Bronchial tree of the human embryo: Examination based on a mammalian model.

The symmetry of the right and left bronchi, proposed in a previous comparative anatomical study as the basic model of the mammalian bronchial tree, was examined to determine if it applied to the embryonic human bronchial tree. Imaging data of 41 human embryo specimens at Carnegie stages (CS) 16-23 (equivalent to 6-8 weeks after fertilization) belonging to the Kyoto collection were obtained using phase-contrast X-ray computed tomography. Three-dimensional bronchial trees were then reconstructed from these images. Bronchi branching from both main bronchi were labeled as dorsal, ventral, medial, or lateral systems based on the branching position with numbering starting cranially. The length from the tracheal bifurcation to the branching point of the labeled bronchus was measured, and the right-to-left ratio of the same labeled bronchus in both lungs was calculated. In both lungs, the human embryonic bronchial tree showed symmetry with an alternating pattern of dorsal and lateral systems up to segmental bronchus B9 as the basic shape, with a more peripheral variation. This pattern is similar to that described in adult human lungs. Bronchial length increased with the CS in all labeled bronchi, whereas the right-to-left ratio was constant at approximately 1.0. The data demonstrated that the prototype of the human adult bronchial branching structure is formed and maintained in the embryonic stage. The morphology and branching position of all lobar bronchi and B6, B8, B9, and the subsegmental bronchus of B10 may be genetically determined. On the other hand, no common structures between individual embryos were found in the peripheral branches after the subsegmental bronchus of B10, suggesting that branch formation in this region is influenced more by environmental factors than by genetic factors.

Formation of tendinous intersections in the human fetal rectus abdominis.

Previous studies have poorly described the initial development process of the tendinous intersections of the rectus abdominis muscle (RAM). The present study aimed to observe the formation of tendinous intersections in the RAM during the early fetal period using diffusion tensor imaging (DTI). Fifteen human fetal specimens (crown-rump length [CRL]: 39.5-93.7 mm) were selected. Three-dimensional measurements revealed that Zone-4 (i.e., the zone between the pubic symphysis and the caudal base of the umbilical ring in the RAM) had a smaller width and was thicker than Zone-1 and Zone-2 (i.e., the zones between the costal arch and the cranial base of the umbilical ring) and Zone-3 (i.e., the zone at the umbilical ring). Characteristics of tendinous intersections in the RAM during the early fetal period were assessed according to number, size, type, laterality, and sex. The mean number of tendinous intersections on both sides was 3.1 (range: 2.0-4.0), and 21% of specimens had only two tendinous intersections, which was higher than that reported in previous adult studies. The present data suggest that the formation of tendinous intersections was still in progress in specimens with two tendinous intersections in the RAM and that the third tendinous intersection was formed in Zone-2. Ordinal logistic regression via generalized estimating equations revealed that the odds for a higher type of tendinous intersections in Zone-1 and Zone-2 were significantly higher than those in Zone-4 (adjusted odds ratio: 14.85, 8.84). The odds for the presence of incomplete types (tendinous intersections that could not completely transverse the RAM) in Zone-3 were significantly higher than those in Zone-1 (adjusted odds ratio: 7.4). The odds for missing tendinous intersections in Zone-4 were significantly higher than those in Zone-1 (adjusted odds ratio: 20.5). These zonal differences in the formation of tendinous intersections were consistent with those observed in previous adult studies. In this study, DTI detected tendinous intersections in a sample with a CRL of 45.8 mm (approximately 11 weeks of gestation), which is earlier than that in previous histological findings, indicating that the RAM does not have mature tendinous intersections until the 17th week of gestation. In conclusion, DTI could detect the premature differentiation of tendinous intersection formation. Our data may aid in elucidating the developmental processes of tendinous intersections in the RAM.

Morphogenesis of the pulmonary vein and left atrial appendage in human embryos and early fetuses.

