Abstract
A classical study has revealed the general growth of the bronchial tree and its variations up to Carnegie stage (CS) 19. In the present study, we extended the morphological analysis CS by CS until the end of the embryonic period (CS23). A total of 48 samples between CS15 and CS23 belonging to the Kyoto Collection were used to acquire imaging data by performing phase‐contrast X‐ray computed tomography. Three‐dimensionally reconstructed bronchial trees revealed the timeline of morphogenesis during the embryonic period. Structures of the trachea and lobar bronchus showed no individual difference during the analyzed stages. The right superior lobar bronchus was formed after the generation of both the right middle lobar bronchus and the left superior lobar bronchus. The speed of formation of the segmental bronchi, sub‐segmental bronchi, and further generation seemed to vary among individual samples. The distribution of the end‐branch generation among five lobes was significantly different. The median branching generation value in the right middle lobe was significantly low compared with that of the other four lobes, whereas that of the right inferior lobe was significantly larger than that of both the right and left superior lobes. Variations found between CS20 and CS23 were all described in the human adult lung, indicating that variation in the bronchial tree may well arise during the embryonic period and continue throughout life. The data provided may contribute to a better understanding of bronchial tree formation during the human embryonic period.
Reconstructed embryonic bronchial tree formation during CS18 and CS23 was shown. Distribution of the end‐branch generation among five lobes significantly differed. We showed the morphological change of the bronchial tree and the end‐branch generation in each lobe during the human embryonic period.
1. INTRODUCTION
The bronchial tree of the human lung is composed of both conducting and respiratory airways (Standring, 2015). In particular, the conducting airway, located in the proximal portion of the lung, includes the main bronchus, lobar bronchus, segmental bronchus, and sub‐segmental bronchus. Although there is a prevailing typical structure of these central bronchi, many researchers have reported variations of branching in the human adult lung (Boyden and Hartmann, 1946; Boyden and Scannell, 1948; Boyden and Hamre, 1951; Yamashita, 1978; Ghaye et al., 2001; Nagashima et al., 2015; Nagashima et al., 2017). Recently, a large sample analysis implied that variations in the central airway, especially the segmental bronchus, are associated with chronic obstructive pulmonary disease (Smith et al., 2018). Thus, it is important to reveal the developmental mechanism of variations of the bronchial tree to understand potential disease mechanisms.
Although comprehensive changes in central bronchi formation have been revealed, few researchers have addressed both the timing and degree of individual variations in the human embryonic bronchial tree. Several morphological studies have investigated the development of the human lung during the embryonic period (Palmer, 1936; Streeter, 1942; 1945; Wells and Boyden, 1954; O'Rahilly and Boyden, 1973; Belle et al., 2017). The pulmonary primordium bud appears from the middle of the foregut at approximately Carnegie stage (CS) 12 (Ueno et al., 2016). The bud divides into the right and left primary bronchial buds at CS13 (Streeter, 1945). Subsequently, lobar buds are generated in each primary bronchial bud between CS15 and CS16 (Streeter, 1948). Segmental buds on branching trees appear at CS17, and the first generation of sub‐segmental bronchus is complete at CS19 (Wells and Boyden, 1954). Recently, a detailed three‐dimensional (3D) bronchial tree was prepared in a study; however, the study did not sufficiently analyze the morphogenesis of the proximal bronchi (Belle et al., 2017).
The present study aimed to describe the morphogenesis of the bronchial tree until the end of the human embryonic period. We observed in detail the altered morphology of the bronchial tree according to CS and showed individual variation on the proximal bronchial tree until the segmental bronchus.
2. METHODS
2.1. Human embryo specimens
Approximately 44,000 human embryos, constituting the Kyoto Collection, are stored at the Congenital Anomaly Research Center of Kyoto University (Nishimura et al., 1968; Shiota, 1991). In most cases, the pregnancies were terminated during the first trimester for socioeconomic reasons under the Maternity Protection Law of Japan. The samples were collected from 1963 to 1995 according to the regulations of each period. Aborted embryos brought to the laboratory were measured, examined, and staged using the criteria provided by O'Rahilly and Müller (1987). A total of 48 human embryos between CS15 and CS23 were selected from the Kyoto Collection (n = 7 each at CS15, CS16, CS17, CS18, and CS19; n = 4 each at CS20 and 21; n = 3 at CS22; n = 2 at CS23). None of the samples had overt damage or any anomalies.
