help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on September 20, 2007, doi:10.1165/rcmb.2007-0246OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
2007-0246OCv1
38/3/269    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Burns, A. J.
Right arrow Articles by Barlow, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Burns, A. J.
Right arrow Articles by Barlow, A. J.
American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 269-275, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0246OC

Development of the Neural Crest–Derived Intrinsic Innervation of the Human Lung

Alan J. Burns1, Nikhil Thapar1 and Amanda J. Barlow1

1 Neural Development Unit, UCL Institute of Child Health, London, United Kingdom

Correspondence and requests for reprints should be addressed to Alan J. Burns, Ph.D., Neural Development Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. E-mail: A.Burns{at}ich.ucl.ac.uk


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The formation of neural tissue, in association with airway smooth muscle (ASM), is a feature of normal lung development and function. Intrinsic neuronal tissue has recently been shown, in animal models, to be derived from neural crest cells (NCC). Since defects in NCC development underlie a range of disease states (neurocristopathies), it is important to determine the spatiotemporal development of NCC in the human lung, as defects in their development could have pathophysiologic implications. The aims of this study were to: (1) establish a time course for the formation of ASM and neural tissue within the embryonic and fetal human lung, (2) investigate whether intrinsic neural tissue within the lung is derived from NCC, and (3) gain insight into the possible signaling mechanisms underlying the development of the intrinsic lung innervation. Using human lung tissue from Weeks 6 to 12 of gestation, we analyzed the formation of ASM, NCC, neuronal and glial tissue, and the expression of Gfr{alpha}1, a receptor component of the RET (rearranged during transfection) tyrosine kinase signaling pathway. Our results showed that NCC accumulated along the branching airways, in close association with the ASM, and differentiated into neurons and glia. Neural crest–derived neural tissue within the lung strongly expressed membrane-bound Gfr{alpha}1, and soluble Gfr{alpha}1 was expressed within the lung mesenchyme, but only at early developmental stages. Together these findings indicate that the intrinsic innervation of the human lung is derived from the neural crest.

Key Words: neural crest cells • lung • intrinsic innervation • neurons • RET signaling



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We have shown that the development of intrinsic neural tissue in the human lung is derived from neural crest cells. This could lead to further investigations concerning neural crest–related defects in lung innervation that may be important in lung disease.

 
During embryogenesis, the lung buds arise by a process of evagination from a presumptive respiratory territory in the ventral wall of the primitive foregut. After initial bifurcation of the primordia, the entire bronchial tree and associated lung lobes are formed during a prolific branching period (1). From early in development (i.e., embryonic day [E] 11 in the mouse [2], Week 7.5–8 in humans [3]), the lung buds become invested with mesenchyme-derived airway smooth muscle (ASM) that encircles the epithelial buds and the bronchial tree (reviewed in Ref. 4). ASM rapidly becomes functionally active, exhibiting phasic contractions that persist throughout the prenatal period (5). This prenatal phasic activity is thought to play a role in the maturation of the lung by encouraging further myogenesis and lung growth (4–6).

Closely associated with the formation of ASM around the growing airways is the development of extensive networks of neural tissue in the form of ganglia, which contain neurons, glial cells, and nerve processes. Although the development of neural tissue in the lung has been documented in a range of species including chick (7), mouse (2), rabbit (8), pig (9), and human (3), its embryologic origin remained unclear for many years. Since the lung bud primordia undergo evagination and begin branching concomitant with the rostrocaudal migration of vagal neural crest cells (NCC) along the foregut to form the enteric nervous system (ENS) of the gastrointestinal tract (reviewed in Ref. 10), it was postulated that some of these NCC may also colonize the lung and subsequently form neurons (11). We recently confirmed this hypothesis using quail-chick interspecies grafting to fate map NCC. Experiments showed that neural tissue within the lung originates from vagal NCC and that these cells migrate from the foregut into the developing lung buds, where they differentiate into neurons and glia (7).

To date, the signaling cues that direct a subpopulation of vagal NCC to colonize the lung buds have not been clearly identified. However, there is extensive information concerning the molecular mechanisms involved in vagal NC–derived ENS formation along the gut. These include transcription factors (e.g., Sox10, Phox2b), and the RET (rearranged during transfection) and EdnrB signaling pathways (reviewed in Ref. 12). Based on this published data, and specifically on experiments demonstrating that the ligand for the RET receptor, glial cell line–derived neurotrophic factor (GDNF), acts as a chemoattractant for NCC within the gut (13, 14) and lung (15), signaling through this pathway is likely to influence NCC colonization of the lung.

Interestingly, RET has also been implicated in the development of the neural pathway of respiratory carbon dioxide (CO2) chemosensitivity, since Ret knockout mice, which have a range of NCC-derived autonomic nervous system defects, also have a depressed ventilatory response to inhaled CO2 (16). Further, a significant proportion of human patients with congenital central hypoventilation syndrome (CCHS), a rare syndrome defined as the failure of automatic control of breathing (reviewed in Ref. 17), also have Hirschsprung's disease (HSCR) (reviewed in Ref. 18), a congenital defect resulting from the premature arrest of NCC migration along the gastrointestinal tract (19–21). The primary causative gene in HSCR is RET (22–24), and mutations in RET have been documented in patients with both CCHS and HSCR (25). It is therefore possible that not only is RET signaling important for NCC development in the lung, but that developmental defects in the NC-derived intrinsic innervation of the lung could play as yet unidentified key roles in a range of respiratory disease conditions.

