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.

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.

Morphology and morphometry of the human early foetal brain: A three-dimensional analysis.

Morphometric analyses in the early foetal phase (9-13 postconceptional week) are critical for evaluating normal brain growth. In this study, we assessed sequential morphological and morphometric changes in the foetal brain during this period using high-resolution T1-weighted magnetic resonance imaging (MRI) scans from 21 samples preserved at Kyoto University. MRI sectional views (coronal, mid-sagittal, and horizontal sections) and 3D reconstructions of the whole brain revealed sequential changes in its external morphology and internal structures. The cerebrum's gross external view, lateral ventricle and choroid plexus, cerebral wall, basal ganglia and thalamus, and corpus callosum were assessed. The development of the cerebral cortex, white matter microstructure, and basal ganglia can be well-characterized using MRI scans. The insula became apparent and deeply impressed as brain growth progressed. A thick, densely packed cellular ventricular/subventricular zone and ganglionic eminence became apparent at high signal intensity. We detected the emergence of important landmarks which may be candidates in the subdivision processes during the early foetal period; the corpus callosum was first detected in the sample with crown-rump length (CRL) 62 mm. A primary sulcus on the medial part of the cortex (cingulate sulcus) was observed in the sample with CRL 114 mm. In the cerebellum, the hemispheres, posterolateral fissure, union of the cerebellar halves, and definition of the vermis were observed in the sample with CRL 43.5 mm, alongside the appearance of a primary fissure in the sample with CRL 56 mm and the prepyramidal fissure in the sample with CRL 75 mm. The volumetric, linear, and angle measurements revealed the comprehensive and regional development, growth, and differentiation of brain structures during the early foetal phase. The early foetal period was neither morphologically nor morphometrically uniform. The cerebral proportion (length/height) and the angle of cerebrum to the standard line at the lateral view of the cerebrum, which may reflect the growth and C-shape formation of the cerebrum, may be a candidate for subdividing the early foetal period. Future precise analyses must establish a staging system for the brain during the early foetal period. This study provides insights into brain structure, allowing for a correlation with functional maturation and facilitating the early detection of brain damage and abnormal development.

Design, synthesis and biological evaluation of simplified analogues of MraY inhibitory natural product with rigid scaffold.

Muraymycins and caprazamycins are strong inhibitors of MraY, which is responsible for peptidoglycan biosynthesis. Although they are promising antibacterial agents with a novel mode of action, their chemical structures are rather complex. This study investigated the simplification of these natural products by structure-based drug design, synthesis, and biological evaluation. We developed a simplified rigid scaffold with an arylalkyne moiety, which shows sub-micromolar MraY inhibitory activity. The scaffold is suitable for further investigating the structure-activity relationship by virtue of our synthetic strategy, where the substituent of interest is installed in the last stage of synthesis. This scaffold shows the potential for further use in optimizing MraY inhibitory and antibacterial activities.

Development of cyclic peptide derivatives from the N-terminal region of LANA for targeting the nucleosome acidic patch.

Kaposi's sarcoma-associated herpesvirus (KSHV) is known to be a carcinogenic agent that causes AIDS-associated Kaposi's sarcoma (KS). When KSHV infects host's cells, one of the virus's proteins, latency-associated nuclear antigen 1 (LANA), binds to the host's nucleosomes to retain episomes and create latency circumstances. Although the infectious mechanism of KSHV is partly elucidated, the development of drug candidates for targeting KS is ongoing. In this study, we developed cyclic peptides corresponding to an N-terminal LANA sequence that disrupt the LANA-nucleosome interaction. The cyclic peptides showed a different secondary structure compared to their corresponding linear peptide derivatives, which suggests that our cyclization strategy imitates the N-terminal LANA binding conformation on nucleosomes.

Return of the intestinal loop to the abdominal coelom after physiological umbilical herniation in the early fetal period.

