The role of Hoxa3 gene in parathyroid gland organogenesis of the mouse.

Mice with a targeted deletion of the Hoxa3 gene have defects of derivatives of the third branchial arch and pouch. To address the role of the Hoxa3 gene in parathyroid organogenesis, we examined the third pharyngeal pouch development by immunohistochemistry (IHC) using the secretory protein (SP)-1/chromogranin A antiserum, which recognizes the parathyroid from its initial formation onward. At embryonic day (E) 11.5, the SP-1/chromogranin A-immunoreactive primary rudiment of the parathyroid appeared in the cranial region of the third pharyngeal pouch of wild-type embryos. In Hoxa3-null mutants, the third pharyngeal pouch was normally formed but failed to differentiate into the parathyroid rudiment, showing no immunoreactivity for SP-1/chromogranin A. Classic studies using chick-quail chimeras have demonstrated that the ectomesenchymal neural crest cells are required for proper development of the pharyngeal pouch-derived organs, including the thymus and parathyroid glands. To visualize the migration and development of mesenchymal neural crest cells in Hoxa3 mutants, the heterozygotes were crossed with connexin43-lacZ transgenic mice in which beta-galactosidase expression was specific to the neural crest cells. In Hoxa3 homozygotes and in wild types, ectomesenchymal neural crest cells densely populated the pharyngeal arches, including the third one, and surrounded the third pouch epithelium. These results indicate that lack of the Hoxa3 gene affects the intrinsic ability of the third pharyngeal pouch to form the parathyroid rudiment and has no detectable effect on the migration of neural crest cells.     

Expression of the epithelial marker E-cadherin by thyroid C cells and their precursors during murine development.

Studies of chick-quail chimeras have reported that avian ultimobranchial C cells originate from the neural crest. It has consequently been assumed, without much supporting evidence, that mammalian thyroid C cells also originate from the neural crest. To test this notion, we employed both Connexin43-lacZ and Wnt1-Cre/R26R transgenic mice, because their neural crest cells can be marked. We also examined the immunohistochemical expression of a number of markers that identify migratory or postmigratory neural crest cells, namely, TuJ1, neurofilament 160, nestin, P75NTR, and Sox10. Moreover, we examined the expression of E-cadherin, an epithelial cell marker. At embryonic day (E)10.5, the neural crest cells densely populated the pharyngeal arches but were not distributed in the pharyngeal pouches, including the fourth pouch. At E11.5, the ultimobranchial rudiment formed from the fourth pouch and was located close to the fourth arch artery. At E13.0, this organ came into contact with the thyroid lobe, and at E13.5, it fused with this lobe. However, the ultimobranchial body was not colonized by neural crest-derived cells at any of these developmental stages. Instead, all ultimobranchial cells, as well as the epithelium of the fourth pharyngeal pouch, were intensely immunoreactive for E-cadherin. Furthermore, confocal microscopy of newborn mouse thyroid glands revealed colocalization of calcitonin and E-cadherin in the C cells. The cells, however, were not marked in the Wnt-Cre/R26R mice. These results indicated that murine thyroid C cells are derived from the endodermal epithelial cells of the fourth pharyngeal pouch and do not originate from neural crest cells.     

<Emphasis Type="Italic">Hoxa3</Emphasis> regulates the proliferation and differentiation of the third pharyngeal arch mesenchyme in mice

Genetic disruption of Hoxa3 results in bilateral defects of the common carotid artery, which is derived from the third branchial arch artery. The tunica media of the great arteries derived from the arch arteries is formed by the ectomesenchymal neural crest cells. To examine the etiology of the regression of the third arch artery, we generated Hoxa3 homozygous null mutant embryos that expressed a lacZ marker transgene driven by a connexin43 (Cx43): promoter in the neural crest cells. The expression of -galactosidase in these mouse embryos was examined by both whole-mount X-gal staining and immunohistochemistry with the monoclonal -galactosidase antibody on sections. The migration of neural crest cells from the neural tube to the third branchial arch was not affected in the Hoxa3 homozygotes. The initial formation of the third arch artery was also not disturbed. The artery, however, regressed at embryonic day 11.5 (E11.5), when differentiation of the third pharyngeal arch began. The internal and external carotid arteries arose from the dorsal aorta in E12.5 null mutants, which showed an abnormal persistence of the ductus caroticus. The third pharyngeal arch of wild-type mice fuses with the fourth and second arches at E12.0. In the Hoxa3 null mutants, however, the fusion was delayed, and the hypoplastic third pharyngeal arch was still discerned at E12.5. Moreover, the number of proliferating cells in the third arch of the null mutants was small compared with that in the wild-type. Thus, Hoxa3 is required for the growth and differentiation of the third pharyngeal arch. The defective development of the third pharyngeal arch may induce the anomalies of the carotid artery system. This work was supported in part by a grant (no. 14570026) from the Ministry of Education of Japan to Y.K.     