The following study was undertaken to: (1) establish a time course for the development of ASM and neural tissue within the embryonic and fetal human lung, (2) investigate whether intrinsic neural tissue within the lung is derived from NCC, and (3) gain insight into the possible signaling mechanisms underlying the development of the innervation of the lung. To our knowledge, this is the first article addressing the origin and potential developmental mechanisms of the intrinsic innervation of the human lung.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Human Embryonic and Fetal Material
Human material, acquired from ultrasound-guided surgical terminations of pregnancy, was obtained from the Human Developmental Biology Resource (HDBR), a tissue bank funded jointly by the Medical Research Council (MRC) and the Wellcome Trust. Consent and ethical approval from local committees was in place to collect human embryonic (up to Week 8) and fetal (Weeks 9–36) tissue up to Week 12 of development from consenting healthy mothers over 18 years of age. At least three different lung samples were examined for each time point, apart from Week 6, where two samples were analyzed, since it was rare to obtain tissue at such an early stage. However, the reproducibility of staining was found to be very consistent between the different samples examined at each stage of development.

Staging of embryos was performed according to the Carnegie system (26) using anatomical features and foot length as guides to age, which was translated into the number of weeks of development. In embryos and fetuses aged between 6 and 12 weeks, the lung buds and adjoining trachea were dissected free from surrounding tissue and processed for frozen sectioning and immunohistochemistry. Lung tissues were chemically fixed in 4% paraformaldehyde (PFA) in PBS for 3 to 4 hours to overnight, depending on size. They were then rinsed repeatedly in PBS (3 x 5 min) and placed overnight in 15% sucrose solution in PBS, then transferred to a solution containing 15% sucrose, 5% gelatin in PBS at 37°C. Tissues were then placed in blocks, oriented appropriately in the cooling gelatin solution, and frozen in isopentane, precooled in liquid nitrogen to –60°C. Frozen blocks were stored at –80°C until required. Cryosections, at a thickness of 15 µm, were cut from whole embryos and dissected lungs, collected on Superfrost Plus microscope slides (VWR International, Lutterworth, UK), and then stored at –20°C.

Immunohistochemistry
Frozen sections were incubated for 30 minutes in PBS at 37°C to remove the cryoprotectant then treated with PBS + 10% heat-inactivated sheep serum (HISS) + 0.1% Triton-X-100 (blocking solution) for 30 minutes at room temperature (RT). Primary antibodies (Table 1), diluted in blocking solution, were applied for 4 hours at RT in a humid box. Slides were then washed in PBS (3 x 5 min) and incubated with the appropriate fluorescently tagged secondary antibodies (Table 2) for 1.5 hours at RT. After further washing in PBS (3 x 5 min), the slides were mounted with Vectashield with DAPI (Vector Laboratories, Peterborough, UK). Images were captured on a Zeiss Axiophot microscope (Zeiss, Welwyn Garden City, UK) equipped with a Leica DC-500 digital camera, using Leica Firecam software (Leica, Milton Keynes, UK). Figures were assembled and annotated using Adobe Photoshop CS software (Adobe, San Jose, CA).


View this table:
[in this window]
[in a new window]

  		TABLE 1. PRIMARY ANTIBODIES

 

View this table:
[in this window]
[in a new window]

  		TABLE 2. SECONDARY ANTIBODIES

 

   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Development of ASM
ASM was identified within the developing lung buds using anti–{alpha} smooth muscle actin (SMA) immunolabeling. At Week 6 of development, a band of diffuse SMA staining was evident along the branching bronchi and surrounding epithelial tubules in transverse sections (Figure 1A). Epithelial tubules were not afforded with SMA along their entire length, since in more distal lung tissue, some tubules had no SMA-associated staining (Figure 1B). Similar patterns of SMA staining were also observed at Week 8 (Figures 1C and 1D), as were described at Week 6. At Week 10, SMA immunoreactivity was more discrete and occurred as a fine layer along branching bronchi (Figure 1E). High magnification revealed SMA+ fine muscle bands immediately underneath the epithelium of the epithelial tubules (Figure 1F). Similar staining patterns were apparent at Week 12 (Figure 1G). When epithelial tubules were sectioned obliquely, discrete SMA+ muscle bundles were seen to encircle the tubules (Figure 1H), although such staining was not apparent along their entire length as reflected by the absence of SMA staining on occasional tubule cross-sections (Figure 1G).