2.2. Image acquisition and 3D reconstruction
The 3D phase‐contrast X‐ray computed tomography (PXCT) image acquisition conditions are described elsewhere (Yoneyama et al., 2011). Briefly, specimens were visualized with a phase‐contrast imaging system fitted with a crystal X‐ray interferometer. The system was set up at the vertical wiggler beamline (PF BL14C) of the Photon Factory in Tsukuba, Japan. PCXT data from selected embryos were analyzed in detail as serial two‐dimensional (2D) and reconstructed three‐dimensional (3D) images. The structure of the bronchial tree was reconstructed in all samples using amira software version 6.2.0 (Visage Imaging GmbH, Berlin, Germany) (Figure 1). The center of the airway was linearly shown with the centerline module. The coordinates were analyzed using matlab v. R2018a (MathWorks, Inc., Natick, MA, USA) to calculate the generation of all branches and the number of branches.
FIGURE 1.
Three‐dimensional reconstruction of the bronchial tree using 3D phase‐contrast X‐ray CT. (a) Representative 3D phase‐contrast X‐ray CT image in the transverse section at CS18 embryo (ID 18071). R, right; L, left; H, heart; Li, liver. (b) The bronchial tree was reconstructed. (c) Centerline was calculated based on reconstruction
2.3. Definition of the developmental phase for the bronchial tree during CS15 and CS19
All 35 samples during CS15 and CS19 were divided into the following five phases according to the degree of bronchus formation (Figure 2). In phase 1, the primary bronchus had no lobar swellings. The primary bronchus formed an almost symmetric Y‐shape. In phase 2, the bronchus had lobar swellings that emerged from the middle of each bud. The bronchial trees still exhibited almost total symmetry. In phase 3, the bronchus had all five distinct lobar swellings. The right and left primary bronchi showed characteristic asymmetry. In phase 4, the segmental bronchi began to emerge from the lobar bronchi, without any sub‐segmental bronchi. In phase 5, the sub‐segmental bronchi appeared in the segment. Samples that had any sub‐segmental bronchi were divided into phase 5.
FIGURE 2.
Five phases set based on the bronchial morphology. Idealized diagram of each phase is shown. Sample of phase 1 has two primary bronchial buds. In phase 2, the sample generates two lobar bronchi on each side. All lobar bronchi are completed in phase 3. Any sample with segmental bronchi was classified into phase 4. The sample in phase 5 contains any of the sub‐segmental bronchi. IB, intermediate bronchus; LIB, left inferior bronchus; LMB, left main bronchus; LPBB, left primary bronchial bud; RIB, right inferior bronchus; RMB, right main bronchus; RPBB, right primary bronchial bud; temRMB, temporary RMB, branch from the tracheal bifurcation to the base of the right middle lobe. The right superior lobar bronchus was given off at the middle of temRMB
2.4. Statistics
We calculated all end‐branch generations and median of the parameters. We used the Wilcoxon signed‐rank test to determine the deviation of the distributions among the five lobes. The test was run on SPSS v. 22 (IBM Corp., Armonk, NY, USA). A p‐value <.05 was considered statistically significant.
3. RESULTS
3.1. During CS15 and CS19
3.1.1. Distribution of the bronchial tree phases according to CS
Representative reconstructions in each phase are shown in Figure 3a. No samples showed an abnormal appearance of the branching tree, such as a tracheal bronchus.
FIGURE 3.
Representative reconstructions of the bronchial tree from phases 1 to 5 and variation of the embryonic bronchial tree. (a) Representation of the bronchial tree from phases 1 to 5 (frontal view). (b) The right inferior lobe with probable B8 (i) and with definite B8 (ii) (frontal view). When the bifurcation was only below B6 or B7, the lateral bronchus was probably classified into B8 and the medial bronchus into the bronchus that divided into B9 and B10. The bronchus was definitely classified into B8 when B8, B9, and B10 were generated. (c) The right middle lobe with trifurcate bronchus (i) and with bifurcate bronchus (ii) (lateral view). RMLB, the right middle lobar bronchus. Scale bar: 500 μm
Among the seven samples at CS15, we classified five into phase 1 and the remaining two into phase 2 (Table 1). From the seven samples in CS16, only one was classified into phase 2; the remaining six were classified into phase 3. All seven samples at CS17 were classified into phase 4. Regarding CS18, two samples were classified into phase 4 and the remaining five into phase 5. All seven samples at CS19 were classified into phase 5.
TABLE 1.
Distribution of five phases of the bronchial tree according to Carnegie stage (CS)
| Phase 1 | Phase 2 | Phase 3 | Phase 4 | Phase 5 | |
|---|---|---|---|---|---|
| CS15 | 5 | 2 | – | – | – |
| CS16 | – | 1 | 6 | – | – |
| CS17 | – | – | – | 7 | – |
| CS18 | – | – | – | 2 | 5 |
| CS19 | – | – | – | – | 7 |
| Total | 5 | 3 | 6 | 9 | 12 |
The developmental bronchial tree showed variations within the same phase (Tables 2 and 3). During phases 1 and 3, no variation between samples of any phase was observed. The samples were similar in shape at each phase.