The left atrium wall has several origins, including the body, appendage, septum, atrial-ventricular canal, posterior wall, and venous component. Here, we describe the morphogenesis of left atrium based on high-resolution imaging (phase-contrast X-ray computed tomography and magnetic resonance imaging). Twenty-three human embryos and 19 fetuses were selected for this study. Three-dimensional cardiac images were reconstructed, and the pulmonary veins and left atrium, including the left atrial appendage, were evaluated morphologically and quantitatively. The positions of the pericardial reflections were used as landmarks for the border of the pericardial cavity. The common pulmonary vein was observed in three specimens at Carnegie stages 17-18. The pericardium was detected at the four pulmonary veins (left superior, left inferior, right superior, and right inferior pulmonary veins) at one specimen at Carnegie stage 18 and all larger specimens, except the four samples. Our results suggest that the position of the pericardial reflections was determined at two pulmonary veins (right and left pulmonary vein) and four pulmonary veins almost simultaneously when the dorsal mesocardial connection between the embryo and heart regressed. The magnetic resonance images and reconstructed heart cavity images confirmed that the left atrium folds were present at the junction between the body and venous component. Three-dimensional reconstruction showed that the four pulmonary veins entered the dorsal left atrium tangentially from the lateral to the medial direction. More specifically, the right pulmonary veins entered at a greater angle than the left pulmonary veins. The distance between the superior and inferior pulmonary veins was shorter than that between the left and right pulmonary veins. Three-dimensional reconstruction showed that the venous component increased proportionally with growth. No noticeable differences in discrimination between the right and left parts of the venous component emerged, while the junction between the venous component and body gradually became inconspicuous but was still recognizable by the end of the observed early fetal period. The left superior pulmonary vein had the smallest cross-sectional area and most flattened shape, whereas the other three were similar in area and shape. The left atrial appendage had a large volume in the center and extended to the periphery as a lobe-like structure. The left atrial appendage orifice increased in the area and tended to become flatter with growth. The whole left atrium volume^(1/3) increased almost proportionally with growth, parallel to the whole heart volume. This study provided a three-dimensional and quantitative description of the developmental process of the left atrium, comprising the venous component and left atrial appendage formation, from the late embryonic to the early fetal stages.

Three-Dimensional Imaging Analysis of the Developmental Process of Posterior Meniscofemoral Ligaments in Rat Embryos.

INTRODUCTION: The posterior meniscofemoral ligament (pMFL) of knee joint is a ligament that runs posterior to the posterior cruciate ligament and it is known that the height of the pMFL attachment site causes meniscus avulsion. Therefore, understanding the three-dimensional (3D) structure of the pMFL attachment site is essential to better understand the pathogenesis of meniscus disorders. However, the developmental process of pMFL has not been well investigated. The purpose of this study was to analyze pMFL development in rat knee joints using 3D reconstructed images produced from episcopic fluorescence image capture (EFIC) images and examine its relationship with other knee joint components. METHODS: Knee joints of Wistar rat embryos between embryonic day (E) 16 and E21 were observed with HE-stained tissues. Serial EFIC images of the hind limbs of E17-E21 were, respectively, captured from which 3D images were reconstructed and the features of pMFL structure: length and angle were measured. Besides, the chronological volume changes and the volume ratio of the knee joint components compared to E17 were calculated to identify the differences in growth by components. RESULTS: pMFL was observed from E17 and was attached to the medial femoral condyle and lateral meniscus at all developmental stages, as in mature rats. The lack of marked variation in the attachment site and angle of the pMFL with the developmental stage indicates that the pMFL and surrounding knee joint components developed while maintaining their positional relationship from the onset of development. CONCLUSION: Current results may support to congenital etiology of meniscus disorder.

Three-dimensional morphogenesis of the human diaphragm during the late embryonic and early fetal period: Analysis using T1-weighted and diffusion tensor imaging.