Figure 1
View larger version (84K):
[in this window]
[in a new window]

  		Figure 1. Smooth muscle actin (SMA) immunostaining of human lung sections from Weeks 6, 8, 10, and 12. At Week 6 (A, B) and Week 8 (C, D) a diffuse band of SMA+ staining occurs adjacent to the branching bronchi, and surrounds some epithelial tubules in cross-section. Not all epithelial tubules are surrounded by SMA staining (D, arrow). (E, F) At Week 10, more discrete SMA+ staining is evident adjacent to the bronchi. High magnification reveals SMA+ fibers (F, arrowheads) underneath the epithelium of the epithelial tubules. (G, H) At Week 12, the majority of epithelial tubule profiles are SMA+ (G). Epithelial tubules sectioned obliquely reveal SMA+ muscle fiber bundles running circumferentially around the tubules (H, arrows). BB, branching bronchi; EP, epithelium; ET, epithelial tubules. Scale bar = 200 µm in A, C, E, and G; 100 µm in B, D, F, and H.

 
Development of NCC and Neural Tissue
At Week 6 of development, immunostaining with the low-affinity neurotrophin receptor p75NTR, which labels cells of neural crest origin (7, 27), revealed major fiber tracts adjacent to the primitive branching bronchi, and finer fibers in close association with the epithelial tubules (Figures 2A and 2B). This staining pattern was co-incident with anti-neuronal class III β-Tubulin (TuJ1), which revealed nerve fibers in similar locations (Figures 2C and 2D). At Week 8, an extensive network of p75+ nerve bundles and fibers surrounded the bronchi, where they entered the lungs (Figure 2E). These nerve bundles were often associated with groups of neuronal cell bodies (ganglia) in the region underlying the epithelial layer (Figure 2H). The distribution of these p75+ cells was mirrored by the expression of TuJ1 in nerve trunks, ganglia, and neuronal cell bodies (Figures 2H and 2I). In transverse sections that exposed large numbers of epithelial tubules in profile, the majority of tubules were encircled by very fine caliber TuJ1+ nerve fibers (Figure 2J). However, in these same regions, p75 immunostaining was less discrete and appeared as a diffuse ring of fluorescently labeled cells surrounding some of the epithelial tubules (Figure 2G).


Figure 2
View larger version (74K):
[in this window]
[in a new window]

  		Figure 2. p75 and TuJ1 immunolabeling of human lung sections at Week 6 and Week 8. (A, B) p75+ nerve fibers (arrows) occur adjacent to branching bronchi and surrounding epithelial tubules (arrowheads). (C, D) TuJ1+ fibers are present in a similar pattern to p75 immunostaining. (E, F) p75+ nerve fibers and ganglia (arrows) associate closely with branching bronchi. (G) A diffuse ring of p75+ staining (arrowheads) surrounds epithelial tubules in transverse section. (H) TuJ1+ ganglia (arrows) and nerve fibers are adjacent to branching bronchi. (I) Individual TuJ1+ neurons (arrows) are apparent within ganglia, located in the layers underlying the epithelium of the bronchi. (J) Fine-caliber TuJ1+ nerve fibers (arrowheads) surround the majority of epithelial tubules. BB, branching bronchi; EP, epithelium; ET, epithelial tubules. Scale bar = 200 µm in all panels except B, D, and F (100 µm) and I (50 µm).

 
Similar patterns of p75 and TuJ1 immunostaining were observed through Weeks 10 (Figure 3) and 12 (Figure 4). p75+ and TuJ1+ nerve trunks and ganglia were present adjacent to branching bronchi within the lungs (Figures 3A, 3B, 3D, 3E, 4A, 4B, 4D, and 4E). Oblique sections revealed that the bronchi were encircled by an intricate interconnected network of TuJ1+ fibers (Figure 4E). However, TuJ1+ nerve fibers surrounding the epithelial tubules became more sparse from Week 10 to Week 12. At Week 10, TuJ1+ nerve fibers encircled the majority of tubules (Figure 3F), whereas at Week 12 TuJ1 staining was only apparent around occasional tubules (Figure 4F). These general patterns of staining were mirrored by p75 at Week 10 (Figure 3F) and Week 12 (Figure 4F), although p75 immunolabeling was generally more diffuse than TuJ1.


Figure 3
View larger version (139K):
[in this window]
[in a new window]

  		Figure 3. p75 and TuJ1 immunolabeling of human lung sections at Week 10. (A, B) p75+ nerve fibers and ganglia (arrows) occur in close association with branching bronchi. (C) Punctate (arrowheads) and diffuse (*) p75 labeling surrounds epithelial tubules in transverse section. (D) TuJ1+ nerve fibers (arrows) and ganglia (E, arrows) lie adjacent to branching bronchi. (F) Fine-caliber TuJ1+ nerve fibers surround many epithelial tubules (arrowheads), although some tubules have no associated staining. BB, branching bronchi; ET, epithelial tubules. Scale bar = 200 µm in D, C, and F; 100 µm in A, B, and E.