TABLE 2.
Development of the lobular and segmental bronchus classified by the phases of the bronchial tree (right lung)
| Phase | Sample ID | Lobar bronchus | Segmental bronchus | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Superior | Middle | Inferior | B1 | B2 | B3 | B4 | B5 | B6 | B7 | B8 | B9 | B10 | ||
| 1 | 15001 | − | − | − | − | − | − | − | − | − | − | − | − | − |
| 15027 | − | − | − | − | − | − | − | − | − | − | − | − | − | |
| 15080 | − | − | − | − | − | − | − | − | − | − | − | − | − | |
| 15103 | − | − | − | − | − | − | − | − | − | − | − | − | − | |
| 15118 | − | − | − | − | − | − | − | − | − | − | − | − | − | |
| 2 | 15025 | − | + | + | − | − | − | − | − | − | − | − | − | − |
| 15041 | − | + | + | − | − | − | − | − | − | − | − | − | − | |
| 16009 | − | + | + | − | − | − | − | − | − | − | − | − | − | |
| 3 | 16020 | + | + | + | − | − | − | − | − | − | − | − | − | − |
| 16057 | + | + | + | − | − | − | − | − | − | − | − | − | − | |
| 16066 | + | + | + | − | − | − | − | − | − | − | − | − | − | |
| 16095 | + | + | + | − | − | − | − | − | − | − | − | − | − | |
| 16097 | + | + | + | − | − | − | − | − | − | − | − | − | − | |
| 16101 | + | + | + | − | − | − | − | − | − | − | − | − | − | |
| 4 | 17082 | + | + | + | − | + | + | − | − | + | − | p* | − | − |
| 17100 | + | + | + | − | − | − | − | − | + | − | p* | − | − | |
| 17022 | + | + | + | − | + | + | + | + | + | − | p* | − | − | |
| 17052 | + | + | + | + | + | + | + | + | + | + | p* | − | − | |
| 17118 | + | + | + | + | + | + | + | + | + | − | p* | − | − | |
| 18041 | + | + | + | + | + | + | + | + | + | − | − | − | − | |
| 18083 | + | + | + | − | + | + | + | + | + | + | p* | − | − | |
| 17055 | + | + | + | + | + | + | + | + | + | + | + | − | − | |
| 17057 | + | + | + | + | + | + | + | + | + | − | + | + | + | |
| 5 | 18105 | + | + | + | + | + | + | + | ++ | ++ | ++ | p* | − | − |
| 18010 | + | + | + | + | ++ | ++ | tri | tri | ++ | ++ | ++ | ++ | + | |
| 18005 | + | + | + | + | + | ++ | ++ | ++ | ++ | － | ++ | ++ | ++ | |
| 18031 | + | + | + | ++ | ++ | ++ | tri | tri | ++ | － | ++ | + | ++ | |
| 18071 | + | + | + | ++ | + | +++ | + | +++ | +++ | ++ | ++ | + | +++ | |
| 19006 | + | + | + | + | ++ | ++ | ++ | ++ | +++ | ++ | ++ | + | + | |
| 19020 | + | + | + | +++ | ++ | ++ | n.d. | n.d. | +++ | ++ | ++ | ++ | +++ | |
| 19023 | + | + | + | ++ | ++ | +++ | ++ | +++ | +++ | ++ | +++ | ++ | +++ | |
| 19040 | + | + | + | + | + | ++ | ++ | ++ | ++ | ++ | ++ | + | + | |
| 19049 | + | + | + | +++ | +++ | +++ | +++ | ++ | +++ | ++ | ++ | +++ | +++ | |
| 19054 | + | + | + | +++ | +++ | ++ | ++ | ++ | ++ | + | +++ | ++ | ++ | |
| 19057 | + | + | + | ++ | ++ | ++ | + | ++ | +++ | ++ | ++ | + | ++ | |
First two digits of sample ID represent Carnegie Stage (CS). −, absent; +, existing; ++, with sub‐segmental bronchus; +++, with second or more generation of sub‐segmental bronchus; p*, probable; n.d., not distinguished, Candidate bronchi were existing but could not be determined correctly; tri, trifurcation.
TABLE 3.
