Histomorphometric analysis of the epithelial lumen, mesenchyme, smooth muscle cell layers, and mesentery of the mouse developing duodenum in relation with the macroscopic morphogenesis.

Integral analysis of the development of the epithelium, mesenchyme, and smooth muscle cell (SMC) layers, i.e., the inner circular (IC) and outer longitudinal layers, as well as their relation with the mesentery is necessary to understand macroscopic gut development. We here focused on the proximal duodenum with the characteristic "C"-shaped loop and analyzed the duodenum down to the duodenojejunal flexure in C57BL/6J mouse embryos at embryonic days (E) 13.5, 15.5, and 17.5 by histomorphometric analysis. We examined the angle of the axis of the epithelial lumen, which was oval at E13.5 against the mesentery, along with the epithelial cell nuclear shape, the adjacent mesenchymal cell density in relation to the epithelial lumen axis, and the development of SMC layers. The luminal axis of the oval epithelial lumen at E13.5 rotated clockwise against the mesentery in the proximal duodenum. The shape of epithelial nuclei was longer and thinner at the long axis but shorter and broader at the short axis, whereas mesenchymal density was significantly lower in the area on the luminal long axis than that on the short axis. The number of SMC layers in the IC at E13.5, E15.5, and E17.5 showed a regional difference in relation to the mesentery, but no regional difference along the long axis of the duodenum. These findings suggest that epithelial lumen winding against the mesentery and the corresponding changes in the epithelial cell shape and surrounding mesenchymal density may be involved in the formation of the "C" loop of the proximal duodenum.

Development of the smooth muscle layer in the ileum of mouse embryos.

The smooth muscle layer (SML) comprises a significant portion of the intestines and other tubular organs. Whereas epithelial development has recently been extensively studied, SML development has drawn relatively less attention. Previous morphological reports revealed that the inner circular layer (IC) differentiates earlier than the outer longitudinal layer (OL), but detailed development of the SML, including chronological changes in the cell layer number, precise cell orientation, and regional differences in relation to the mesentery, has not been reported. We here observed the development of the SML in the C57BL/6J mouse ileum near the ileocecal junction at embryonic day (E) 13.5, 15.5, and 17.5. By histo-morphometric analyses, in IC, smooth muscle cells (SMCs) were oval-shaped and irregularly arranged in 3-4 layers at E13.5, then adopted an elongated spindle shape and decreased to two cell layers at E15.5 and E17.5. The IC SMC nuclear angle was not vertical, but oriented at 60-80° against the mid-axis of the intestinal lumen. The single SMC layer in OL was observed at E17.5, and the SMC nuclear angle was parallel to the luminal mid-axis. No clear regional difference against the mesentery was observed. Collectively, the findings suggest that development and differentiation of the ileal SML is not simple but regulated in a complex manner and possibly related to the macroscopic organogenesis.

Maternal undernutrition during early pregnancy inhibits postnatal growth of the tibia in the female offspring of rats by alteration of chondrogenesis.

Epidemiological research has suggested that birth weights are correlated with adult leg lengths. However, the relationship between prenatal undernutrition (UN) and postnatal leg growth remains controversial. We investigated the effects of UN during early pregnancy on postnatal hindlimb growth and determined whether early embryonic malnutrition affects the functions of postnatal chondrocytes in rats. Undernourished Wistar dams were fed 40% of the daily intake of rats in the control groups from gestational days 5.5-11.5, and femurs, tibias, and trunks or spinal columns were morphologically measured at birth and at 16 weeks of age in control and undernourished offspring of both sexes. We evaluated cell proliferation and differentiation of cultured chondrocytes derived from neonatal tibias of female offspring and determined chondrocyte-related gene expression levels in neonatal epiphysis and embryonic limb buds. Tibial lengths of undernourished female, but not male, offspring were longer at birth and shorter at 16 weeks of age (p < .05) compared with those of control rats. In chondrocyte culture studies, stimulating effects of IGF-1 on cell proliferation (p < .01) were significantly decreased and levels of type II collagen were lower in female undernourished offspring (p < .05). These phenomena were accompanied by decreased expression levels of Col2a1 and Igf1r and increased expression levels of Fgfr3 (p < .05), which might be attributable to the decreased expression of specificity protein 1 (p < .05), a key transactivator of Col2a1 and Igf1r. In conclusion, UN stress during early pregnancy reduces postnatal tibial growth in female offspring by altering the function of chondrocytes, likely reflecting altered expression of gene transactivators.

