4 -9 Life Science
Three-Dimensional Reconstruction of the Human EmbryonicBronchial Tree Using Phase-Contrast X-Ray ComputedTomography (PXCT) Images
Details of the bronchial tree formation remain unknown because of the difficulty of analyzing extremely small embryos.
We aimed to elucidate the morphogenesis of the human embryonic bronchial tree using phase-contrast X-ray computed tomography (PXCT) images. The three-dimensional (3D) branching of bronchial trees was reconstructed using
PXCT images of a sample of embryos (between 5–8 weeks after fertilization) from the Kyoto Collection. The images
revealed that branching variants arose during the embryonic period and continued throughout life. All proximal bronchi,
except the lingular bronchi, were formed by a monopodial branching mode. The 3D reconstructions of the embryonicbronchial trees provided novel insights into how bronchial trees are generated in small embryos.
The bronchial tree of the human lung is composedof conducting and respiratory airways [1]. This organ
has a highly ramified structure in the lungs. An understanding of branching morphogenesis is essential forthe diagnosis and treatment of congenital anomalies.
However, how such complicated branching networks
are generated during development remains unclear
because of the difficulty of analyzing extremely small
embryos. Recently, we provided new insights into theformation of the branching tree in the human embryoniclung by analyzing 3D reconstructions of the human embryonic bronchial tree [2, 3].
First, we extended the morphological analysis to theend of the embryonic period [2]. The embryonic period
is from the time of fertilization to 8 weeks post-fertilization. It begins with the formation of the body structures,
generally described by a standardized system of 23
stages called the Carnegie stages (CS) [4]. Previous
morphological studies have demonstrated the generalmorphogenetic processes of the human bronchial treeabFigure 1: Image processing of the bronchial tree and illustration
showing the change of bronchus length with monopodial anddipodial branching. a (i) Transverse section using phase-contrast
X-ray computed tomography. Heart (H). (ii) Reconstructedbronchial tree. Scale bar: 1 mm. (iii) The centerline was calculated
based on reconstruction. b The parent bronchus (PBr) length may
shrink with monopodial branching just after generation of childbronchus (CBr) (i), but may not shrink or elongate with dipodial
branching (ii).

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HIGHLIGHTS

during the embryonic period. The primordium of the treebuds extends from the middle of the foregut at approximately CS 12 (at approximately 4 weeks after fertilization) [5]. After that, this pulmonary primordium bud continues to extend and branches out continuously to form
the lobar, segmental, and more peripheral branches.
The first generation of sub-segmental bronchi is complete at CS19 [6]. However, morphological changes inthe trees after CS20 have not been elucidated.
Second, we analyzed how the proximal bronchus of
the human lung branches off [3]. Previous studies haveproposed two simple branching modes: monopodial
and dipodial [7, 8]. With monopodial branching, the childbranches extend from the sidewall of the parent branch.
With dipodial branching, the tip of the bronchus bifurcates. Previous studies estimated the branching mode
based only on visual assessments.
Thus, we aimed to describe the morphogenesis ofbronchial trees during the human embryonic period. We
reconstructed 3D branching trees using phase-contrast
X-ray computed tomography (PXCT) images, observed
the morphological changes in the trees in detail, andcategorized the branching mode as monopodial and dipodial based on the bronchus length.
A total of 48 embryos between CS15 and CS23
(about 5–8 weeks after fertilization, 8–30 mm crownrump length) [4] of the Kyoto Collection were selected[9]. Imaging data of all samples were acquired using
PXCT. The system was set up at the vertical wiggler
beamline (BL-14C). The PXCT imaging data provided
a resolution of ≥ 18 μm/pixel [10], which enabled nondestructive observation of intrabody structures in detail,and highly sensitive morphometry of the embryos. The
structure of the bronchial tree was reconstructed for all
samples using Amira software (version 6.2.0; Visage
Imaging GmbH, Berlin, Germany) (Fig. 1a). The centerof the airway was observed linearly 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 branch lengths.
We categorized the branching modes of the lobar,segmental, and subsegmental bronchi. After calculating the length of each bronchus, we categorized theabc

Figure 2: 3D reconstructions enable novel analyses of the human embryonic bronchial tree. a Representative reconstructions of the bronchialtree. Scale bar: 1 mm. b (left) Generation of end-branching (rainbow color) at CS18, CS21, and CS23. Colored circles indicate generations ofeach end-branch when the lobar bronchus was defined as the 0th branch. Color bars indicate the corresponding generations. (right) Changeof generation in each lobe by Carnegie stage during CS18 and CS23. LIL, left inferior lobe; LSL, left superior lobe; RIL, right inferior lobe;
RML, right middle lobe; RSL, right superior lobe. c Branching mode of the right superior and middle lobar bronchi. The length changes of theright proximal bronchi are shown. Compared with the right primary bronchial bud (RPBB) length before branching, temRMB length and total
length of right main bronchus (RMB) and intermediate bronchus (IB) were shorter. RIB, right inferior bronchus; temRMB, temporary RMB
branch from the tracheal bifurcation to the base of the right middle lobe.

branching mode of the analyzed bronchi based onwhether the parent bronchus was divided after generating the analyzed bronchi (Fig. 1b).
Three-dimensionally reconstructed bronchial trees
revealed a timeline of morphogenesis during the embryonic period (Fig. 2a). The right superior lobar bronchus
was formed after the generation of both the right middle
and left superior lobar bronchi. The distribution of theend-branch generation among the five lobes was significantly different (Fig. 2b). The median branching generation value in the right middle lobe was significantly lower
than that in the other four lobes. Variations found between CS20 and CS23 were all described in the humanadult lung, indicating that variation in the bronchial tree
may arise during the embryonic period and continue
throughout life.
All lobar bronchi were formed with monopodial
branching (Fig. 2c). Twenty-five bifurcations were analyzed to categorize the branching mode of the segmental and subsegmental bronchi. Of these, 22 bifurcations
were categorized as monopodial branching, two bifurcations were not categorized as any branching pattern,
and the only lingular bronchus that bifurcated from theleft superior lobar bronchus was categorized as dipodialbranching.
High-resolution imaging data of human embryonicspecimens using PXCT enabled the reconstruction of
the three-dimensional bronchial tree, revealing morpho-genetic changes during the human embryonic period.
Our novel understanding of bronchial tree development
will provide a crucial resource for elucidating congenitalanomalies.

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A. Yoneyama and T. Takakuwa, PLoS ONE 16, e0245558(2021).
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Teratology 1, 281 (1968).
[10] A. Yoneyama, S. Yamada and T. Takeda, “Fine BiomedicalImaging Using X-Ray Phase-Sensitive Technique” inAdvanced Biomedical Engineering, edited by G. D. Gargiulo
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BEAMLINE
BL-14CS. Fujii and T. Takakuwa (Kyoto Univ.)
HIGHLIGHTS

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