A precise understanding of human diaphragm development is essential in fetal medicine. To our knowledge, no previous study has attempted a three-dimensional (3-D) analysis and evaluation of diaphragmatic morphogenesis and development from the embryonic to the early fetal period. This study aimed to evaluate the morphogenesis and fibrous architecture of the developing human diaphragm during the late embryonic and early fetal periods. Fifty-seven human embryos and fetuses (crown-rump length [CRL] = 8-88 mm) preserved at the Congenital Anomaly Research Center of Kyoto University and Shimane University were analyzed. 3-D morphogenesis and fiber orientation of the diaphragm were assessed using phase-contrast X-ray computed tomography, T1-weighted magnetic resonance imaging (T1W MRI), and diffusion tensor imaging (DTI). T1W MR images and DTI scans were obtained using a 7-T MR system. The diaphragm was completely closed at Carnegie stage (CS) 20 and gradually developed a dome-like shape. The diaphragm was already in contact with the heart and liver ventrally in the earliest CS16 specimen observed, and the adrenal glands dorsally at CS19 or later. In the fetal period, the diaphragm contacted the gastric fundus in samples with a CRL ≥41 mm, and the spleen in samples with a CRL ≥70 mm. The relative position of the diaphragm with reference to the vertebrae changed rapidly from CS16 to CS19. The most cranial point of the diaphragm was located between the 4th and 8th thoracic vertebrae, regardless of fetal growth, in samples with a CRL of ≥16 mm. Diaphragmatic thickness was nearly uniform (0.15-0.2 mm) across samples with a CRL of 8-41 mm. The sternal, costal, lumbar parts, and the area surrounding the esophageal hiatus thickened with growth in samples with a CRL of ≥46 mm. The thickness at the center of the diaphragm and the left and right hemidiaphragmatic domes did not increase with growth. Tractography showed that the fiber orientation of the sternal, costal, and lumbar parts became more distinct as growth progressed in CS19 or later. All fibers in the costal and lumbar regions ran toward the left and right hemidiaphragmatic domes, except for those running to the caval opening and esophageal hiatus. The fiber orientation patterns from the right and left crura surrounding the esophageal hiatus were classified into three types. Distinct fiber directions between the costal and sternal and between the costal and lumbar diaphragmatic parts were observable in samples with a CRL of ≥46 mm. Anterior costal and sternal fibers ran toward the center. Fiber tracts around the center and the left and right hemidiaphragmatic domes; between the costal and lumbar orientations; and between the costal and sternal orientations showed a tendency for decreasing fractional anisotropy values with fetal growth and showed less density than other areas. In conclusion, we used 3-D thickness assessment and DTI tractography to identify qualitative changes in the muscular and tendonous regions of the diaphragm during the embryonic and early fetal periods. This study provides information on normal human diaphragm development for the progression of fetal medicine and furthering the understanding of congenital anomalies.

The return process of physiological umbilical herniation in human fetuses: The possible role of the vascular tree and umbilical ring.

The human intestine elongates during the early fetal period, herniates into the extraembryonic coelom (EC), and subsequently returns to the abdominal cavity (AC). The process by which the intestinal loop returns to the abdomen remains unclear. This study aimed to document positional changes in the intestinal tract with the superior mesenteric artery (SMA) and branches in 3D to elucidate the intestinal loop return process (transition phase). Serial histological cross-sections from human fetuses (crown-rump length [CRL] range: 30-50 mm) in the herniation (n = 1), transition (n = 7), and return (n = 2) phases were selected from the Blechschmidt Collection. The distribution of the SMA trunk and all intestinal and sister branches entering the intestines was visualized so that positional changes in branches were continuous from the herniation to return phases. Positional changes in SMA branches proceeded in an orderly and structured manner; this is essential for continuous blood supply via the SMA to the intestine during transition and for safe intestinal return. Changes in the SMA distribution proceeded prior to the detection of initiation of intestinal tract return, which might start earlier and last much longer than our consensus (i.e., that the return of the herniated intestine begins when the CRL is approximately 40 mm and ends within a short time). In the cross-section of the umbilical ring in the herniation and transition phases, one proximal limb and one distal limb were observed with SMA intestinal branches, which were fully packed in the umbilical ring. The SMA branches were aligned from inferior to superior along the SMA main trunk. In the herniation phase, the distribution of 3rd-13th branches aligned from proximal inferior medial to distal superior left with a slight spiral in the EC, the tips of which suggested an orderly running course of the small intestine. In the transition phase, SMA branches running across the umbilical ring that fed the small intestine were observed, suggesting that the intestine was uncoiled and ran across the umbilical ring almost vertically. The estimated curvature value supported the phenomenon of uncoiling at the umbilical ring; the value at the umbilical ring was lesser than that in the AC and EC. During the transition phase, the proximal and distal limbs transversely ran side by side in the AC, umbilical ring, limbs on the cranial side, and mesentery on the caudal side. The SMA trunk and its branches ran in parallel, cranially to caudally aligned in the mesentery. This layout of the umbilical ring was maintained during the transition phase. In the return phase, the SMA trunk was gently curved from the upper left to the lower right of the AC; around 12 branches spread with a winding staircase appearance. The intestinal tract reached its definitive position immediately after all tissues crossed the umbilical ring and released any restriction. Each SMA branch and the corresponding region of the intestinal tract form a unit and change their position, though the conformation may change within each unit when running across the umbilical ring. We suggest that the slide-stack model requires revision.

Morphometric analysis of secondary palate development in human embryos.