 

Figure 4
View larger version (128K):
[in this window]
[in a new window]

  		Figure 4. p75 and TuJ1 immunolabeling of human lung sections at Week 12. (A) p75+ nerve fibers and ganglia (arrows) occur in close association with branching bronchi. (B) p75+ ganglia frequently occur in close association (arrows) with the epithelial layer of the bronchi. (C) p75 labeling occurs as punctate staining (arrowheads) around larger diameter epithelial tubules. More diffuse p75 labeling (*) is also present close to some epithelial tubules. (D) TuJ1+ nerve fibers (arrows) run adjacent to branching bronchi. (E) On oblique sections of bronchi (white dashed outline), TuJ1+ nerve fibers form an interconnected network. (F) Fine-caliber TuJ1+ nerve fibers, occurring in a punctate pattern (arrowheads), surround occasional epithelial tubules. BB, branching bronchi; ET, epithelial tubules; EP, epithelium. Scale bar = 200 µm in A and D; 100 µm in B, C, E, and F.

 
To confirm that the nerve cell bodies and fibers were of NCC origin, double immunohistochemical labeling was performed on lung tissue at Week 10 of development (Figure 5). These experiments revealed that the vast majority of labeled cells and fibers co-expressed TuJ1 and p75 (Figures 5C and 5F). Only a small number of p75+ cells did not express TuJ1 (Figures 5B and 5E, arrows), thus demonstrating that a subpopulation of NCC was either undifferentiated at this developmental stage (Week 10), or that they had differentiated into glia (see Figure E1 in the online supplement), as previously described in the developing chick lung (7).


Figure 5
View larger version (122K):
[in this window]
[in a new window]

  		Figure 5. p75 and TuJ1 double immunolabeling of human lung sections at Week 10. Merging p75 (A, D) and TuJ1 (B, E) reveals the co-expression of these markers (C, F) in ganglia adjacent to larger airways (C), and in fibers surrounding smaller-caliber tubules (D). However, occasional p75+ cells (A, D; arrows) are TuJ1-ve (B, C, E, F; arrows), thus suggesting that not all NC-derived cells differentiate into neurons. Scale bar = 50 µm.

 
To demonstrate the spatial relationship between ASM and neural tissue, double immunohistochemical labeling was performed using anti-SMA to label smooth muscle, along with the pan-neuronal marker TuJ1. Oblique sections of branching bronchi showed that ASM was located close to the airway epithelium, and that neuronal tissue, including nerve fibers and ganglia, was positioned beneath the ASM (Figure 6A). Nerve fibers were occasionally found to penetrate the ASM such that neural tissue was present on both sides of the ASM and between the fiber bundles (Figure 6B). When transverse sections of epithelial tubules were double stained, a ring of SMA+ fibers was shown to encircle the tubules, immediately underneath the airway epithelium (Figures 6C and 6D). Although neural tissue was closely associated with the ASM, it was located adjacent to the outer layers of the ASM, away from the epithelium. The airway epithelium is thus surrounded by ASM, with neural tissue positioned adjacent to the outer layers of the ASM.


Figure 6
View larger version (123K):
[in this window]
[in a new window]

  		Figure 6. SMA and TuJ1 double immunolabeling of human lung sections at Week 10. (A, B) Oblique sections of branching bronchioles show that SMA (A; red staining, arrowheads) is located adjacent to the airway epithelium, and that TuJ1+ neural tissue (green staining, arrows) is external to the SMA. Occasional TuJ1+ nerve fibers penetrate the ASM, and pass from one side of the muscle bundles to the other (B, arrowheads). (C, D) Transverse sections of epithelial tubules reveal SMA immediately underneath the airway epithelium, encircling the tubules, and TuJ1+ neural tissue outside the muscle layers. At higher magnification, neuronal cell bodies (D, arrowheads) are evident within ganglia adjacent to the ASM. Scale bar = 100 µm in A–C, 50 µm in D.

 
Expression of the RET Co-Receptor Gfr{alpha}1
In all stages of human lung examined in this study (i.e., from Week 6 to Week 12), Gfr{alpha}1 immunostaining was present within ganglia, where it labeled neuronal cell bodies, and nerve fibers that occurred in close association with the epithelial layers of the branching bronchi (Figures 7A, 7C, 7E, and 7G). Double immunohistochemical staining using anti-Gfr{alpha}1 and TuJ1 confirmed that Gfr{alpha}1 co-localizes with neural tissue (Figures 7H–7J). However, over the time period examined in this study, the extent of Gfr{alpha}1+ fine nerve fibers associated with the epithelial tubules decreased dramatically. For example, at Week 8, transverse sections revealed that the majority of tubules were encircled by Gfr{alpha}1+ fibers, and occasional small ganglia containing immunopositive nerve cell bodies were also present (Figure 7D). Through Weeks 10 and 12, the level of Gfr{alpha}1+ staining decreased such that at Week 12, only occasional profiles of epithelial tubules had Gfr{alpha}1+ associated nerve fibers (Figure 7H). We also observed a dynamic expression of soluble Gfr{alpha}1 within the lung mesenchyme similar to that described previously in the chick (7). At Week 6, weak Gfr{alpha}1 immunostaining was present throughout the lung mesenchyme (Figure 7B). This staining decreased in intensity by Week 8 (Figure 7D), and was not detected at later stages (Figures 7F and 7H).


