Abstract: The embryonic period is characterized by organogenesis and accompanying dynamic changes in external features. The measurement of human embryos has been limited to whole body dimensions, such as crown-rump length. More detailed measurements would add quantitative information about these characteristic events and provide a better understanding of normal and abnormal embryonic development. In the present study, we defined axes, landmarks, and measurements for human embryos, and measured 250 externally normal human embryos at Carnegie stages 14-23 (6.5-29.3 mm in crown-rump length, approximately 5-8 weeks of estimated ovulation age) that were fixed in Bouin's solution and preserved in 10% formalin solution. The axes, landmarks, and measurements defined for human embryos are corresponding to those in human and primate fetuses. The whole body, head, face, and extremities were measured using a scale attached to a dissecting microscope. Axial length, head height plus ear-shoulder length plus trunk height, was designated as a new measurement of the whole body, which is comparable with crown-rump length. Approximate standards of these measurements were obtained. The ratios of some measurements to trunk height and between the different parts were also obtained, and several different developmental patterns were recognized. The reproducibility of each measurement was evaluated by measuring 50 specimens three times each at intervals of one or two months. As a pilot study for the application of the proposed measurements, 84 human embryos with external anomalies, including holoprosencephaly, anomalies of extremities, and pharyngeal arch anomalies, were measured using the same method, and a few tendencies characteristic to holoprosencephaly were noticed.

Abstract: Previous studies suggest that jaw movement is an important factor in the development of cartilage in the temporomandibular joint during the prenatal and postnatal periods. In the present study, the effects of fetal jaw movement on the articular disk were studied in mice by restraining the opening movement of the mouth using the mouse exo utero development system. At embryonic day 18.5, the articular disk was reduced in size in the embryos whose maxilla and mandible were sutured (sutured group) and there were changes in the cellular morphology of the mesenchymal cells in the disk. The volume of the articular disk, the total number of cells and the number of 5-bromo-2'-deoxyuridine-positive cells in the articular disk were significantly lower in the sutured group than in the non-sutured control group. Our data revealed that fetal jaw movement affects the development of the articular disk in the temporomandibular joint.

Abstract: Morphometric and histological studies of the pons were performed by light microscopy in 28 cases of externally normal human fetuses ranging from 90 to 246 mm in crown-rump length (CRL) and from 13 to 28 weeks of gestation. The brainstems of fetuses were embedded in celloidin or paraffin, and transverse sections were prepared. The pons was divided into two regions at the most ventral margin of the medial lemniscus at the level of the motor trigeminal nucleus. The relationships between the total dorsoventral length, ventral length, and dorsal length of the pons versus CRL and gestational ages were calculated, and empiric formulas were fitted. It was found that the ventral portion increased in size more rapidly than the dorsal portion. The proportion of the ventral portion in the total dorsoventral length was constitutively higher than that of the dorsal portion in the present range of CRL. In the pontine nuclei, from 235 mm in the CRL, some large cells with rich cytoplasm, pale nuclei, and a distinct nucleolus appeared on the dorsal side of the pyramidal tract. According to Weigert stained preparations, the first myelinated fibers in each motor root of the trigeminal, abducent, and facial nerves were recognized at 130-140 mm in CRL and the medial lemniscus at 230-235 mm.

Abstract: In mammals, three ras genes, H-ras, N-ras and K-ras, encode homologous but distinct 21-kDa Ras proteins. We examined the in vivo functional relationship of the three ras genes in mouse embryonic development by investigating the phenotypes of mice deficient in one or multiple ras genes. H-ras(-/-) mice and N-ras(-/-) mice as well as a substantial proportion of H-ras(-/-)/N-ras(-/-) mice expressing only the K-ras gene were viable, while K-ras(-/-) mice were embryonically lethal, as have been reported previously. N-ras(-/-)/K-ras(+/-) mice died neonatally, while H-ras(-/-)/K-ras(-/-) embryos died much earlier than K-ras homozygous mutant fetuses. To further investigate the functional relationship of the ras genes in embryonic development, we introduced a human H-ras transgene into single or multiple ras mutant mice and found that the transgene rescued mice, including triple ras mutants, from embryonic lethality in association with correction of thin ventricular walls of the heart in null K-ras mutant mice. In situ hybridization revealed that the expression of the H-ras transgene on embryonic day E13.5 and E15.5 was more intense in major organs, including the heart, than those of endogenous ras genes. We therefore conclude that the functions of the ras genes are partially overlapping in mouse embryonic development.Oncogene advance online publication, 3 December 2007; doi:10.1038/sj.onc.1210956.