Movement of paraquat in resistant and susceptible biotypes of Erigeron philadelphicus and E. canadensis

Paraquat (1,1'-dimethyl-4,4'-bipyridinium) resistant biotypes of Erigeron philadelphicus and E. canadensis , from fields where paraquat had been used for weed control, showed more than 100 times higher resistance than the susceptible biotype of both plants. Excised leaves of the susceptible biotypes wilted when supplied with more than 5 μ M paraquatat at the cut ends, but those from the resistant biotypes did not wilt even at 500 μ M. Autoradiographs indicated that (¹⁴CH₃)-paraquat taken up through the cut ends was rapidly distributed through the vascular system in leaves of the susceptible biotype, but was barely translocated in leaves of the resistant biotype. The amount of paraquat taken up during 48 h in the resistant biotype was 0.5% of that in the susceptible biotype in light. This difference in paraquat movement may be correlated with paraquat resistance in Erigeron.     

Drinking Citrus Fruit Juice Inhibits Vascular Remodeling in Cuff-Induced Vascular Injury Mouse Model

Citrus fruits are thought to have inhibitory effects on oxidative stress, thereby attenuating the onset and progression of cancer and cardiovascular disease; however, there are few reports assessing their effect on vascular remodeling. Here, we investigated the effect of drinking the juice of two different citrus fruits on vascular neointima formation using a cuff-induced vascular injury mouse model. Male C57BL6 mice were divided into five groups as follows: 1) Control (water) (C), 2) 10% Citrus unshiu (CU) juice (CU10), 3) 40% CU juice (CU40), 4) 10% Citrus iyo (CI) juice (CI10), and 5) 40% CI juice (CI40). After drinking them for 2 weeks from 8 weeks of age, cuff injury was induced by polyethylene cuff placement around the femoral artery. Neointima formation was significantly attenuated in CU40, CI10 and CI40 compared with C; however, no remarkable preventive effect was observed in CU10. The increases in levels of various inflammatory markers including cytokines such as monocyte chemotactic protein-1, interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α in response to vascular injury did not differ significantly between C, CU10 and CI10. The increases in cell proliferation and superoxide anion production were markedly attenuated in CI10, but not in CU10 compared with C. The increase in phosphorylated ERK expression was markedly attenuated both in CU10 and CI10 without significant difference between CU10 and CI10. Accumulation of immune cells did not differ between CU10 and CI10. These results indicate that drinking citrus fruit juice attenuates vascular remodeling partly via a reduction of oxidative stress. Interestingly, the preventive efficacy on neointima formation was stronger in CI than in CU at least in part due to more prominent inhibitory effects on oxidative stress by CI.     

Adhesive subdivisions intrinsic to the epithelial somites.

Developing somites express two subtypes of classic cadherin adhesion receptors, N-cadherin and cadherin-11 (cad11). To investigate the role of these adhesion molecules in somite morphogenesis, we analyzed the somites of mice whose N-cadherin and cad11 genes were disrupted. The epithelial somites of N-cadherin null mutant mice were fragmented as reported, whereas those of cad11(-/-) mice showed no structural anomaly. In mice double homozygous for N-cadherin and cad11 mutation, however, somites were further fragmented into smaller clusters than in the N-cadherin-deficient mice, suggesting that these two cadherins cooperate in the maintenance of epithelial somites. Despite the disorganization of epithelial structures, dorsoventral polarity markers were expressed in their correct patterns in all of these mutant somites. Uncx4.1, whose expression is localized only in the caudal region of each somite, was also expressed in a normal pattern in the mutant somites. However, the staining for Uncx4.1 revealed that, in the N-cadherin mutants, each somite tended to be cleaved at the border between the Uncx4. 1-positive and -negative regions and that the cleaved subunits maintained the clustered state, often exhibiting epithelioid morphology. This separation of the rostral and caudal regions was observed as soon as the epithelial somites had been formed. In the N-cadherin/cad11 double-homozygous mutants, this tendency was also observed, although each half of the somite further disintegrated into randomly arranged cell clusters. These results suggest that cells of the rostral and caudal regions of each epithelial somite have an activity to aggregate independently or separate from one another and that one role of N-cadherin and cad11 is to connect the two halves into a single unit.     