Figure 7
View larger version (65K):
[in this window]
[in a new window]

  		Figure 7. Gfr{alpha}1 immunostaining of human lung sections from weeks 6, 8, 10, and 12. Immunolabeling of ganglia, adjacent to the epithelium of bronchi and epithelial tubules, is consistent through Weeks 6–12 (A, C, E, F), such that individual cells (presumptive neurons) can be identified (A, C, E, F; arrows). The extent of Gfr{alpha}1 labeling of nerve fibers changes over this time period; at Weeks 6 and 8, extensive Gfr{alpha}1+ fibers surround epithelial tubes (D, arrowheads). By Week 10 (F) and Week 12 (H), only extremely fine-caliber Gfr{alpha}1+ fibers are present adjacent to occasional epithelial tubules (F, H; arrowheads). Low-level Gfr{alpha}1+ staining is also present within the lung mesenchyme at Weeks 6 and 8 (B, D; diffuse red staining), but is absent at Weeks 10 and 12. Double immunohistochemical labeling at Week 10 confirms that Gfr{alpha}1 is expressed by neural tissue. Gfr{alpha}1+ (I) and TuJ1+ (J) tissue entirely overlap when merged (J). BB, branching bronchi; EP, epithelium; ET, epithelial tubules. Scale bar = 100 µm in all panels except for A and G (50 µm), and I (100 µm).

 
Development of Glial Cells
Glial fibrillary acidic protein (GFAP) was used to identify glial cells within the developing lung. At Week 6, extrinsic nerve trunks, located outside the lung tissue, were strongly GFAP+ (Figure E1A). This tissue may represent the vagus nerve, based on its location adjacent to the foregut and bronchi (Figure E1A). Within the lung, GFAP staining was only apparent in weakly immunopositive fibers in close proximity to the epithelial tubules (Figure E1B). At Week 8, GFAP staining increased, and groups of immunopositive cells, and fine fibers, were apparent in close association with epithelial tubules (Figures E1C and E1D). Through Weeks 10 and 12, GFAP staining became progressively more discrete. At Week 10, GFAP immunolabeling was restricted to a small number of cells and fibers adjacent to large and small diameter epithelial tubules (Figures E1E and E1F), while at Week 12, only occasional cells and very fine cell extension processes were GFAP+ (Figures E1G and E1H).


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In this study of early human lung development we have shown that: (1) rapidly maturing ASM is closely associated with the branching bronchi and epithelial tubules from Week 6 of development; (2) an extensive neural network, consisting of nerve cell bodies and glia organized into ganglia, forms around the bronchi and epithelial tubules in close association with the ASM; (3) neural tissue co-localizes with a marker for NCC, indicating that the intrinsic neural tissue of the lung is derived from the neural crest; and (4) the NC-derived tissue expresses Gfr{alpha}1, a receptor component of the RET signaling pathway. Our findings therefore suggest that the intrinsic neurons and glial cells that closely associate with the ASM of the human lung arise from NCC, and that Gfr{alpha}1, a co-receptor component of the RET signaling pathway, may be involved in their development.

Development of the Airway Neuromusculature
ASM has been shown to envelop the developing bronchial tree in a range of species, including the mouse (2), pig (28), and human (3). Initially, branching epithelial tubules become covered with a thin layer of ASM that extends to the base of the terminal sacs (3), with muscle bundles occurring in a circumferential orientation, perpendicular to the long axis of the tubules (2–4). Such bundles, stained with {alpha}-SMA and calponin, have been described as early as Embryonic Day (E)11 in the mouse (2), and using {alpha}-SMA, at Day 53 of gestation (Week 7+) in the human (3). Previous studies in animals such as the mouse (2) and pig (28) have also demonstrated the close association between the development of neural tissue and of ASM. For example, in the E12 mouse, Tollet and coworkers (2) identified abundant neural tissue in the proximal lung and showed that nerve fibers followed the ASM-covered tubules. In the human, at Day 53 of gestation, Sparrow and colleagues (3) demonstrated an extensive plexus of nerve trunks, ganglia, and Schwann cells surrounding the ASM. Here we have demonstrated strong p75 (NCC) and TuJ1 (neural tissue) immunostaining in the human lung at Week 6 of development, a stage at which {alpha}-SMA–immunopositive cells are already present. Since we were not able to obtain human embryonic tissue earlier than Week 6, we could not ascertain when the first {alpha}-SMA+, p75+, or TuJ1+ cells appear in the human lung. However, our findings, and results from the studies mentioned above, strongly suggest that the development of ASM and NC-derived neural tissue is virtually concomitant.