Rapid shelf elevation and contact of the secondary palate and fusion reportedly occur due to a growth-related equilibrium change in the structures within the oro-nasal cavity. This study aimed to quantitatively evaluate complex three-dimensional morphological changes and their effects on rapid movements, such as shelf elevation and contact, and fusion. Morphological changes during secondary palate formation were analyzed using high-resolution digitalized imaging data (phase-contrast X-ray computed tomography and magnetic resonance images) obtained from 22 human embryonic and fetal samples. The three-dimensional images of the oro-nasal structures, including the maxilla, palate, pterygoid hamulus, tongue, Meckel's cartilage, nasal cavity, pharyngeal cavity, and nasal septum, were reconstructed manually. The palatal shelves were not elevated in all the samples at Carnegie stage (CS)21 and CS22 and in three samples at CS23. In contrast, the palatal shelves were elevated but not in contact in one sample at CS23. Further, the palatal shelves were elevated and fused in the remaining four samples at CS23 and all three samples from the early fetal period. For each sample, 70 landmarks were subjected to Procrustes and principal component (PC) analysis. PC-1 accounted for 67.4% of the extracted gross changes before and after shelf elevations. Notably, the PC-1 values of the negative and positive value groups differed significantly. The PC-2 value changed during the phases in which the change in the PC-1 value was unnaturally slow and stopped at CS22 and the first half of CS23. This period, defined as the "approach period", corresponds to the time before dynamic changes occur as the palatal shelves elevate, the tongue and mandibular tip change their position and shape, and secondary palatal shelves contact and fuse. During the "approach period", measurements of PC-2 changes showed that structures on the mandible (Meckel's cartilage and tongue) and maxilla (palate and nasal cavity) did not change positions, albeit both groups of structures appeared to be compressed anterior-posteriorly. However, during and after shelf elevation, measurements of PC-1 changes showed significant changes between maxillary and mandibular structures, particularly positioning of the shelves above the tongue and protrusion of the tongue and mandible. These results suggest an active role for Meckel's cartilage growth in repositioning the tongue to facilitate shelf elevation. The present data representing three distinct phases of secondary palate closure in humans can advance the understanding of morphological growth changes occurring before and after the horizontal positioning of palatal shelves and their fusion to close the secondary palate in humans successfully.

Three-Dimensional Analysis of Human Laryngeal and Tracheobronchial Cartilages during the Late Embryonic and Early Fetal Period.

Laryngeal and tracheobronchial cartilages are present as unique U-shaped forms around the respiratory tract and contribute to the formation of rigid structures required for the airway. Certain discrepancies still exist concerning cartilage formation in humans. To visualize the accurate timeline of cartilage formation, tracheobronchial and laryngeal cartilages were 3D reconstructed based on serial tissue sections during the embryonic period (Carnegie stage [CS] 18-23) and early fetal period (crown rump length [CRL] = 35-45 mm). The developmental phases of the cartilage were estimated by histological studies, which were performed on the reconstructed tissue sections. The hyoid greater horns were recognizable at CS18 (phase 2). Fusion of 2 chondrification centers in the mid-sagittal region was observed at CS19 in the hyoid bone, at CS20 in the cricoid cartilage, and in the specimen with CRL 39 mm in the thyroid cartilage. Phase 3 differentiation was observed at the median part of the hyoid body at CS19, which was the earliest among all other laryngeal and tracheobronchial cartilages. Most of the laryngeal cartilages were in phase 3 differentiation at CS22 and in phase 4 differentiation at CS23. The U-shaped tracheobronchial cartilages with phase 2 differentiation covered the entire extrapulmonary region at CS20. Phase 3 differentiation started on the median section and propagates laterally at CS21. The tracheobronchial cartilages may form simultaneously during the embryonic period at CS22-23 and early fetal periods, similar to adults in number and distribution. The spatial propagation of the tracheal cartilage differentiation provided in the present study indicates that cartilage differentiation may have propagated differently on phase 2 and phase 3. This study demonstrates a comprehensible timeline of cartilage formation. Such detailed information of the timeline of cartilage formation would be useful to improve our understanding of the development and pathophysiology of congenital airway anomalies.

Early development of the cortical layers in the human brain.