Development of the lobular and segmental bronchus classified by the phases of the bronchial tree (left lung)
| Phase | Sample ID | Lobar bronchus | Superior division bronchus | Lingular bronchus | Segmental bronchus | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Superior | Inferior | B1 + 2 | B3 | B4 | B5 | B6 | B7 + 8 | B9 | B10 | ||||
| 1 | 15001 | − | − | − | − | − | − | − | − | − | − | − | − |
| 15027 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 15080 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 15103 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 15118 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 2 | 15025 | + | + | − | − | − | − | − | − | − | − | − | − |
| 15041 | + | + | − | − | − | − | − | − | − | − | − | − | |
| 16009 | + | + | − | − | − | − | − | − | − | − | − | − | |
| 3 | 16020 | + | + | − | − | − | − | − | − | − | − | − | − |
| 16057 | + | + | − | − | − | − | − | − | − | − | − | − | |
| 16066 | + | + | − | − | − | − | − | − | − | − | − | − | |
| 16095 | + | + | − | − | − | − | − | − | − | − | − | − | |
| 16097 | + | + | − | − | − | − | − | − | − | − | − | − | |
| 16101 | + | + | − | − | − | − | − | − | − | − | − | − | |
| 4 | 17082 | + | + | + | + | − | − | − | − | − | − | − | − |
| 17100 | + | + | + | + | − | − | − | − | + | p* | − | − | |
| 17022 | + | + | + | + | − | − | − | − | + | p* | − | − | |
| 17052 | + | + | + | + | − | − | − | − | − | p* | − | − | |
| 17118 | + | + | + | + | − | − | − | − | + | p* | − | − | |
| 18041 | + | + | + | + | − | − | − | − | + | p* | − | − | |
| 18083 | + | + | + | + | − | − | − | − | + | p* | − | − | |
| 17055 | + | + | + | + | + | + | − | − | + | p* | − | − | |
| 17057 | + | + | + | + | + | + | − | − | + | + | + | + | |
| 5 | 18105 | + | + | + | + | + | + | − | − | + | + | + | + |
| 18010 | + | + | + | + | ++ | + | + | + | ++ | ++ | + | + | |
| 18005 | + | + | + | + | ++ | +++ | + | ++ | ++ | + | + | ++ | |
| 18031 | + | + | + | + | +++ | ++ | + | + | ++ | ++ | ++ | + | |
| 18071 | + | + | + | + | +++ | +++ | + | + | +++ | ++ | ++ | ++ | |
| 19006 | + | + | + | + | ++ | ++ | + | ++ | +++ | － | +++ | +++ | |
| 19020 | + | + | + | + | ++ | ++ | ++ | ++ | ++ | +++ | ++ | +++ | |
| 19023 | + | + | + | + | +++ | + | ++ | + | +++ | +++ | ++ | +++ | |
| 19040 | + | + | + | + | + | + | + | + | ++ | ++ | + | + | |
| 19049 | + | + | + | + | n.d. | n.d. | tri | tri | +++ | +++ | ++ | ++ | |
| 19054 | + | + | + | + | +++ | +++ | tri | tri | +++ | ++ | ++ | +++ | |
| 19057 | + | + | + | + | +++ | + | + | + | ++ | +++ | + | + | |
First two digits of sample ID represent Carnegie Stage (CS). −, absent; +, existing; ++, with sub‐segmental bronchus; +++, with second or more generation of sub‐segmental bronchus; p*, probable; n.d., not distinguished. Candidate bronchi were existing but could not be determined correctly; tri, trifurcation.
The following variations were observed in phase 4 (Tables 1, 2, 3):
The right superior lobe: formation of any segmental bronchi of the superior lobe occurred in all but one sample. In five of the nine samples, the right superior lobar bronchus (RSLB) was divided into three segmental bronchi. Three samples showed a bifurcate pattern that had B2 and B3.
The right middle lobe: seven of every nine samples had a B4 and B5 bronchus and the remaining two only had swellings at the right middle lobar bronchus (RMLB).
The right inferior lobe: all samples had B6. B7 was absent in six of the nine samples. Six samples presented bifurcation below B6 or B7. One of the bronchi was directed laterally and another medially (Figure 3b). It was difficult to assign a segmental bronchus correctly (B8, B9 or B10) to these bronchi. Based on their orientation, the lateral bronchus was classified into probably B8 and the medial bronchus as the parent bronchus of B9 and B10.
The left superior lobe: the superior division bronchus (SDB) and lingular bronchus (LB) could be identified in all embryos at this phase. Only two samples formed segmental bronchi. In these two samples, B1 + 2 and B3 could be observed, but B4 and B5 were absent.
The left inferior lobe: in all but two samples, B6 was found. Some segmental bronchi were generated below B6; their appearance was similar to that of the right inferior lobe. These bronchi were classified into probable B8 and parent of B9 and B10, similar to the classification in the right inferior lobe.
The following variation was observed in phase 5 (Tables 2 and 3):
The right superior lobe: all samples had prevalent three segmental bronchi of the right superior lobe. In six of the 12 samples, all three bronchi divided into a sub‐segmental bronchus. The sample with ID 18105 generated no sub‐segmental bronchi in this lobe, whereas the sample with ID 19049 formed two or more bifurcations of all segmental bronchi.