NCC: Origin of the Intrinsic Innervation of the Lung
Here we have shown that p75+ NCC are present within the human lung at Week 6 of development, and at this and all subsequent stages of development examined, TuJ1+ neural tissue is co-incident with the spatiotemporal distribution of p75+ NCC, indicating a neural crest origin for neural tissue within the human lung. Previous studies of the development of neural tissue in the embryonic mouse lung have described the presence of p75+ cells within the vagus nerves and the lung buds (2). These authors suggested that in addition to innervating lung ganglia, the vagus nerves provided a conduit into the lung for migrating NCC. It is difficult to differentiate between potential migration routes of NCC into the lungs using fixed tissue sections and immunostaining with p75, since this antibody identifies all neural tissue of neural crest origin (vagal NCC in the gut, NCC from the vagus, fibers from sensory ganglia). Possibilities include: (1) NCC may migrate from the foregut into the lung buds, as suggested by the findings of our previous study using quail–chick interspecies grafting (7); (2) NCC may migrate from the vagus nerves into the lungs as suggested by Tollet and coworkers (2) and supported by the findings of Baetge and colleagues (29), who showed that neural precursors are present in the vagus nerve and that in vitro these precursors are capable of migration, ganglion formation, and differentiation into neuronal phenotypes; and (3) a combination of (1) and (2). An in vivo imaging approach, for example, using gene electroporation or DiI injection in chick embryos to selectively label NCC (30, 31), or organotypic cultures using foregut and lung tissues from transgenic mice (e.g., ROSA26 YFPStop;Wnt1Cre, in which all NCC express yellow fluorescent protein) (32), would greatly facilitate fate analysis of the NCC that colonize the lung and would help to distinguish between the possible migration pathways outlined above.

Colonization of the Lung Buds by NCC: Possible Signaling Mechanisms
Although there is little information about the signaling mechanisms that direct NCC into the lung, mechanisms underlying the development of the NC-derived precursors that migrate along the gut have received much attention in the literature (for review see Ref. 12). One of the most likely candidates as a chemoattractant for NCC into the lung is GDNF, the ligand for the tyrosine kinase receptor RET (33). Support for this idea comes from experiments which have shown that GDNF, expressed in the gut wall, is a chemoattractant for RET-expressing NCC (13, 14), and that GDNF-impregnated beads, placed in mouse lung buds grown in organ culture, attracted neuronal precursors and influenced the direction of neurite extension (15). In our previous study in the chick (7), RET and its co-receptor Gfr{alpha}1 were found to be expressed on NCC within the lung throughout development, thus supporting this hypothesis, although GDNF was not detected in the lung mesenchyme (7). In our current study of the human lung, following exhaustive investigation using different antibodies, we did not detect RET within NCC in the lung (data not shown), and also for technical reasons we were not able to analyze the expression of GDNF within the lung. However, Gfr{alpha}1 was found to be strongly expressed in the neural tissue of the human lung throughout the developmental stages examined, and it was weakly expressed in the lung mesenchyme at the earlier stages of development (Weeks 6 and 8). Thus, in our previous (7) and current study, we observed secreted Gfr{alpha}1 to be expressed at low levels in the lung mesenchyme. Since Gfr{alpha}1 has been shown to act as a long-range directional cue for axons of peripheral neurons that express RET (34), it is possible that a developmental signaling mechanism may be operating through a receptor component of the RET pathway, largely mediated by Gfr{alpha}1, and possibly in combination with GDNF and/or other as yet unidentified ligands.

Implications for NCC Origin of Neural Tissue in the Lung: Potential Roles in Lung Disease
To our knowledge, this is the first study to demonstrate that intrinsic neural tissue within the human lung originates from NCC. This, and our previous findings (7) concerning the possible role of the RET signaling pathway for NC-derived formation of intrinsic ganglia in the lung, could open the door to a host of further investigations concerning potential neural crest–related defects in lung innervation that may be important in lung disease. For example, within the lung, neuroepithelial bodies (NEBS) (35), which have been suggested to act as hypoxia-sensitive airway chemoreceptors (36), have been shown to be extensively innervated by different nerve fibers. These include extrinsic vagal sensory afferents and unmyelinated sensory nerve fibers (37), and nitrergic terminals that originate in nNOS-containing intrinsic neurons (38). The latter authors have proposed that in healthy lungs, NO may modulate the afferent transmission of NEBS and/or be involved in homeostatic regulation of local blood flow by influencing release of neuroendocrine peptides from NEBS (38). Thus, a congenital deficiency of intrinsic neurons in the lung could have profound effects in NEBS-mediated oxygen sensing and/or pulmonary vasoconstriction and hypertension. Indeed, recent attention has focused on the potential involvement of NEBS in Sudden Infant Death Syndrome (SIDS), since hypertrophy and hyperplasia of NEBS have been documented in infants who have died from SIDS compared with control subjects (39).

As mentioned previously, in the developmental disorder Congenital Central Hypoventilation Syndrome (CCHS), there is an absence of adequate autonomic control of respiration with decreased sensitivity to hypoxia and hypercapnia. Although recent reports have identified mutation of the paired homeobox protein 2b (PHOX2B) gene as playing a major role in CCHS (40), a significant percentage of patients with CCHS have the NC-derived aganglionic gut disorder HSCR and mutations in RET (20, 41, 42). Ret-null mice, which have gut aganglionosis, also have a depressed ventilatory response to inhaled CO2 (16). Together, these data highlight the influence of genetic factors in breathing control. Close examination of the developing lungs of mice with mutations in Ret, Grf{alpha}1, or other genes necessary for NCC development, will not only give insight to the signaling mechanisms underlying the development of the NC-derived intrinsic ganglia of the lung, but may also address their functional significance in health and disease before and after birth.


