupting morphogenesis in the lung and skeleton. They indicate that increased Shh function due to lack of hip1 function causes a misregulation of fgf10 expression resulting in failure of secondary branching.21 The sum of these observations implicates Hip1 as a negative feedback regulator of Hh signaling, crucial to normal development of the lung, as well as other organ systems.
Turning attention to the downstream transcription factors of the Shh pathway, we can see further evidence denoting the importance of Shh signaling in lung development. The Gli family of transcription factors are a group of three genes which encode proteins containing DNA binding zinc finger motifs. Early analysis suggested these genes would function as transcription factors with a relationship to cellular proliferation. The glis were found to be expressed in the splanchnic component of the lateral mesoderm in the developing gut amongst other places.22 More detailed analysis of expression patterns revealed that all three gli genes are strongly expressed in different but over-lapping domains in the lung mesenchyme during the pseudoglandular stage with expression declining towards birth.9 Gli1 is expressed in the distal mesenchyme, mostly concentrated around the developing endodermal lung buds. Gli2 on the other hand has a more dispersed mesenchymal expression pattern which is still more spatially restricted towards the distal regions of the lung, but has strong expression near the trachea as well. Gli3 is not particularly concentrated in either proximal or distal mesoderm, however is not as widely dispersed as Gli2, lying in between the expression domains of Gli1 and Gli2.9 Expression of all three gli genes is dynamic and seems to correspond with branching morphogenesis in the developing lung lobes. The temporal down-regulation of the gli transcripts towards birth appears to occur in three separate phases. While expression of each gli is elevated early in lung development, gli2 and gli3 show a decrease in expression from 12.5 dpc to 16.5 dpc, at this point gli2 mRNA expression stabilizes. Gli1 and gli3 continue to decrease (along with shh expression) until just prior to birth when gli2 will also further diminish, resulting in down-regulation of all gli genes just before birth.9
Removal of the zinc finger coding region of the gli1 gene from mice results in a loss-of-function mutation in the Gli1 protein which can no longer signal to other Shh targets. These mice are viable, show no physical abnormalities and display no observable behavioral traits.23 Gli2 null mice on the other hand have a very severe lung phenotype. While a heterozygous gli2 deletion has no detectable effect on lung development, complete removal of gli2 results in a lethal phenotype, with mice dying in-utero during late gestation.24 The lungs of the gli2 null mice are very hypoplastic in appearance and most notably show defective branching in the right lung with only one lobe forming. The left lung still forms one lobe but has a severe reduction in wet weight of approximately 60% at 13.5 dpc. This developmental trend continued to 18.5 dpc, when the left lung weight was 50% lighter than its wild-type counter part. The lungs show little sign of apoptosis but bromodeoxyuridine (BrdU) incorporation experiments demonstrated a 40% and 25% reduction in cellular proliferation in the mesenchyme and epithelium, respectively. Histological analysis revealed that the trachea and esophagus are both hypoplastic, but still separate. Smaller air sacs were also evident and they were surrounded by thicker than normal mesenchyme. When lung development associated growth factors such as fgf1, fgf7, fgf10, bmp2, bmp4, and bmp6 were examined for changes in gene expression, no deviations were found. However, in-situ hybridization was able to detect decreases in both ptc and gli1 expression, further demonstrating a reduced response to Shh signaling.25 Gli3 null mice have been around for many years. The null allele designated Gli3Xtj, was discovered in the “Extra toes” mouse mutant to be a viable homozygous deletion.26 Unlike the viable gli1 null mouse, the gli3 null mouse does show an altered pulmonary phenotype. While the homozygous gli3Xtj mouse embryo is actually heavier than its wild-type littermates at all stages of gestation, the lungs are typically smaller with an altered shape, most noticeably a reduction in lung width.9 The wet lung weight when measured at 18.5 dpc was 35% lower than wild-type littermates. The gli3 heterozygous mice did not show any altered lung phenotype. When gene expression for other Shh pathway members was tested in the gli3 null lung, no changes in expression levels or localization was detected for shh, gli1, gli2, or ptc, as well as bmp4, and wnt2.9