The right middle lobe: two of every 12 samples showed trifid bifurcation and the bronchi of the sample with ID 19020 were not classified into any sub‐segmental bronchus (Figure 3c). In the remaining nine samples, six formed sub‐segmental bronchi of both B4 and B5 and the other three had B4 without sub‐segmental bronchus.
The right inferior lobe: a sub‐segmental bronchus of B6 was present in all 12 samples. In two of the 12 samples, B7 was absent. In contrast, B7 of nine samples formed a sub‐segmental bronchus. All but one sample presented B8, B9, and B10, in which some segmental bronchi subdivided into a sub‐segmental bronchus. In the sample with ID 18105, probable B8 and parent of B9 and B10 were generated below B7. A sub‐segmental bronchus of B8 was formed in all samples excluding the sample with ID 18105. B9 without a sub‐segmental bronchus was observed in five samples. In eight samples, B10 divided into a sub‐segmental bronchus but the remaining three samples formed no sub‐segmental bronchus of B10.
The left superior lobe: In the superior segment, nine of every 12 samples showed a sub‐segmental bronchus of B1 + 2. In six samples, B3 divided into the sub‐segmental bronchus. The bronchus that bifurcated from the SDB in the sample with ID 19049 was not classified into any bronchus. In the lingular segment, trifid bifurcation from the LB was found in two samples. Only two samples among the 12 formed the sub‐segmental bronchus of B4. The sub‐segmental bronchus of B5 was also observed in only three samples. Both B4 and B5 were absent in the sample with ID 18105.
The left inferior lobe: all but one sample formed the following typical segmental bronchus; B6, B7 + 8, B9, and B10. However, these samples had different sub‐segmental bronchi. The sub‐segmental bronchus of B6 occurred in all but one sample. B7 + 8 subdivided to form a sub‐segmental bronchus in nine of the 12 samples. This segmental bronchus was not observed in the sample with ID 19006. There were seven samples with the sub‐segmental bronchus of B9. The sub‐segmental bronchus of B10 was also found in seven samples.
3.1.2. Timing of the bronchus formation for the right upper lobe
The lobar buds were formed between phases 1 and 3 (Figure 3a). The buds of the right middle and left superior lobar bronchi branched off at the symmetrical position of each trunk in phase 2. The right and left trunks first showed asymmetry in phase 3. In this phase, the right middle bud and the left superior bud were apparently in symmetrical positions. It is noted that RSLB first appeared in phase 3.
3.2. During CS20 and CS23
We show representative reconstructions of the bronchial tree at each CS in Figure 4. All samples had a five‐lobe structure and did not show either a supernumerary or a displaced bronchus originating from the trachea, carina or main bronchus. As described above, the sub‐segmental bronchi began to appear in the segment in CS19. In the bronchial trees during CS20 and 23, all segmental bronchi except the right B7 in the sample with ID 21120 formed a sub‐segmental bronchus and bronchioles. Both maximum generation and the number of branches increased according to CS (Table 4). Similarly, the median of generations in all lobes, excluding the right inferior lobe between CS20 and CS21, increased according to CS when the lobar bronchus was defined as the 0th generation (Table 4, Figure 4). In CS23, the median generation value was 6.5, 6.0, 7.5, 7.0, and 7.5 in the right superior, right middle, right inferior, left superior, and left inferior lobes, respectively. Distribution of the end‐branch generation among five lobes differed significantly (p < .05; Table S1). The median branching generation value in the right middle lobe was significantly lower than that of the other four lobes (Figure S1). The median branching generation value of the right inferior lobe was significantly greater than that of both the right and left superior lobes. Similarly, the median branching generation value of the left inferior lobe was significantly higher than that of the right superior lobe.
FIGURE 4.
Representative reconstructions of the bronchial tree during CS18 and CS23. The bronchial tree showing representative reconstructions (purple) and generation of end‐branching (rainbow color) during CS18 and CS23. The colored circle indicates generations of each end‐branch when the lobar bronchus was defined as 0th branch. Color bar indicates the corresponding colors. Scale bar: 1 mm
TABLE 4.