   Acknowledgments
 
The authors thank the Medical Research Council (MRC) and the Wellcome Trust-funded Human Developmental Biology Resource (HDBR) for provision of human embryonic and fetal material.


   Footnotes
 
This work was partly funded by a research grant from The Royal Society awarded to A.J.B.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2007-0246OC on September 20, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form June 28, 2007

Accepted in final form September 7, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Chinoy MR. Lung growth and development. Front Biosci 2003;8:d392–d415.[Medline]
  2. Tollet J, Everett AW, Sparrow MP. Spatial and temporal distribution of nerves, ganglia, and smooth muscle during the early pseudoglandular stage of fetal mouse lung development. Dev Dyn 2001;221:48–60.[CrossRef][Medline]
  3. Sparrow MP, Weichselbaum M, McCray PB. Development of the innervation and airway smooth muscle in human fetal lung. Am J Respir Cell Mol Biol 1999;20:550–560.[Abstract/Free Full Text]
  4. Sparrow MP, Lamb JP. Ontogeny of airway smooth muscle: Structure, innervation, myogenesis and function in the fetal lung. Respir Physiolo Neurobiol 2003;137:361–372.[CrossRef][Medline]
  5. Schittny JC, Miserocchi G, Sparrow MP. Spontaneous peristaltic airway contractions propel lung liquid through the bronchial tree of intact and fetal lung explants. Am J Respir Cell Mol Biol 2000;23:11–18.[Abstract/Free Full Text]
  6. McCray PB Jr. Spontaneous contractility of human fetal airway smooth muscle. Am J Respir Cell Mol Biol 1993;8:573–580.[Medline]
  7. Burns AJ, Delalande JM. Neural crest cell origin for intrinsic ganglia of the developing chicken lung. Dev Biol 2005;277:63–79.[CrossRef][Medline]
  8. Hung KS. Innervation of rabbit fetal lungs. Am J Anat 1980;159:73–83.[CrossRef][Medline]
  9. Weichselbaum M, Sparrow MP. A confocal microscopic study of the formation of ganglia in the airways of fetal pig lung. Am J Respir Cell Mol Biol 1999;21:607–620.[Abstract/Free Full Text]
  10. Burns AJ. Migration of neural crest-derived enteric nervous system precursor cells to and within the gastrointestinal tract. Int J Dev Biol 2005;49:143–150.[CrossRef][Medline]
  11. Dey R, Hung K-S. Development of innervation in the lung. In: McDonald J, editor. Lung growth and development. New York: Dekker; 1997. pp. 244–265.
  12. Young HM, Newgreen D, Burns AJ. The development of the enteric nervous system in relation to hirschsprung's disease. In: Ferretti P, Copp AJ, Tickle C, Moore G, editors. Embryos, genes and birth defects, 2nd ed. Chichester: John Wiley and Sons; 2006. pp. 263–300.
  13. Young HM, Hearn CJ, Farlie PG, Canty AJ, Thomas PQ, Newgreen DF. Gdnf is a chemoattractant for enteric neural cells. Dev Biol 2001;229:503–516.[CrossRef][Medline]
  14. Natarajan D, Marcos-Gutierrez C, Pachnis V, de Graaff E. Requirement of signalling by receptor tyrosine kinase ret for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis. Development 2002;129:5151–5160.[Medline]
  15. Tollet J, Everett AW, Sparrow MP. Development of neural tissue and airway smooth muscle in fetal mouse lung explants: a role for glial-derived neurotrophic factor in lung innervation. Am J Respir Cell Mol Biol 2002;26:420–429.[Abstract/Free Full Text]
  16. Burton MD, Kawashima A, Brayer JA, Kazemi H, Shannon DC, Schuchardt A, Costantini F, Pachnis V, Kinane TB. Ret proto-oncogene is important for the development of respiratory co2 sensitivity. J Auton Nerv Syst 1997;63:137–143.[CrossRef][Medline]
  17. Chen ML, Keens TG. Congenital central hypoventilation syndrome: not just another rare disorder. Paediatr Respir Rev 2004;5:182–189.[CrossRef][Medline]
  18. Swenson O. Hirschsprung's disease. Rev Pediatr 2002;109:914–918.
  19. Haddad GG, Mazza NM, Defendini R, Blanc WA, Driscoll JM, Epstein MA, Epstein RA, Mellins RB. Congenital failure of automatic control of ventilation, gastrointestinal motility and heart rate. Medicine 1978;57:517–526.[Medline]
  20. Croaker GD, Shi E, Simpson E, Cartmill T, Cass DT. Congenital central hypoventilation syndrome and hirschsprung's disease. Arch Dis Child 1998;78:316–322.[Abstract/Free Full Text]
  21. Rohrer T, Trachsel D, Engelcke G, Hammer J. Congenital central hypoventilation syndrome associated with hirschsprung's disease and neuroblastoma: case of multiple neurocristopathies. Pediatr Pulmonol 2002;33:71–76.[CrossRef][Medline]