Disruption of the Hoxa3 homeobox gene results in anomalies of the carotid artery system and the arterial baroreceptors

Homeobox gene Hoxa3 is expressed in the third pharyngeal arch and pouch and is required for development of the third arch artery in addition to the thymus, parathyroid gland and carotid body. We therefore statistically analyzed malformations of the carotid artery system in Hoxa3 homozygous mutant mice, in comparison with wild-type and heterozygous littermates. To identify the carotid artery system, red carbon ink was injected, or vascular casts were made by injection of Mercox resin and observed by scanning electron microscopy. Furthermore, innervation of the carotid sinus and baroreceptor regions in the aortic arch and right subclavian artery were studied in the Hoxa3 null mutants having an abnormal carotid artery system by immunohistochemistry with TuJ1 and protein gene product (PGP) 9.5 antibodies, which recognize nerve fibers and neurons. The common carotid artery of Hoxa3 homozygous mutants was absent or very short and therefore the internal and external carotid artery arose from a more proximal level than those of wild types. The baroreceptor innervation, however, persisted in the mutants, although vascular targets were changed. These results indicate that Hoxa3 gene is crucial for the formation of the common carotid artery and the null mutant mice are the first useful animal models to show that the third arch arteries on both sides specifically degenerate but the fourth and sixth arch arteries are normal.     

Morphological changes of human neuroblastoma cells induced by human gamma interferon

The treatment of GOTO cells, originated from human neuroblastoma, with recombinant human interferon-gamma (rHuIFN-gamma) induced the morphological changes: the extension and bifurcation of neurites and the multinucleated giant cell formation. The treatment of KP-N-RT cells, originated from human neuroblastoma, with rHuIFN-gamma also induced the similar morphological changes. The treatment of these cells with natural HuIFN-gamma also induced the same morphological changes, but those with recombinant human leukocyte interferon (rHuIFN-alpha A), recombinant human fibroblast interferon (rHuIFN-beta) and recombinant murine interferon-gamma (rMuIFN-gamma) did not induce it. The rHuIFN-gamma and the rHuIFN-beta inhibited more strongly the growth of GOTO and KP-N-RT cells than the rHuIFN-alpha A. This suggests that the morphological changes of these neuroblastoma cells are not simply due to the cell growth inhibition, but due to the property which only the rHuIFN-gamma possesses.     

The essential region for assembly and particle formation in hepatitis B virus surface antigen produced in yeast cells

The hepatitis B virus (HBV) genome carries a HBV surface antigen (HBsAg) gene that can encode a polypeptide of 226 amino acids (aa). This gene can be expressed in the yeast, Saccharomyces cerevisiae, and the products can be assembled into 22-nm particles indistinguishable from those recovered from a patient's serum. We constructed a set of deletion derivatives of the HBsAg gene, and examined the particle-forming ability of the resulting polypeptides by expressing the gene in yeast. Elimination of 9 aa from the N terminus had no effect, whereas the elimination of 21-80 aa decreased the ability to form particles, and the particles formed were correspondingly smaller. Elimination of 100 aa that delete the major hydrophobic domain of the molecule abolished the ability to form particles completely. Deletion of 53 aa from the C terminus showed little effect. However, deletions proceeding further toward the center of the molecule rendered the polypeptides unstable.     

Early flowering and increased expression of a FLOWERING LOCUS T-like gene in chrysanthemum transformed with a mutated ethylene receptor gene mDG-ERS1(etr1-4)

Ethylene has an inhibitory effect on flowering in a short-day (SD) plant chrysanthemum (Chrysanthemum morifolium Ramat.). In this study, we used a hexaploid chrysanthemum ??Sei-Marine?? and found that its transgenic lines transformed with a mutated ethylene receptor gene mDG-ERS1(etr1?C4), which conferred reduced ethylene sensitivity (J. Plant Biol. 51: 424?C427, 2008), opened flowers earlier than the non-transformed control. We examined whether the accelerated flower induction in the transformant occurred through the enhanced expression of chrysanthemum genes homologous to FLOWERING LOCUS T (FT), a floral inducer gene in Arabidopsis. We cloned three cDNAs for FT homologs (CmFTL1, CmFTL2, and CmFTL3) from ??Sei-Marine??. CmFTL2 putatively encodes a non-functional gene product due to a frame shift caused by a 2 bp-deletion in the coding region. RT-PCR analysis revealed differential expression patterns of CmFTL genes in the transgenic and control lines, suggesting that these genes might be under the control of ethylene. CmFTL1/2 mRNA level was lower in a SD condition than a long-day (LD) condition. CmFTL3 mRNA accumulated abundantly under SD condition as compared with LD condition in the transgenic line. These results suggest the association of increased expression of CmFTL3 gene with the accelerated flowering in the transgenic line with reduced ethylene sensitivity.