Interkinetic nuclear migration in the mouse embryonic ureteric epithelium: Possible implication for congenital anomalies of the kidney and urinary tract.

Interkinetic nuclear migration (INM) is a phenomenon in which progenitor cell nuclei migrate along the apico-basal axis of the pseudostratified epithelium, which is characterized by the presence of apical primary cilia, in synchrony with the cell cycle in a manner of apical mitosis. INM is suggested to regulate not only stem/progenitor cell proliferation/differentiation but also organ size and shape. INM has been reported in epithelia of both ectoderm and endoderm origin. We examined whether INM exists in the mesoderm-derived ureteric epithelium. At embryonic day (E) 11.5, E12.5 and E13.5, C57BL/6J mouse dams were injected with 5-bromo-2'-deoxyuridine (BrdU) and embryos were killed 1, 2, 4, 6, 8, 10 and 12 h later. We immunostained transverse sections of the ureter for BrdU, and measured the position of BrdU (+) nuclei in the ureteric epithelia along the apico-basal axis at each time point. We analyzed the distribution patterns of BrdU (+) nuclei in histograms using the multidimensional scaling. Changes in the nucleus distribution patterns suggested nucleus movement characteristic of INM in the ureteric epithelia, and the mode of INM varied throughout the ureter development. While apical primary cilia are related with INM by providing a centrosome for the apical mitosis, congenital anomalies of the kidney and urinary tract (CAKUT) include syndromes linked to primary ciliary dysfunction affecting epithelial tubular organs such as kidney, ureter, and brain. The present study showed that INM exists in the ureteric epithelium and suggests that INM may be related with the CAKUT etiology via primary ciliary protein function.

Fetal jaw movement affects Ihh signaling in mandibular condylar cartilage development: the possible role of Ihh as mechanotransduction mediator.

OBJECTIVE: Jaw movement is an important mechanical factor for prenatal development of the condylar cartilage of mandible. Fetal jaw movement restriction has been shown to cause deformity of the mandibular condyle. We hypothesized that this treatment affects the expression of mechanosensitive molecules, namely Indian hedgehog (Ihh) and Parathyroid hormone related protein (PTHrP) in the condyle. EXPERIMENTAL METHODS: We restrained jaw movement by suturing the jaw of E15.5 mouse embryos and allowed them to develop until E18.5 using exo utero system, and analyzed them by immunohistochemistry and in situ hybridization methods. RESULTS: Morphological, histomorphometric and immunohistochemical study showed that the mandibular condylar cartilage was reduced and deformed, the volume and total cell numbers in the condylar cartilage were also reduced, and number and/or distribution of 5-bromo-2'-deoxyuridine-positive cells, Ihh-positive cells in the mesenchymal and pre-hypertrophic zones were significantly and correspondingly decreased in the sutured group. Using in situ hybridization, reduced expression of Ihh, PTHrP and their related receptors were observed in condylar cartilage of the sutured embryos. CONCLUSIONS: Our results revealed that the altered mechanical stress induced by prenatal jaw movement restriction decreased proliferating cells, the amount of cartilage, and altered expression of the Ihh and PTHrP, suggesting that Ihh act as mechanotransduction mediators in the development of mandibular condylar cartilage.

In vivo analysis of Arg-Gly-Asp sequence/integrin α5ß1-mediated signal involvement in embryonic enchondral ossification by exo utero development system.