Development of the branching tree in each lobe between CS18 and CS23
| CS | n | Median of generation | Maximum generation | Number of branches | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| RSL | RML | RIL | LSL | LIL | RSL | RML | RIL | LSL | LIL | RSL | RML | RIL | LSL | LIL | |||
| 18 | 7 | Mean | 2.0 | 1.8 | 2.9 | 2.2 | 2.6 | 2.3 | 2.0 | 3.6 | 2.9 | 3.1 | 7.1 | 5.6 | 11.9 | 9.3 | 9.9 |
| (± SD) | (0.5) | (0.5) | (0.9) | (0.9) | (0.4) | (0.7) | (0.8) | (1.4) | (1.5) | (0.8) | (2.7) | (2.1) | (5.5) | (5.3) | (4.1) | ||
| 19 | 7 | Mean | 3.1 | 2.2 | 4.0 | 3.1 | 3.7 | 3.6 | 2.7 | 5.3 | 3.7 | 4.7 | 14.4 | 8.4 | 23.3 | 15.3 | 21.0 |
| (± SD) | (0.6) | (0.4) | (0.8) | (0.7) | (0.5) | (0.9) | (0.7) | (1.2) | (0.9) | (0.9) | (5.0) | (2.1) | (5.4) | (4.9) | (6.6) | ||
| 20 | 4 | Mean | 4.3 | 3.3 | 5.8 | 4.0 | 4.8 | 5.0 | 4.0 | 7.8 | 5.3 | 6.3 | 30.5 | 15.3 | 50.3 | 31.8 | 42.5 |
| (± SD) | (0.4) | (0.4) | (0.4) | (0.7) | (0.4) | (0.7) | (0.7) | (0.4) | (0.8) | (0.8) | (6.7) | (3.5) | (13.4) | (8.3) | (12.4) | ||
| 21 | 4 | Mean | 5.3 | 4.5 | 5.8 | 5.5 | 5.8 | 6.8 | 7.3 | 8.5 | 7.3 | 8.3 | 61.0 | 35.5 | 92.8 | 65.0 | 93.0 |
| (± SD) | (0.4) | (0.9) | (0.8) | (0.5) | (0.4) | (0.8) | (1.1) | (0.5) | (0.8) | (0.8) | (17.5) | (9.1) | (23.6) | (22.7) | (29.6) | ||
| 22 | 3 | Mean | 6.0 | 5.2 | 6.7 | 6.3 | 6.7 | 7.7 | 7.3 | 10.7 | 8.3 | 9.7 | 87.0 | 49.3 | 126.3 | 99.7 | 132.7 |
| (± SD) | (0.0) | (0.2) | (0.5) | (0.5) | (0.5) | (0.5) | (0.9) | (1.2) | (0.5) | (0.5) | (17.3) | (4.0) | (30.8) | (20.7) | (34.1) | ||
| 23 | 2 | Mean | 6.5 | 6.0 | 7.5 | 7.0 | 7.5 | 10.0 | 8.0 | 10.5 | 9.0 | 11.0 | 146.5 | 85.5 | 244.0 | 173.0 | 222.0 |
| (± SD) | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | ||
Generation of the lobar bronchus was counted as the 0th generation.
CS, Carnegie stage; LIL, the left inferior lobe; LSL, the left superior lobe; RIL, the right inferior lobe; RML, the right middle lobe; RSL, the right superior lobe.
3.3. Variations of the segmental bronchus
A total of 14 branching variations of the segmental bronchus were identified in the right superior (n = 4), middle (n = 2), inferior (n = 2), left superior (n = 4), and inferior lobes (n = 2). We describe below the variations of the segmental bronchus for each lobe.
3.3.1. Right superior lobe
In 10 of 13 samples, RSLB divided into three segmental bronchi (Table S2). In these 10 samples, five showed the prevailing trifurcate pattern in which B1, B2, and B3 were equally divided (Table S3, Figure 5a[i]). The remaining five samples showed bifurcate patterns such as B1 + 2 and B3 or B2 + 3 and B1. Two samples (ID 21056 and 21079) appeared to lack B1 (Figure 5a[ii]). Although there was no apical segmental bronchus, the bronchi that bifurcated from B2 or B3 extended apically, resulting in the shape of the right superior lobe being similar to that of a typical trifurcate pattern. In the two samples with ID 21056 and 21079, B1 seemed to be defective. RSLB in the sample with ID 20035 had four segmental bronchi as described in Figure 5a(iii).
FIGURE 5.
Variation of the segmental bronchus in the right lobe. (a) Variations found in the right superior lobe, the prevailing trifurcation pattern (i), bifurcation pattern by absent B1 (ii), and quadrivial pattern (iii). (b) Variations found in the right middle lobe. B4 and B5 extended dorsally and ventrally (i). These bronchi divided into superior and inferior bronchi (ii). (c) Variations found in the right inferior lobe. The prevailing pattern with B6, B7, B8, B9, and B10 (i). Frontal view (left) and right inferior lobe of the same sample eliminating B7 (right). Absent B7 pattern (ii). B* originated from stem bronchus of B9 and B10 (iii). RILB, the right inferior lobar bronchus; RMB, the right main bronchus; RMLB, the right middle lobar bronchus; RSLB, the right superior lobar bronchus. Scale bar: 500 µm
3.3.2. Right middle lobe
All 13 samples had both B4 and B5 (Table S2). B4 and B5, respectively, extended dorsally and ventrally in all but one sample (Table S3, Figure 5b[i]). The sample with ID 20054 showed a superior‐inferior pattern (Figure 5b[ii]). No samples showed the trifurcation patterns that were seen in samples at CS18 (Figure 3c[i]).