  22. Edery P, Lyonnet S, Mulligan LM, Pelet A, Dow E, Abel L, Holder S, Nihoul-Fekete C, Ponder BA, Munnich A. Mutations of the ret proto-oncogene in hirschsprung's disease. Nature 1994;367:378–380.[CrossRef][Medline]
  23. Pelet A, Geneste O, Edery P, Pasini A, Chappuis S, Atti T, Munnich A, Lenoir G, Lyonnet S, Billaud M. Various mechanisms cause ret-mediated signaling defects in hirschsprung's disease. J Clin Invest 1998;101:1415–1423.[Medline]
  24. Lantieri F, Griseri P, Ceccherini I. Molecular mechanisms of ret-induced hirschsprung pathogenesis. Ann Med 2006;38:11–19.[CrossRef][Medline]
  25. Sakai T, Wakizaka A, Nirasawa Y. Congenital central hypoventilation syndrome associated with hirschsprung's disease: mutation analysis of the ret and endothelin-signaling pathways. Eur J Pediatr Surg 2001;11:335–337.[CrossRef][Medline]
  26. O'Rahilly R. Guide to the staging of human embryos. Anat Anz 1972;130:556–559.[Medline]
  27. Wallace AS, Burns AJ. Development of the enteric nervous system, smooth muscle and interstitial cells of cajal in the human gastrointestinal tract. Cell Tissue Res 2005;319:367–382.[CrossRef][Medline]
  28. Sparrow MP, Warwick SP, Everett AW. Innervation and function of the distal airways in the developing bronchial tree of fetal pig lung. Am J Respir Cell Mol Biol 1995;13:518–525.[Abstract]
  29. Baetge G, Schneider KA, Gershon MD. Development and persistence of catecholaminergic neurons in cultured explants of fetal murine vagus nerves and bowel. Development 1990;110:689–701.[Abstract/Free Full Text]
  30. Kulesa PM, Fraser SE. Neural crest cell dynamics revealed by time-lapse video microscopy of whole embryo chick explant cultures. Dev Biol 1998;204:327–344.[CrossRef][Medline]
  31. McLennan R, Kulesa PM. In vivo analysis reveals a critical role for neuropilin-1 in cranial neural crest cell migration in chick. Dev Biol 2007;301:227–239.[CrossRef][Medline]
  32. Druckenbrod NR, Epstein ML. The pattern of neural crest advance in the cecum and colon. Dev Biol 2005;287:125–133.[CrossRef][Medline]
  33. Manie S, Santoro M, Fusco A, Billaud M. The ret receptor: function in development and dysfunction in congenital malformation. Trends Genet 2001;17:580–589.[CrossRef][Medline]
  34. Ledda F, Paratcha G, Ibanez CF. Target-derived gfralpha1 as an attractive guidance signal for developing sensory and sympathetic axons via activation of cdk5. Neuron 2002;36:387–401.[CrossRef][Medline]
  35. Linnoila RI. Functional facets of the pulmonary neuroendocrine system. Lab Invest 2006;86:425–444.[CrossRef][Medline]
  36. Cutz E, Jackson A. Neuroepithelial bodies as airway oxygen sensors. Respir Physiol 1999;115:201–214.[CrossRef][Medline]
  37. Brouns I, Van Genechten J, Hayashi H, Gajda M, Gomi T, Burnstock G, Timmermans JP, Adriaensen D. Dual sensory innervation of pulmonary neuroepithelial bodies. Am J Respir Cell Mol Biol 2003;28:275–285.[Abstract/Free Full Text]
  38. Brouns I, Van Genechten J, Scheuermann DW, Timmermans JP, Adriaensen D. Neuroepithelial bodies: A morphologic substrate for the link between neuronal nitric oxide and sensitivity to airway hypoxia? J Comp Neurol 2002;449:343–354.[CrossRef][Medline]
  39. Cutz E, Perrin DG, Pan J, Haas EA, Krous HF. Pulmonary neuroendocrine cells and neuroepithelial bodies in sudden infant death syndrome: potential markers of airway chemoreceptor dysfunction. Pediatr Dev Pathol 2007;10:106–116.[CrossRef][Medline]
  40. Amiel J, Laudier B, Attie-Bitach T, Trang H, de Pontual L, Gener B, Trochet D, Etchevers H, Ray P, Simonneau M, et al. Polyalanine expansion and frameshift mutations of the paired-like homeobox gene phox2b in congenital central hypoventilation syndrome. Nat Genet 2003;33:459–461.[CrossRef][Medline]
  41. Bajaj R, Smith J, Trochet D, Pitkin J, Ouvrier R, Graf N, Sillence D, Kluckow M. Congenital central hypoventilation syndrome and hirschsprung's disease in an extremely preterm infant. Pediatrics 2005;115:e737–e738.[Abstract/Free Full Text]
  42. Fitze G, Paditz E, Schlafke M, Kuhlisch E, Roesner D, Schackert HK. Association of germline mutations and polymorphisms of the ret proto-oncogene with idiopathic congenital central hypoventilation syndrome in 33 patients. J Med Genet 2003;40:E10.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
2007-0246OCv1
38/3/269    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Burns, A. J.
Right arrow Articles by Barlow, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Burns, A. J.
Right arrow Articles by Barlow, A. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2008 American Thoracic Society.