The intestine elongates during the early fetal period, herniates into the extraembryonic coelom, and subsequently returns to the abdominal coelom. The manner of herniation is well-known; however, the process by which the intestinal loop returns to the abdomen is not clear. Thus, the present study was designed to document and measure intestinal movements in the early fetal period in three dimensions to elucidate the intestinal loop return process. Magnetic resonance images from human fetuses whose intestinal loops herniated (herniated phase; n = 5) while returning to the abdominal coelom [transition phase; n = 3, crown-rump length (CRL)] 37, 41, and 43 mm] and those whose intestinal loops returned to the abdominal coelom normally (return phase; n = 12) were selected from the Kyoto Collection. Intestinal return began from proximal to distal in samples with CRL of 37 mm. Only the ileum ends were observed in the extraembryonic coelom in samples with CRLs of 41 and 43 mm, whereas the ceca were already located in the abdominal coeloms. The entire intestinal tract had returned to the abdominal coelom in samples with CRL > 43 mm. The intestinal length increased almost linearly with fetal growth irrespective of the phase (R2  = 0.90). The ratio of the intestinal length in the extraembryonic coelom to the entire intestinal length was maximal in samples with CRLs of 32 mm (77%). This ratio rapidly decreased in three of the samples that were in the transition phase. The abdominal volumes increased exponentially (to the third power) during development. The intestinal volumes accounted for 33-41% of the abdominal volumes among samples in the herniated phase. The proportion of the intestine in the abdominal cavity increased, whereas that in the liver decreased, both without any break or plateau. The amount of space available for the intestine by the end of the transition phase was approximately 200 mm3 . The amount of space available for the intestine in the abdominal coelom appeared to be sufficient at the beginning of the return phase in samples with CRLs of approximately 43 mm compared with the maximum intestinal volume available for the extraembryonic coelom in the herniated phase, which was 25.8 mm3 in samples with CRLs of 32 mm. A rapid increase in the space available for the intestine in the abdominal coelom that exceeded the intestinal volume in the extraembryonic coelom generated an inward force, leading to a 'sucked back' mechanism acting as the driving force. The height of the hernia tip increased to 8.9 mm at a maximum fetal CRL of 37 mm. The height of the umbilical ring increased in a stepwise manner between the transition and return phases and its height in the return phase was comparable to or higher than that of the hernia tip during the herniation phase. We surmised that the space was generated in the aforementioned manner to accommodate the herniated portion of the intestine, much like the intestine wrapping into the abdominal coelom as the height of the umbilical ring increased.

Femoral posture during embryonic and early fetal development: An analysis using landmarks on the cartilaginous skeletons of ex vivo human specimens.

The pre-axial border medially moves between the fetal and early postnatal periods, and the foot sole can be placed on the ground. Nonetheless, the precise timeline when this posture is achieved remains poorly understood. The hip joint is the most freely movable joint in the lower limbs and largely determines the lower-limb posture. The present study aimed to establish a timeline of lower-limb development using a precise measurement of femoral posture. Magnetic resonance images of 157 human embryonic samples (Carnegie stages [CS] 19-23) and 18 fetal samples (crown rump length: 37.2-225 mm) from the Kyoto Collection were obtained. Three-dimensional coordinates of eight selected landmarks in the lower limbs and pelvis were used to calculate the femoral posture. Hip flexion was approximately 14° at CS19 and gradually increased to approximately 65° at CS23; the flexion angle ranged from 90° to 120° during the fetal period. Hip joint abduction was approximately 78° at CS19 and gradually decreased to approximately 27° at CS23; the average angle was approximately 13° during the fetal period. Lateral rotation was greater than 90° at CS19 and CS21 and decreased to approximately 65° at CS23; the average angle was approximately 43° during the fetal period. During the embryonic period, three posture parameters (namely, flexion, abduction, and lateral rotation of the hip) were linearly correlated with each other, suggesting that the femoral posture at each stage was three-dimensionally constant and exhibited gradual and smooth change according to growth. During the fetal period, these parameters varied among individuals, with no obvious trend. Our study has merits in that lengths and angles were measured on anatomical landmarks of the skeletal system. Our obtained data may contribute to understanding development from anatomical aspects and provide valuable insights for clinical application.

Less invasive treatment for broad ligament hernia: A case report.