3.3.3. Right inferior lobe
The prevailing pattern of the right inferior lobe is shown in Figure 5c(i). All samples during CS20 and CS23 had B6 (Table S2). B7 occurred in all but one of the 13 samples. In the sample with ID 22007, B7 was absent (Figure 5c[ii]). Anterior, lateral, and posterior basal bronchi (B8, B9, and B10) were defined in 12 of 13 samples. In the sample with ID 21056, it was difficult to assign B9 and B10 to a specific bronchus because basal branches that bifurcated below B8 did not determinately extend laterally and posteriorly. Sub‐superior bronchus B*, which is generally located posteriorly, was observed in approximately half of the samples. In three samples, B* originated from the bronchus between B7 and B8. Another B* variation, found in five samples, was a bifurcation of this bronchus from the parent trunk of B9 and B10 (Figure 5c[iii]). Both absent B7 (medial basal bronchus) and B* were reported as variations that have been significantly associated with chronic obstructive pulmonary disease (COPD) in the adult lung (Smith et al., 2018).
3.3.4. Left superior lobe
In 10 of 13 samples, LSLB bifurcated into SDB and LB (Figure 6a[i], Table S4). LSLB in the remaining three samples seemed to trifurcate (Figure 6a[ii]).
FIGURE 6.
Variation of the segmental bronchus in the left lobe. (a) Variations in the left superior lobe, the prevailing pattern (i), trifurcation of LSLB (ii), and trifurcation of LB (iii). Frontal view. (b) Variations observed in the left inferior lobe. The prevailing pattern with B6, B7 + 8, B9, and B10 (i). B* originated from common bronchus of B9 and B10 (ii). Medial view. LB, lingular bronchus; LILB, the left inferior lobar bronchus; LSLB, the left superior lobar bronchus; SDB, superior division bronchus. Scale bar: 500 µm
Seven of 10 samples with the bifurcation patterns had the prevailing pattern of the left superior lobe (Figure 6a[i], Table S5); that is, they had the apicoposterior and anterior bronchus (B1 + 2 and B3) in the superior division and the superior and inferior bronchus (B4 and B5) in the lingular division. In the remainder, for reasons unique to each sample, classification of the segmental bronchi was difficult. In the sample with ID 20053, the branch of the left lingular division connected to the branch of the left inferior lobe. The lingular division of the sample with ID 21039 had an excess bronchus that originated from LB and extended dorsolaterally. LB divided into three bronchi in the sample with ID 23008 (Figure 6a[iii]).
In the trifurcation pattern, one bronchus extended dorsosuperiorly, another laterally, and the third bronchus ventromedially (Figure 6a[ii]). The trifurcated bronchus made it difficult to classify the segmental bronchus because the lateral bronchus of the trifurcated bronchus was not correctly classified into the specific segmental bronchus.
3.3.5. Left inferior lobe
All samples had B6 (Table S4). Anteromedial, lateral, and posterior basal bronchi (B7 + 8, B9, and B10) were formed in all samples except the sample with ID 20053, which we failed to reconstruct. Additionally, their branching pattern was typical: B7 + 8 arose anteromedially, and then a common bronchus bifurcated into B9 and B10 (Figure 6b[i]). B* in seven of 13 samples extended lateroposteriorly and originated from the common trunk of B9 and B10 (Figure 6b[ii]).
4. DISCUSSION
In the present study, we extended the morphological analysis according to CS until the end of the embryonic period (CS23). The precise 3D reconstruction of the bronchial tree was demonstrated by each CS, which provided new knowledge about the development of the bronchial tree during the human embryonic period.
The proximal part of the bronchial tree until lobar bronchi in human was formed stereotypically, as indicated in the previous study in mice (Short and Smyth, 2016). No bronchial abnormalities were found in the present study. The result of the shrinkage of bronchi is proposed as the hypothetical mechanism for congenital bronchial abnormalities (the reduction theory) (Ghaye et al., 2001). The 3D reconstructions in our study did not support this theory, which is based on the notion that the developed bronchi may shrink at a later period.
Streeter (1948) observed that lobar buds were present at CS15 and elongated at CS16. They suggested that all lobar bronchi were formed at the same time. However, our present data indicate that the formation of the lobar bronchi may not be simultaneous but may occur in consecutive order. Namely, both RMLB and LSLB emerged first, followed by RSLB. A branching tree having a symmetrical structure between the left and the right sides was found during phases 1 and 2 (at CS15 and one sample at CS16). Delayed generation of RSLB may trigger a characteristic asymmetrical structure in phase 3.