The evolution of the Glis from their common ancestor cubitus interruptus (Ci) in Drosophila melanogaster to the mammalian three part signaling system, suggests the evolution of separate roles for each Gli. Double mutant combinations of the gli genes have been created to help elucidate the possible functional roles for each Gli transcription factor during pulmonary development. Different combinations of gli1 and gli2 null alleles show that there is some level of redundancy between the two genes. While neither the gli1-/-, gli2+/-, nor the double gli1/gli2 heterozygous mouse, show an altered lung phenotype, the combined gli1-/-;gli2+/-; genetic condition results in a variable lung phenotype with minor alterations in size and shape relative to its wild-type sibling.23 While not as severe as the gli2 null lung phenotype, this signifies some functional redundancy between Gli1 and Gli2 activities. Further supporting this notion is the fact that when one copy of gli1 is removed from a gli2 null lung which already has a severe phenotype on its own, the resulting lung is even smaller. Finally, if all functional Gli1 and Gli2 protein is removed from the developing lung, the result is a lung with two very small single lobes, smaller than the gli2 null lung.23 To date, the only gli1/gli3 double mutant mouse analyzed has been the gli1-/-;gli3+/- mouse. These mice show an identical polydactyly phenotype to the gli3 null mouse, and analysis of pulmonary phenotype effects were not performed.23 So it appears that there is functional redundancy between Gli1 and Gli2, and not Gli1 and Gli3. However, the most severe lung phenotype indicating further functional redundancy between Glis reveals itself in the gli2/gli3 double null mutant. While the gli2/gli3 heterozygous double mutant shows no observable foregut malformations, the gli2/gli3 double null mouse suffers an early embryonic lethal phenotype typically not surviving past 10.5 dpc. A few of the double null embryos will survive to 14.5 dpc and these mice fail to show any lung formation. In fact, no evidence of any trachea or lung primordia is found past 9.5 dpc.25 While the gli2+/-;gli3-/- mouse has been generated, no comment on the pulmonary phenotype has been reported thus far. However, the gli2-/-;gli3+/- mutant mouse has a severe lung phenotype, resulting in an extremely hypoplastic, single lung lobe. This is suggested to result from an ectopic lung bud developing between the left and right lung buds fusing them together after the primary branching at the posterior end of the lung. These mice also develop a single tracheo-oesophageal tube connecting the lung directly to the stomach.25 While each of the Gli proteins has evolved to function independently, as evident from unique expression patterns and null phenotypes, there is also a level of redundancy between them. This is evident from the observation of increased severity of developmental defects in combination null mutants. One interesting double knockout recently published was the combined shh-/- and gli3-/- double knockout mouse.27 These mice actually show a pulmonary phenotype that is less severe than the shh null lung. The shh/gli3 double null lungs showed enhanced vasculogenesis and growth potential. There was also an increase in Cyclin D1 expression in both the epithelium and mesenchyme compared with the shh null lung. Wnt2, fox1, tbx2 and tbx3 (via fox1), have also been shown to be de-repressed by the removal of gli3 from the shh null lung. The de-repression of these genes could explain the less severe growth defects seen in these double transgenic lungs. Perhaps the most interesting finding was that the levels of the truncated repressor form of Gli3 (Gli3R), were much higher in shh null lungs than wild-type. This could explain the reduction in phenotypic severity of the shh-/-;gli3-/- lung as the Gli3R level would no longer increase but be absent. Therefore, since no more Gli3R is present, the effect of increase in Gli3 repressor function in the shhnull lung over wild-type levels is abrogated, thus decreasing phenotypic severity.