INTRODUCTION: Among diseases that cause small bowel obstruction, internal hernia may result in severe outcomes. Therefore, emergency laparotomy has been often performed, but it may be invasive. We experienced a case of a broad ligament hernia of the uterus, which is relatively rare among the cases of internal hernia that were treated by less invasive laparoscopic surgery. CASE PRESENTATION: A 71-year-old woman came to our hospital because of abdominal pain and vomiting. Abdominal computed tomography (CT) revealed a broad ligament hernia of the uterus without intestinal ischemia. Intestinal decompression was done by inserting an ileus tube (® Long Intestinal Tube, CLINY), followed by elective laparoscopic surgery. DISCUSSION: If intestinal ischemia can be ruled out, a less invasive laparoscopic surgery may be performed after intestinal decompression. Further, by detecting the cause of internal hernia, more safe and smooth surgery can be performed. These findings highlighted the importance of accurate CT diagnosis. CONCLUSION: In order to perform laparoscopic surgery for internal hernia, evaluation of the presence or absence of intestinal ischemia and detection of the cause of obstruction by preoperative abdominal CT are important.

Relationship between rectal abdominis muscle position and physiological umbilical herniation and return: A morphological and morphometric study.

The herniation of the intestinal loop (IL) in the extraembryonic coelom and its return to abdominal cavity is in parallel with the formation of the rectal abdominis muscle (RAM). Using high-resolution magnetic resonance imaging data of human fetuses (n = 19, CRL22-69 mm; stored at Kyoto Collection), this study aimed to analyze the relationship between the development of RAM and phase of IL herniation. The RAM runs at the lateral part of the abdominal wall in the small samples in the herniation phase. The position was shifted to the midline area in the larger samples in the herniation phase. According to fetal growth, the caudal ends of the muscles extended along the umbilical ring towards the pubis, though the caudal part of the RAMs were thin and faint in most of the samples. Length measurements related with the growth of the abdominal wall including RAM and abdominal circumference showed positive correlation with fetal growth. On the contrary, diastasis of RAMs and the width and area of the umbilical ring were almost constant according to fetal growth. Such morphometric value showed no obvious changes regardless of the phases of herniation. The ratio of the width and diastasis of the RAMs to the circumference was decreased, indicating that the closure of the ventral body wall was influenced by growth differences. The present data indicate that the formation of the abdominal wall including RAM is independent of the phase of IL herniation, whether in the extraembryonic coelom or in the abdominal cavity.

Spatial Relationship Between the Metanephros and Adjacent Organs According to the Carnegie Stage of Development.

The morphological changes in the metanephros and its spatial relationship to the adjacent organs was evaluated based on the Carnegie stages (CSs) from 14 through 23. The imaging modalities used included magnetic resonance imaging (N = 4), phase-contrast X-ray computed tomography (N = 11), and serial histological sections (N = 40), supplemented by three-dimensional image reconstruction. The orientation of the hilus of the metanephros changed significantly between CS17 (34.4 ± 13.7 degrees) and 18 (122.3 ± 38.1 degrees), with an increase in the number of branches of the urinary collecting system, from 1.61 ± 0.42 at CS17 to 3.20 ± 0.35 at CS18. This increase in the number of branches influenced the growth of the metanephros and the orientation of its hilus. The right and left metanephroses were in proximity throughout the embryonic period. The local maximum interpole distances were observed at CS18 (0.87 ± 0.11 mm for the upper and 0.50 ± 0.25 mm for the lower pole). Mesenchymal tissue was observed between the metanephros and iliac arteries, as well as between the right and left metanephros. Throughout development, the position of the lower pole of the metanephros remained adjacent to the aortic bifurcation. The position of the upper pole, referenced with respect to the aortic bifurcation, increased by >2.0 mm, reflecting the longitudinal growth of the metanephros. Our findings provide a detailed description of the morphogenesis of the metanephros and of its hilus, which might contribute to our understanding of congenital malformations and malpositions of the kidneys. Anat Rec, 302:1901-1915, 2019. © 2019 American Association for Anatomy.