Enchondral ossification is a fundamental mechanism for longitudinal bone growth during vertebrate development. In vitro studies suggested that functional blockade with RGD peptides or with an antibody that interferes with integrin α5ß1-ligand interactions inhibited pre-hypertrophic chondrocyte differentiation. The purpose of this study is to elucidate in vivo the roles of the integrin α5ß1-mediated signal through the Arg-Gly-Asp (RGD) sequence in the cell-extracellular matrix (ECM) interaction in embryonic enchondral ossification by an exo utero development system. We injected Arg-Gly-Asp-Ser (RGDS) peptides and anti-integrin α5ß1 antibody (α5ß1 ab) in the upper limbs of mouse embryos at embryonic day (E) 15.5 (RGDS-injected limbs, α5ß1 ab-injected limbs), and compared the effects on enchondral ossification with those found in the control limbs (Arg-Gly-Glu-Ser peptide-, mouse IgG-, or vehicle-injected, and no surgery) at E16.5. In the RGDS-injected limbs, the humeri were shorter and there were fewer BrdU-positive cells than in the control limbs. The ratios of cartilage length and area to those of the humerus were higher in the RGDS-injected limbs. The ratios of type X collagen to type 2 collagen mRNA and protein (Coll X/Coll 2) were significantly lower in the RGDS-injected limbs. In those limbs, TUNEL-positive cells were hardly observed, and the ratios of fractin to the Coll X/Coll 2 ratio were lower than in the control limbs. Furthermore, the α5ß1 ab-injected limbs showed results similar to those of RGDS-injected limbs. The present in vivo study by exo utero development system showed that RGDS and α5ß1 ab injection decreased chondrocyte proliferation, differentiation, and apoptosis in enchondral ossification, and suggested that the integrin α5ß1-mediated ECM signal through the RGD sequence is involved in embryonic enchondral ossification.

Mathematical analysis of mandibular morphogenesis by micro-CT-based mouse and alizarin red S-stained-based human studies during development.

Prenatal development of the mandible is an important factor in its postnatal function. To examine quantitatively normal and abnormal developmental changes of the mandible, we here evaluated morphological changes in mineralizing mandibles by thin-plate spline (TPS) including bending energy (BE) and Procrustes distance (PD), and by Procrustes analyses including warp analysis, regression analysis, and discriminant function analysis. BE and PD were calculated from lateral views of the mandibles of mice or of human fetuses using scanned micro-computed tomography (CT) images or alizarin red S-stained specimens, respectively. BE and PD were compared (1) between different developmental stages, and further, to detect abnormalities in the data sets and to evaluate the deviation from normal development in mouse fetuses, (2) at embryonic day (E) 18.5 between the normal and deformed mandibles, the latter being caused by suturing the jaw at E15.5, (3) at E15.5 and E18.5 between normal and knockout mutant mice of receptor tyrosine kinase-like orphan receptor (Ror) 2. In mice, BE and PD were large during the prenatal period and small after postnatal day 3, suggesting that the mandibular shape changes rapidly during the prenatal and early postnatal periods. In humans, BE of the mandibles peaked at 16-19 weeks of gestation, suggesting the time-dependent change in the mandibular shape. TPS and Procrustes analyses statistically separated the abnormal mandibles of the sutured or Ror2 mutant mouse fetuses from the normal mandible. These results suggest that TPS and Procrustes analyses are useful for assessing the morphogenesis and deformity of the mandible.

Effects of restriction of fetal jaw movement on prenatal development of the temporalis muscle.

Jaw movement affects masticatory muscles during the postnatal period. Prenatal jaw movement has also been implicated in the development of the temporomandibular joint; however, its effect on prenatal development of the masticatory muscles has not been extensively analysed. In the present study, we examined the effects of the restriction of fetal jaw movement on the temporalis muscle, a major masticatory muscle, in mice by suturing the maxilla and mandible (sutured group) using an exo utero development system. We compared the morphology of the temporalis muscle between sutured, sham-operated and normal in utero groups. At embryonic day (E) 18.5, the volume of muscle fibres, but not that of connective tissue, in the temporalis muscle was decreased in the sutured group. The E18.5 temporalis muscle in the sutured group appeared morphologically similar to that of the E17.5 in utero group, except for frequent muscle fibre irregularities. By transmission electron microscopy, in the sutured group, the myofibrils were immature and scattered, the nuclei appeared comparatively immature, the mitochondria were expanded in volume with fewer cristae, and cytoplasmic inclusion bodies were frequently observed. Expression of Myf-6, a late myogenic transcription factor, by real-time RT-PCR was not significantly different between the sutured and sham-operated groups. These findings demonstrated approximately 1-day delay in the morphological development of the temporalis muscle in the sutured group, and some abnormalities were observed, although Myf-6 level was not affected in the sutured group. The present study revealed that the prenatal jaw movement influences the development of the temporalis muscle.