It is worth taking a closer look at a few of the other signaling factors in how they relate to certain aspects of lung development and the potential for interaction with the Hedgehog signaling network. Several Bmps, members of the transforming growth factor-β (Tgf-β) superfamily, have been found in the lung and they include Bmp4, Bmp5, and Bmp7. Bmp5 is expressed throughout the embryonic lung mesenchyme, however null mutants show no pulmonary aberrations, and over-expression of Bmp5 in the lung has not yet been examined.28Bmp7 was found to be ubiquitously expressed in the lung endoderm and the null phenotype is quite severe, however no lung defects were reported.29 On the other hand, Bmp4 has been shown to have significant effects on proper lung formation. Bmp4 expression is similar to shh expression in that it is found primarily at the developing tips in the distal endoderm, but its expression has also been established in the adjacent mesenchyme.4 A bmp4 null mutant has been created, however the embryo does not live long enough to see potential effects on lung development as it dies between 6.5 and 9.5 dpc.30,31 Conversely, an over-expression model for bmp4 has been created in which bmp4 is mis-expressed by the SP-C promoter. These lungs are smaller, show a reduction in the amount of branching, have distended terminal buds and also show defects in differentiation with a lower proportion of type II alveolar cells later in gestation.4 Closer inspection revealed no differences in shh expression suggestive that BMP4 has no direct regulatory effects on shh. The Fgf family and its receptors have also been shown to be major players in lung branching morphogenesis. Both Fgf1 and Fgf7 have been shown to have effects on lung development in lung culture systems.32,33 While both induce lung growth, Fgf7 appears to have greater proliferative effects. Fgf1 was also shown to have some minor effects on lung differentiation, but Fgf7 again proved to be the more potent inducer of differentiation by inducing both surfactant proteins A and B, as well as the appearance of clusters of lamellar bodies.34 This was also shown to be true in the in vivo system, as mice containing a construct over-expressing Fgf7 in the lung by the SP-C promoter, produce cyst-like structures and show differentiation markers.35 However, an Fgf7 null mouse has been created and no lung phenotype was evident, suggesting it is not essential to lung development or can be compensated by other growth factors.36 Fgf10 on the other hand, is a crucial growth factor pertaining to lung development. Expressed as early as 9.75 dpc, fgf10 is localized to the distal mesenchyme surrounding the developing lung buds. Expression is quite dynamic, appearing to precede lung bud growth in that it is expressed in areas of the mesenchyme where the next lung bud will form, suggesting interactions between the developing epithelium and adjacent mesenchyme regulate its expression.37 Recent studies have implicated a couple T-box genes in regulating the expression of Fgf10. Several T-box genes have been found to be expressed in the lung, with tbx1 restricted to the epithelium and tbx2-5 in the surrounding mesenchyme.38 By using antisense oligonucleotides to hinder gene expression, it was found that inhibition of tbx4 and tbx5 resulted in a dramatic reduction in branching of early embryonic lung cultures, whereas inhibition of tbx2 and tbx3 failed to show any effect on branching morphogenesis.39 Further inspection revealed that there was a loss of shh expression in the lung epithelium and that mesenchymal fgf10 expression was severely reduced in the lung cultures. Reintroduction of exogenous Fgf10 into the culture restored most of the branching defects suggesting that inhibition of tbx4 and tbx5 disrupts branching morphogenesis through Fgf10.39 Removal of fgf10 from the developing lung results in severe complications. Fgf10 null mice survive to birth but will quickly die due to lack of proper lung formation. The trachea forms in these mice, but ends in a mass of disorganized mesenchymal cells in which no primary lung buds are visible.40 An interesting connection between Fgf10 and Shh has been uncovered. Expression levels of fgf10 increase as development proceeds towards birth.6 This follows the opposite trend of shh expression, although recent studies do not agree with a reduction in shh expression at later lung gestation.7,8 Interestingly, over-expression of shh in the distal epithelium causes a reduction in fgf10 expression.37 This pattern of interaction suggests that Shh is a potential negative regulator of fgf10. However, the fgf10 null mouse shows no lung formation, with shh expression only in the rudimentary trachea, and not in the distal lung endoderm, indicating Fgf10 is upstream of shh.40 When experiments testing the effect of Fgf10 on shh expression using beads soaked with Fgf10 implanted in wild-type 11.5 dpc lung explants were performed, no changes in shh expression were detected.41 This is in contrast to more a recent finding in murine palate formation in which exogenous Fgf10 was shown to induce shh expression in wild-type palatal epithelium. 