Rib Cage Morphogenesis in the Human Embryo: A Detailed Three-Dimensional Analysis.

Formation of the skeletal structure in the human embryo has important consequences in terms of support, protection, and function of organs and other systems. We aimed to describe the formation of the rib cage during the embryonic period, in order to detect prominent features and identify the possible factors affecting rib cage morphology. We employed high-resolution digitized imaging data (n = 34) obtained in human embryos with Carnegie stage (CS) between 17 and 23. The rib cage became detectable as cartilage formation at CS17, expanding outward from the dorsal side of the chest-abdominal region. Ribs elongated progressively to surround the chest, differentiating into the upper and lower rib cage regions by CS20. The ends of corresponding ribs in the upper region elongated toward each other, leading to their joining and sternum formation between CS21 and CS23, while the lower region of the rib cage remained widely open. The rib cage area with the largest width shifted from the 5th rib pair at CS17 to the 9th pair at CS23. The depth of the rib cage was similar across the upper region at CS17, with the major portion remaining in the middle part after CS20. The heart was located beneath the rib pairs providing the largest depth, while the liver was located beneath the rib pairs providing the largest width. Formation of the sternum, development of spinal kyphosis, and organization of larger internal organs within the thoracic and abdominal cavity are possible factors affecting rib cage morphology. Anat Rec, 302:2211-2223, 2019. © 2019 American Association for Anatomy.

Morphogenesis of the Middle Ear during Fetal Development as Observed Via Magnetic Resonance Imaging.

Recently, our research group has utilized serial histological sections to investigate the morphogenesis of the middle ear, which corresponds to the period of middle ear ossicle (MEO) cartilage formation. However, research regarding middle ear development during the post-embryonic period has been limited. In the present study, we investigated morphogenesis of the middle ear in human fetuses with a crown-rump length (CRL) between 37 and 197 mm using high-resolution magnetic resonance imaging (MRI). Our findings indicated that the morphology of the MEOs is similar during fetal development and adulthood; further, growth of the MEOs nearly ceases once a CRL of 150 mm is attained. In each MEO, ossification spreads from a single center. The malleus and Meckel's cartilage could be discriminated in samples exhibiting a CRL of 145 mm based on differences in MRI signal intensity. In samples with a CRL of 86 mm, the tympanic cavity (TC) appeared as a thin yet distinct structure attached to the external auditory meatus at the convex surface. Only the handle of the malleus was covered by the TC, while the incus and stapes contacted the cavity at the region of articulation between the two ossicles only, even after a CRL of 145 mm had been attained. Thus, although the TC increased in both diameter and thickness, coverage did not extend across all three MEOs during the observation period. These data are expected to provide a useful standard for morphogenesis and may aid researchers in distinguishing between normal and abnormal development. Anat Rec, 301:757-764, 2018. © 2017 Wiley Periodicals Inc.

Variations of the Circle of Willis at the End of the Human Embryonic Period.

Variations of the circle of Willis (CW) influence blood supply to the brain and adjacent structures in adults. We examined the formation of the CW in 20 human embryo samples at the end of the embryonic period using 3-D reconstructions of serial histological sections. The CW was closed in all samples, and did not form in a single plane, but was composed of multiple stair-like planes. The artery acutely curved at the caudal part of the CW, namely, at the inlet of the basilar artery and bifurcation of the P1 segment of the posterior cerebral artery (PCA), reflecting flexure of the mesencephalon and diencephalon at this stage. Variations were observed in 17 of 20 samples-only anterior parts (anterior communicating artery [Acom] and anterior cerebral artery [ACA]) in 10 samples, only posterior parts (posterior communicating artery [Pcom]) in one sample, and both anterior and posterior parts in six samples. Variations included the Acom formed as partially duplicated in three samples, duplicated in four, plexiform in three, and no channel as a result of a single azygos ACA in one. The ACA formed as duplicated in two, median ACA in two, and right hypoplasia in one. The Pcom formed in hypoplasia of either side in six samples. Variations observed in this study are similar to those observed in fetuses, neonates, and adults. The P1 segment of PCA was very large in all samples. The present observations indicate that variations in the CW are present from the initiation of CW formation. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Formation of the Periotic Space During the Early Fetal Period in Humans.