The cortical plate (CP) first appears at seven postconceptional weeks (pcw), when it splits the preexisting preplate into two layers, the marginal zone and the presubplate (pSP). Although three-dimensional (3D) analysis using fetal magnetic resonance imaging and two-dimensional tissue observations have been reported, there have been no studies analyzing the early development of the layer structure corresponding to the pSP stage in 3D. Here, we reconstructed 3-D models of the brain with a focus on the cortical layers in pSP stage. To achieve this, we digitized serial tissue sections of embryos between CS20 and CS23 from the Kyoto Collection (n = 7, approximately 7-8.5 pcw), and specimens at early fetal phase from the Blechschmidt Collection (n = 2, approximately 9.5-12 pcw, crown rump length [CRL] 39 and 64 mm). We observed tissue sections and 3D images and performed quantitative analysis of the thickness, surface area, and volume. Because the boundary between pSP and the intermediate zone (IZ) could not be distinguished in hematoxylin and eosin-stained sections, the two layers were analyzed together as a single layer in this study. The histology of the layers was observed from CS21 and became distinct at CS22. Subsequently, we observed the 3-D models; pSP-IZ was present in a midlateral region of the cerebral wall at CS21, and an expansion centered around this region was observed after CS22. We observed it over the entire cerebral hemisphere at early fetal phase (CRL 39 mm). The thickness of pSP-IZ was visible in 3D and was greater in the midlateral region. At the end of the pSP stage (CRL 64 mm), the thick region expanded to lateral, superior, and posterior regions around the primordium of the insula. While, the region near the basal ganglia was not included in the thickest 10% of the pSP-IZ area. Middle cerebral artery was found in the midlateral region of the cerebral wall, near the area where pSP-IZ was observed. Feature of layer structure growth was revealed by quantitative assessment as thickness, surface area, and volume. The maximum thickness value of pSP-IZ and CP increased significantly according to CRL, whereas the median value increased slightly. The layer structure appeared to grow and spread thin, rather than thickening during early development, which is characteristic during pSP stages. The surface area of the cerebral total tissue, CP, and pSP-IZ increased in proportion to the square of CRL. The surface area of CP and pSP-IZ approached that of the total tissue at the end of the pSP stage. Volume of each layer increased in proportion to the cube of CRL. pSP-IZ and CP constituted over 50% of the total tissue in volume at the end of the pSP stages. We could visualize the growth of pSP-IZ in 3D and quantify it during pSP stage. Our approach allowed us to observe the process of rapid expansion of pSP-IZ from the midlateral regions of the cerebral wall, which subsequently becomes the insula.

Nascent nephrons during human embryonic development: Spatial distribution and relationship with urinary collecting system.

The two major components of the metanephros, the urinary collecting system (UCS) and nephron, have different developmental courses. Nephron numbers vary widely between species and individuals and are determined during fetal development. Furthermore, the development of nascent nephrons may contribute to the expansion of the proximal part of the UCS. This study investigated the distribution of nascent nephrons and their interrelationship with UCS branches during human embryogenesis. We obtained samples from 31 human embryos between Carnegie stages (CSs) 19 and 23 from the Kyoto Collection at the Congenital Anomaly Research Center of Kyoto University in Japan. Serial histological sections of the metanephros with the UCS were digitalized and computationally reconstructed for morphological and quantitative analyses. The three-dimensional (3D) coordinates for the positions of all UCS branch points, end points, attachment points to nascent nephrons (APs), and renal corpuscles (RCs) were recorded and related to the developmental phase. Phases were categorized from phase 1 to phase 5 according to the histological analysis. The UCS branching continued until RCs first appeared (at CS19). End branches with attached nascent nephrons (EB-AP[+]) were observed after CS19 during the fifth generation or higher during the embryonic period. The range of end branch and EB-AP(+) generation numbers was broad, and the number of RCs increased with the embryonic stage, reaching 273.8 ± 104.2 at CS23. The number of RCs connected to the UCS exceeded the number not connected to the UCS by CS23. The 3D reconstructions revealed RCs to be distributed around end branches, close to the surface of the metanephros. The RCs connected to the UCS were located away from the surface. The APs remained near the end point, whereas connecting ducts that become renal tubules were found to elongate with maturation of the RCs. Nascent nephrons in RC phases 3-5 were preferentially attached to the end branches at CS22 and CS23. The mean generation number of EB-AP(-) was higher than that of EB-AP(+) in 19 of 22 metanephros and was statistically significant for eight metanephros at CS22 and CS23. The ratio of the deviated branching pattern was almost constant (29%). The ratio of the even branching pattern with EB-AP(+) and EB-AP(+) to the total even branching pattern increased with CS (9.2% at CS21, 19.2% at CS22, and 45.4% at CS23). Our data suggest the following: EB-AP(+) may not branch further at the tip (i.e., by tip splitting), but branching beneath the AP (lateral branching) continues throughout the embryonic stages. Our study provides valuable data that can further the understanding of the interactions between the UCS and nascent nephrons during human embryogenesis.