Abstract: Leptin is a hormone that reduces food intake and increases energy expenditure by acting on the arcuate nucleus in the hypothalamus. Recent studies indicated that the neuronal circuit related to food intake in the hypothalamus is formed in the neonatal period and that leptin is necessary for the formation of this circuit. Our studies have further suggested that leptin may act on the fetal cerebral cortex, including the cingulate cortex, which is involved in motor and cognitive processes, and that leptin may affect maintenance and differentiation of neural stem cells, glial-restricted progenitor cells and/or neuronal lineage cells. These recent studies showed that leptin not only has homeostatic functions in adults, but also regulates brain development in the prenatal and neonatal periods. These findings suggest that leptin is related to formation of the normal brain structure and regenerative potency of neural cells as well as the predisposition to homeostatic dysfunction, low locomotor activity or impairment of cognitive function.

Abstract: Leptin is detected in the sera, and leptin receptors are expressed in the cerebrum of mouse embryos, suggesting that leptin plays a role in cerebral development. Compared with the wild type, leptin-deficient (ob/ob) mice had fewer cells at embryonic day (E) 16 and E18 and had fewer 5-bromo-2'-deoxyuridine(+) cells at E14 and E16 in the neuroepithelium. Intracerebroventricular leptin injection in E14 ob/ob embryos increased the number of neuroepithelium cells at E16. In cultured neurosphere cells, leptin treatment increased Hes1 mRNA expression and maintained neural progenitors. Astrocyte differentiation was induced by low-dose (0.1 microg/ml) but not high-dose (1 microg/ml) leptin. High-dose leptin decreased Id mRNA and increased Ngn1 mRNA in neurosphere cells. The neuropeptide Y mRNA level in the cortical plate was lower in ob/ob than the wild type at E16 and E18. These results suggest that leptin maintains neural progenitors and is related to glial and neuronal development in embryos.

Abstract: In the present study to analyze the role of ACTH in fetal tissues and organs, we observed the expression of melanocortin type 2 (MC2) and 5 (MC5) receptors in ICR mouse embryos from E11.5 to E18.5 by immunohistochemistry. In the adrenal gland and testis, both receptors were expressed from E13.5 to E18.5. In the genital ridge and the ovary, melanocortin type 2 receptors (MC2R) was detected from E11.5 to E12.5 and from E13.5 to E18.5, respectively, while melanocortin type 5 receptors (MC5R) was not detected. In the mesonephros, MC2R and MC5R were expressed from E11.5 to E12.5, and in the metanephros, MC2R and MC5R were expressed from E12.5 to E18.5 and from E14.5 to E18.5, respectively. In the lung, MC2R was expressed from E11.5 to E14.5, but MC5R was not expressed at all. In blood cells, MC5R was detected at all stages examined, while MC2R was detected at none. MC2R was observed in the brain and spinal cord from E11.5 to E13.5, while MC5R was detected only in the telencephalon and only from E16.5 to E18.5. At different temporal patterns, MC2R, but not MC5R, was detected in the choroid plexus, while MC5R, but not MC2R, was expressed in the liver and in the nasal epithelium, and both MC2R and MC5R were expressed in the dorsal root ganglion and the trigeminal ganglion. These findings show the spatio-temporal specific expression patterns of MC2R and MC5R in the mouse embryo and suggest that ACTH may be related to histogenesis and/or prenatal function of various tissues and organs via MC2R and/or MC5R.

Abstract: Leukemia inhibitory factor contributes to the self-renewal of neural stem cells in the forebrain. Although the existence of endogenous leukemia inhibitory factor in the brain parenchyma has been controversial, the cerebrospinal fluid is known to be another source of leukemia inhibitory factor. No reports of the measurement of leukemia inhibitory factor concentrations in the cerebrospinal fluid, however, exist. In the present study, we determined the leukemia inhibitory factor concentration in cerebrospinal fluid, amniotic fluid, and sera of embryos and dams in mice by enzyme-linked immunosorbent assay. The leukemia inhibitory factor concentrations were found to be constitutively high in the cerebrospinal fluid from embryonic day 11 to embryonic day 17, with a peak on embryonic day 13 and embryonic day 14. These findings correspond to the timing of cortical neuron production in mouse cerebrum.