The inner ear is a very complicated structure, composed of a bony labyrinth (otic capsule; OC), membranous labyrinth, with a space between them, named the periotic labyrinth or periotic space. We investigated how periotic tissue fluid spaces covered the membranous labyrinth three-dimensionally, leading to formation of the periotic labyrinth encapsulated in the OC during human fetal development. Digital data sets from magnetic resonance images and phase-contrast X-ray tomography images of 24 inner ear organs from 24 human fetuses from the Kyoto Collection (fetuses in trimesters 1 and 2; crown-rump length: 14.4-197 mm) were analyzed. The membranous labyrinth was morphologically differentiated in samples at the end of the embryonic period (Carnegie stage 23), and had grown linearly to more than eight times in size during the observation period. The periotic space was first detected at the 35-mm samples, around the vestibule and basal turn of the cochlea, which elongated rapidly to the tip of the cochlea and semicircular ducts, successively, and almost covered the membranous labyrinth at the 115-mm CRL stage or later. In those samples, several ossification centers were detected around the space. This article thus demonstrated that formation of the membranous labyrinth, periotic space (labyrinth), and ossification of the OC occurs successively, according to an intricate timetable. Anat Rec, 301:563-570, 2018. © 2018 Wiley Periodicals, Inc.

Branching morphogenesis of the urinary collecting system in the human embryonic metanephros.

An elaborate system of ducts collects urine from all nephrons, and this structure is known as the urinary collecting system (UCS). This study focused on how the UCS is formed during human embryogenesis. Fifty human embryos between the Carnegie stage (CS) 14 and CS23 were selected from the Kyoto Collection at the Congenital Anomaly Research Center of Kyoto University, Japan. Metanephroses, including the UCS, were segmented on serial digital virtual histological sections. Three-dimensional images were computationally reconstructed for morphological and quantitative analyses. A CS timeline was plotted. It consisted of the 3-D structural morphogenesis of UCS and quantification of the total amount of end-branching, average and maximum numbers of generations, deviation in the metanephros, differentiation of the urothelial epithelium in the renal pelvis, and timing of the rapid expansion of the renal pelvis. The first UCS branching generation occurred by CS16. The average branching generation reached a maximum of 8.74 ± 1.60 and was already the twelfth in CS23. The total end-branching number squared between the start and the end of the embryonic period. UCS would reach the fifteenth branching generation soon after CS23. The number of nephrons per UCS end-branch was low (0.21 ± 0.14 at CS19, 1.34 ± 0.49 at CS23), indicating that the bifid branching occurred rapidly and that the formation of nephrons followed after. The renal pelvis expanded mainly in CS23, which was earlier than that reported in a previous study. The number of nephrons connected to the UCS in the expanded group (246.0 ± 13.2) was significantly larger than that of the pre-expanded group (130.8 ± 80.1) (P < 0.05). The urothelial epithelium differentiated from the zeroth to the third generations at CS23. Differentiation may have continued up until the tenth generation to allow for renal pelvis expansion. The branching speed was not uniform. There were significantly more branching generations in the polar- than in the interpolar regions (P < 0.05). Branching speed reflects the growth orientation required to form the metanephros. Further study will be necessary to understand the renal pelvis expansion mechanism in CS23. Our CS-based timeline enabled us to map UCS formation and predict functional renal capacity after differentiation and growth.

Positional Changes of the Ocular Organs During Craniofacial Development.