A previous study by Wells and Boyden (1954) described the segment bud formation of the bronchial tree in a CS‐dependent manner. Wells and Boyden (1954) reported that the segmental buds arose on the branching tree at CS17 and a few sub‐segmental bronchi appeared at CS18. The first generation of the sub‐segmental bronchi was complete at CS19; subsequently, the second generation of these bronchi formed at CS20 (Wells and Boyden, 1954; O'Rahilly and Boyden, 1973). On the other hand, the generation speed of the bronchial tree in our study seems to differ among individual samples and is not constant, especially after CS17. Subsequently, during CS18 and CS23, the speed of generation among individuals in the same stage varied.
No previous studies have enumerated the number of generations of the bronchial tree by CS during the embryonic period. Palmer (1936) enumerated the generations of the right superior lobe by crown–rump length (CRL) of the embryos, which showed that the number of generations at embryo with CRL 11 mm was 3, subsequently increasing up to 10 at 35 mm. Although a former embryo might correspond to around CS17 or 18, and the latter to around CS23 or early fetus stage, an exact comparison was not possible. Bucher and Reid (1961) described generations of selected pathways in each lobe, but their 14 specimens were fetuses older than 10 weeks of gestation (CRL of more than 40 mm). Reconstructed bronchial trees in our study enabled us to count all end‐branch generations and demonstrate the distribution of branching for each lobe during the human embryonic period.
Wells and Boyden (1954) analyzed seven embryonic bronchial trees up to CS19 and described variations of branching pattern in the segment, in which all but one variation (B7 originated independently in the left inferior lobe) was observed in our study. Fourteen branching variations of the segmental bronchus were identified in the current study. We found that the RMLB divided into superior and inferior segmental bronchi, which was not reported in the foregoing embryonic study (Wells and Boyden, 1954). The variations observed in our study as well as in the previous study (Wells and Boyden, 1954) are known as a common bronchial variant in the human adult lung (Ghaye et al., 2001). Thus, the fundamental structure of the proximal part of the bronchial tree consisting of the lobar and segmental bronchi seems to be determined at the embryonic period, and may be maintained through a lifetime. In addition, variations in our study included the patterns correlated with COPD (Smith et al., 2018), suggesting that these branching variants could be detected during the embryonic period.
Some limitations of the current study should be noted. First, during CS17 and CS18, we could not accurately define a few branches due to the developmentally immature structure of the bronchial tree. We could accurately classify each segmental bronchus based on its orientation and relative position when analyzing samples that generated after sub‐segmental bronchi during CS19 and 23. Conversely, identifying each sub‐segmental bronchus and further small bronchus was difficult using the present method. Although the distribution of pulmonary vessels is useful to determine the segmental bronchus accurately, we could not observe those branches owing to limited resolution. Second, the number of samples was insufficient to evaluate the occurrence rate of each variation. Third, though the present data indicate that CS may be unsuitable for the developmental index of the bronchial tree, a substitutable developmental index for bronchial development remains to be proposed. Finally, we observed fixed embryos in the collection, which enabled us to understand individual variations of the bronchial tree at the embryonic period, but the growth change of the bronchial tree over time using same individual could not be demonstrated.
5. CONCLUSION
In the present study, 3D‐reconstructed bronchial trees during the embryonic period by each CS were precisely analyzed. The data provided may contribute to a better understanding of bronchial tree formation during the human embryonic period.
CONFLICT OF INTEREST
There are no conflicts of interest to declare.
AUTHOR CONTRIBUTIONS
Sena Fujii: data analysis/interpretation and drafting of the manuscript. Taiga Muranaka, Jun Matsubayashi: data analysis/interpretation. Shigehito Yamada, Akio Yoneyama: acquisition of data. Tetsuya Takakuwa: concept/design, critical revision of the manuscript.
Supporting information
Fig S1
Table S1‐S5
ACKNOWLEDGEMENTS
The authors thank Ms Chigako Uwabe and Dr. Haruyuki Makishima at the Congenital Anomaly Research Center for their technical assistance in handling the human embryos. This study was supported by grants JP26220004, JP16K15535, JP17H05294, and JP18K07876 from the Japan Society for the Promotion of Science. This work was performed under the approval of the Photon Factory Program Advisory Committee (Proposal No. 2017G688, G598).
Fujii S, Muranaka T, Matsubayashi J, Yamada S, Yoneyama A, Takakuwa T. The bronchial tree of the human embryo: an analysis of variations in the bronchial segments. J. Anat. 2020;237:311–322. 10.1111/joa.13199
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Fig S1
Table S1‐S5
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.