Abstract: OBJECT: The authors investigated the effects of heavy water (D2O) on intrameningeal fibrosis and on the expression of cytokine production in mice with kaolin-induced hydrocephalus. METHODS: Mice in which kaolin was injected into the cisterna magna were divided into two groups: 1) Group H, which had free access to H2O as tap water; and 2) Group D, which had free access to 30% D2O as tap water before and after kaolin injection. A distilled water-injected group, which had free access to H2O as tap water was designated the sham-operated group. The authors examined the effects of D2O within 28 days after injection on the development of hydrocephalus and intrameningeal fibrosis, as well as on the expression levels of several inflammatory and fibrogenic cytokines: transforming growth factor-beta1 (TGFbeta1), fibroblast growth factor-2 (FGF2), platelet-derived growth factor (PDGF)-BB, and interleukin (IL)-6. The cerebral ventricles were less expanded, and intrameningeal fibrosis was milder in Group D than in Group H. The proliferation of fibroblasts was assessed by applying the bromodeoxyuridine labeling index, which was lower in Group D than in Group H. Expression of TGFbeta1 in the macrophages, choroid plexus, and meninges was inhibited in Group D but not in Group H. The serum level of total TGFbeta1 was significantly lower in Group D than in Group H on Day 14, whereas the levels of FGF2, PDGF-BB, and IL-6 did not differ significantly among the groups. CONCLUSIONS: Administration of D2O prevented the development of kaolin-induced hydrocephalus in mice and inhibited intrameningeal fibrosis and upregulation of TGFbeta1.

Abstract: Using a mouse exo utero system to examine the effects of fetal jaw movement on the development of condylar cartilage, we assessed the effects of restraint of the animals' mouths from opening, by suture, at embryonic day (E)15.5. We hypothesized that pre-natal jaw movement is an important mechanical factor in endochondral bone formation of the mandibular condyle. Condylar cartilage was reduced in size, and the bone-cartilage margin was ill-defined in the sutured group at E18.5. Volume, total number of cells, and number of 5-bromo-2'-deoxyuridine-positive cells in the mesenchymal zone were lower in the sutured group than in the non-sutured group at E16.5 and E18.5. Hypertrophic chondrocytes were larger, whereas fewer apoptotic chondrocytes and osteoclasts were observed in the hypertrophic zone in the sutured group at E18.5. Analysis of our data revealed that restricted fetal TMJ movement influences the process of endochondral bone formation of condylar cartilage.

Abstract: Effects of stimulation of the maternal immune system on abnormal pregnancy induced with 5-azacytidine (5ACDR) administration at embryonic day 7.5 (E7.5) were examined in mice treated with recombinant interleukin 1beta (IL-1beta) at E6.5 (5ACDR + IL-1 at E6.5) or E9.5 (5ACDR + IL-1 at E9.5), OK432 (5ACDR + OK432) at E7.5 or PSK (5ACDR + PSK) at E7.5. Embryos from these dams were examined at E13.5. The frequency of dead and malformed embryos and number of malformations on each embryo increased in the 5ACDR + IL-1 at E6.5 groups compared with the 5ACDR-alone group. Adverse pregnancy outcomes in the 5ACDR + OK432 and 5ACDR + PSK groups were less frequent than in the 5ACDR-alone group. The frequency of exencephaly, facial cleft, eye anomalies (micro- or anophthalmos), and micrognathia significantly increased in the 5ACDR + IL-1 groups, in contrast, that of exencephaly decreased in the 5ACDR + OK432 and 5ACDR + PSK groups compared with the 5ACDR-alone group. The phagocytes on the exencephalic surface drastically increased in the 5ACDR + IL-1 groups, and they often appeared to ingest the migrating neuroepithelial cells. Such findings, however, were rarely observed in the 5ACDR-alone, 5ACDR + OK432 and 5ACDR + PSK groups. Thus, administration of IL-1beta to the abnormal pregnant dams increased the mortality and severity of the malformations in the embryos caused by 5ACDR, whereas PSK or OK432 decreased them. These results suggest that the different modes of activation of the maternal immune system may exert alternative or opposite effects on teratogenic pregnancy.

Abstract: The role of gp130 in cerebral cortical histogenesis remains unknown. Mice lacking gp130 showed a hypoplastic cortical plate and decreased incorporation of 5-bromo-2'-deoxyuridine (BrdU) in progenitor cells of the developing cerebrum. In contrast, injection of leukemia inhibitory factor (LIF), a gp130 ligand, into the lateral cerebral ventricle of wild-type embryos exo utero induced hyperplasia of the cerebral cortex and increased the incorporation of BrdU in progenitor cells. Furthermore, chronologically controlled injection of LIF followed or preceded by BrdU revealed that gp130-mediated signals promote the progenitor cells to reenter the stem cell cycle without affecting the duration of cell cycle and enhance the migration of postmitotic neurons in the developing cerebrum.