The present study aimed to describe the positional changes of the ocular organs during craniofacial development; moreover, we examined the relationships among the ocular organs and other internal structures. To do this, we traced the positions of the ocular organs in 56 human early fetal samples at different stages of development using high-resolution magnetic resonance imaging and phase-contrast X-ray computed tomography. The eyes were located on the lateral side in the ventral view at Carnegie stage (CS) 16, and then changed their positions medially during development. The eyes remained in the neurocranium until CS17. However, the eyes changed their positions medially and caudally in the viscerocranium after CS18. The positional relationship between the eyes and pituitary gland changed in the lateral view as development progressed. Specifically, they were close to each other at CS17, but moved apart during the later stages of development. These positional changes were also demonstrated quantitatively with morphometric analyses. Based on the present data, the positional changes of the eyes can be categorized into phases, as follows: Phase 1, dramatic positional changes (early fetal period until CS23); and Phase 2, mild positional changes (stabilized; early fetal period after CS23). Notably, all absolute lengths measured in the present study linearly increased as the crown-rump length increased irrespective of the phase, while features of the measured angles and ratios differentially changed in Phases 1 and 2. The present data may help improve our understanding of both the normal and abnormal development of the ocular organs and craniofacial area. Anat Rec, 300:2107-2114, 2017. © 2017 Wiley Periodicals, Inc.

Cartilage formation in the pelvic skeleton during the embryonic and early-fetal period.

The pelvic skeleton is formed via endochondral ossification. However, it is not known how the normal cartilage is formed before ossification occurs. Furthermore, the overall timeline of cartilage formation and the morphology of the cartilage in the pelvis are unclear. In this study, cartilage formation in the pelvic skeletons of 25 human fetuses (crown-rump length [CRL] = 11.9-75.0 mm) was observed using phase-contrast computed tomography and 7T magnetic resonance imaging. The chondrification center of the ilium, ischium, and pubis first appeared simultaneously at Carnegie stage (CS) 18, was located around the acetabulum, and grew radially in the later stage. The iliac crest formed at CS20 while the iliac body's central part remained chondrified. The iliac body formed a discoid at CS22. The growth rate was greater in the ilium than in the sacrum-coccyx, pubis, and ischium. Connection and articulation formed in a limited period, while the sacroiliac joint formed at CS21. The articulation of the pubic symphysis, connection of the articular column in the sacrum, and Y-shape connection of the three parts of the hip bones to the acetabulum were observed at CS23; the connection of the ischium and pubic ramus was observed at the early-fetal stage. Furthermore, the degree of connection at the center of the sacrum varied among samples. Most of the pelvimetry data showed a high correlation with CRL. The transverse and antero-posterior lengths of the pelvic inlet of the lesser pelvis varied among samples (R2 = 0.11). The subpubic angle also varied (65-90°) and was not correlated with CRL (R2 = 0.22). Moreover, cartilaginous structure formation appeared to influence bone structure. This study provides valuable information regarding the morphogenesis of the pelvic structure.

Morphogenesis of the middle ear ossicles and spatial relationships with the external and inner ears during the embryonic period.

We describe the three-dimensional morphogenesis of the middle ear ossicles (MEOs) according to Carnegie stage (CS) in human embryos. Seventeen samples including 33 MEOs from CS18 to 23 were selected from the Kyoto Collection. The primordia of the MEOs and related structures were histologically observed and three-dimensionally reconstructed from digital images. The timing of chondrogenesis was variable among structures. The stapes was recognizable as a vague condensation of the mesenchymal cells in all samples from CS18, whereas the malleus and incus were recognizable at CS19. Chondrogenesis of all MEOs was evident in all samples after CS21. The chondrocranium was recognizable in all samples by CS18, and the perichondrium border of the auricular cartilage and otic capsule was distinct in all samples at CS23. At CS19, the MEOs were positioned in the anterior to posterior direction, following the order malleus, incus, stapes, which adjusted gradually during development. The MEOs connected in all samples after CS22. The stapes was located close to the vestibular part of the inner ear, although the basal part was not differentiated into the "footplate" form, even at CS23. The handles of the malleus were close to the tubotympanic recess at CS23, but were distant from the external auditory meatus. Determining the timeline of the formation of MEOs and connection of the external and inner ears can be informative for understanding hearing loss caused by failure of this connection. These data may provide a useful standard for morphogenesis, and will contribute to distinguishing between normal and abnormal MEO development. Anat Rec, 299:1325-1337, 2016. © 2016 Wiley Periodicals, Inc.