42 While the shh null lung phenotype develops further than the fgf10 null lung, as demonstrated by its ability to form two small lung lobes, the trachea and esophagus fail to separate signifying failure in other areas of pulmonary development. This contrasts the fgf10 null phenotype, in which the trachea and esophagus do manage to separate into individual tubes, but no further lung formation is evident. This suggests that any potential interaction between Shh and Fgf10 would be regulated by intermediate proteins. Furthermore, it has been suggested that the primary developmental functions of these two proteins most likely act in separate parallel pathways during lung development.43
If we now take a closer look at the Fgf receptors (Fgfr), further conclusions into a possible relation with Shh signaling can be drawn. While all four fgfrs are expressed in the postnatal lung, it is difficult to elucidate their functions through the generation of knockouts. Both fgfr-1 and fgfr-2 null mice die very early in development so effects on lung development can not be observed.44-46Fgfr-3 null mice do exhibit some skeletal and inner ear developmental defects, however, no lung phenotype has been reported to date.47 The fgfr-4 null phenotype is by far the most mild, as these animals show no gross developmental abnormalities of any kind.48 However, the combined fgfr-3/fgfr-4 double homozygous null mutant mouse suffers from dwarfism, and failure to complete alveogenesis postnatally.48 Most interesting though, is a special fgfr-2 null mutant, the fgfr2b-/- (IIIb isoform), generated through fusion chimeras in which mutant embryonic stem cells were combined with wild-type tetraploid embryos to allow survival until birth. These mice fail to develop limbs and lungs, quite similar to the fgf10 null mouse.49 When murine fgfr2b-/- palate explants were given exogenous Fgf10, no induction of shh was observed contrary to effects observed in wild-type explants.42 This indicates that Fgf10 signalling from the mesenchyme, through the epithelial located Fgfr2b receptor regulates shh expression in the epithelium. If this holds true in the lung, this could explain previous observations suggesting a relationship between Fgf10 and Shh in the developing lung. While the previously discussed Fgf10 null mutations resulting in a reduction in shh expression fits this model, a possible explanation for shh over-expression causing a reduction in fgf10 now exists. The observed over-expression of shh in the distal epithelium causing a reduction in fgf10 expression could possibly be a form of negative feedback. If mesenchymal fgf10 expression causes an epithelial increase in shh expression signalled through the Fgfr2b receptor, so that Shh can now signal back to the developing mesenchyme to help direct morphogenesis, the prospect of a negative feedback loop to Fgf10 signaling to attenuate the shh induction cue could exist.
In summary, development of the lung is a complex process as it not only entails growth and differentiation processes like many other organs, but also the creation of a complicated series of branched airways with their associated vasculature to create the interface for gas exchange. Members of the Shh pathway are found at the epithelial/mesenchymal border and removal of these proteins can have devastating effects on growth and branching morphogenesis. Cross-talk between epithelium and mesenchyme is essential for cordinating growth and branching signals so that the two tissues will successfully grow to form one cohesive functioning unit (see fig. 2). Feedback mechanisms play an integral part in regulation of developmental signaling mechanisms and the Shh pathway has shown evidence of self-regulation through Ptc and Hip1. While clearly required for pulmonary development, our lack in comprehension of the Hedgehog signaling network and its interactions with other regulatory molecules, clearly demonstrates the need for further investigation.
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Martin Rutter and Martin Post*.
Landes Bioscience, Austin (TX)
Rutter M, Post M. Shh/Gli Signaling During Murine Lung Development. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.