Six1 is indispensable for production of functional progenitor cells during olfactory epithelial development

The rodent olfactory epithelium (OE) is a good model system for studying the principles of stem and progenitor cell biology, because of its capacity for continuous neurogenesis throughout life and relatively well-characterized neuronal lineage. The development of mouse OE is divided into two stages, early and established neurogenesis. In established neurogenesis, which starts at embryonic day (E) 12.5, sustentacular cells and olfactory receptor neurons (ORNs) are produced from apical and basal progenitors, respectively. We previously reported that Six1(-/-) shows a lack of mature ORNs throughout development and disorganization of OE after E12.5. However, the molecular bases for these defects have not been addressed. Here, we show that Six1 is expressed in both apical and basal progenitors. In Six1(-/-) mice, apical proliferating cells were absent and no morphologically identifiable sustentacular cells were observed. Consistently, the expression of Notch2 and Jagged1 in the apical layer was absent in Six1(-/-) mice. On the other hand, basal proliferating cells were observed in Six1(-/-) animals, but the expression of Ngn1, NeuroD, Notch1, and Jagged2 in the basal layer was absent. The expression of Mash1, the determination gene for ORNs, and Hes genes was enhanced in Six1(-/-) mice. The present findings suggest that Six1 regulates production of functional apical and basal progenitors during OE development, through the regulation of various genes, such as neuronal basic helix-loop-helix (bHLH), neuronal repressor bHLH, and genes involved in the Notch signaling pathway.     

Differential Inactivation of Seed Exudate Stimulation of Pythium ultimum Sporangium Germination by Enterobacter cloacae Influences Biological Control Efficacy on Different Plant Species

This study was initiated to understand whether differential biological control efficacy of Enterobacter cloacae on various plant species is due to differences in the ability of E. cloacae to inactivate the stimulatory activity of seed exudates to Pythium ultimum sporangium germination. In biological control assays, E. cloacae was effective in controlling Pythium damping-off when placed on the seeds of carrot, cotton, cucumber, lettuce, radish, tomato, and wheat but failed to protect corn and pea from damping-off. Seeds from plants such as corn and pea had high rates of exudation, whereas cotton and cucumber seeds had much lower rates of exudation. Patterns of seed exudation and the release of P. ultimum sporangium germination stimulants varied among the plants tested. Seed exudates of plants such as carrot, corn, lettuce, pea, radish, and wheat were generally more stimulatory to P. ultimum than were the exudates of cotton, cucumber, sunflower, and tomato. However, this was not directly related to the ability of E. cloacae to inactivate the stimulatory activity of the exudate and reduce P. ultimum sporangium germination. In the spermosphere, E. cloacae readily reduced the stimulatory activity of seed exudates from all plant species except corn and pea. Our data have shown that the inability of E. cloacae to protect corn and pea seeds from Pythium damping-off is directly related to its ability to inactivate the stimulatory activity of seed exudates. On all other plants tested, E. cloacae was effective in suppressing damping-off and inactivating the stimulatory activity of seed exudates.     

Effects of Diet and Time of the Day on Serum and CSF Leptin Levels in Osborne‐Mendel and S5B/Pl Rats

Objective: To characterize the effects of dietary fat on the diurnal variation in serum and cerebrospinal fluid (CSF) leptin levels in Osborne‐Mendel (OM) and S5B/Pl rats and quantitate the dose response to lower doses of leptin administered into the third cerebral ventricle. Research Methods and Procedures: Rats were fitted with implanted vascular ports or third ventricular cannulas and fed either laboratory chow or one of two semipurified high‐fat or low‐fat diets. Leptin and insulin were measured by immunoassay. Results: Serum leptin and insulin levels were positively correlated and had similar patterns of diurnal change. CSF leptin and insulin also had diurnal rhythms, with a peak at 7:00 am, but the diurnal oscillations of leptin and insulin were significantly lower in the S5B/Pl rats than the OM rats. Thus, the ratio of CSF to serum leptin was significantly higher in the S5B/Pl rats than in the OM rats. Dietary fat had no effect on these diurnal patterns. There was a right shift in the dose response to leptin in the OM rats compared with the S5B/P1 rats. S5B/P1 rats treated with leptin had higher signal transduction and translation (STAT‐3) mRNA levels compared with pair‐fed or saline injected S5B/P1 rats. Hypothalamic suppressors of cytokine signaling mRNA levels were not statistically different between the groups. Discussion: The higher CSF‐to‐serum leptin ratio in the S5B/P1 rats, the enhanced suppression of food intake and body weight with leptin injections, and the higher STAT‐3 activity in these animals suggest that S5B/P1 rats are more sensitive to leptin than OM rats.     

Oscillations in notch signaling regulate maintenance of neural progenitors

Expression of the Notch effector gene Hes1 is required for maintenance of neural progenitors in the embryonic brain, but persistent and high levels of Hes1 expression inhibit proliferation and differentiation of these cells. Here, by using a real-time imaging method, we found that Hes1 expression dynamically oscillates in neural progenitors. Furthermore, sustained overexpression of Hes1 downregulates expression of proneural genes, Notch ligands, and cell cycle regulators, suggesting that their proper expression depends on Hes1 oscillation. Surprisingly, the proneural gene Neurogenin2 (Ngn2) and the Notch ligand Delta-like1 (Dll1) are also expressed in an oscillatory manner by neural progenitors, and inhibition of Notch signaling, a condition known to induce neuronal differentiation, leads to downregulation of Hes1 and sustained upregulation of Ngn2 and Dll1. These results suggest that Hes1 oscillation regulates Ngn2 and Dll1 oscillations, which in turn lead to maintenance of neural progenitors by mutual activation of Notch signaling.     

Synthesis and biological activities of 4-trifluoromethylindole-3-acetic acid: a new fluorinated indole auxin

In our studies on the development of new promoters for the root formation of tree cuttings, 4-trifluoromethylindole-3-acetic acid (4-CF(3)-IAA), a new fluorinated auxin, was synthesized via 4-trifluoromethylindole and 4-trifluoromethylindole-3-acetonitrile by using 2-methyl-3-nitrobenzotrifluoride as the starting material. As a control compound for comparing biological activities, 4-methylindole-3-acetic acid (4-CH(3)-IAA) was also synthesized by using 2,3-dimethylnitrobenzene as the starting material. The biological activities of these compounds were compared by three bioassays with those of indole-3-acetic acid and 4-chloroindole-3-acetic acid (4-Cl-IAA), which, like 4-CF(3)-IAA and 4-CH(3)-IAA, has a substituent at the 4-position of the indole nucleus. 4-CF(3)-IAA showed strong root formation-promoting activity with black gram cuttings which was 1.5 times higher than that of 4-(3-indole)butyric acid at 1x10(-4) M. 4-CH(3)-IAA, however, only weakly promoted root formation in spite of its strong inhibition of hypocotyl growth in Chinese cabbage and promotion of hypocotyl swelling and lateral root formation in black gram. On the other hand, 4-CF(3)-IAA demonstrated weaker activities than 4-CH(3)-IAA and 4-Cl-IAA in these two bioassays.     

Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline

The choroid plexus in the brain is unique because it is a non-neural secretory tissue. It secretes the cerebrospinal fluid and functions as a blood-brain barrier, but the precise mechanism of specification of this non-neural tissue has not yet been determined. Using mouse embryos and lineage-tracing analysis, we found that the prospective choroid plexus region initially gives rise to Cajal-Retzius cells, specialized neurons that guide neuronal migration. Inactivation of the bHLH repressor genes Hes1, Hes3 and Hes5 upregulated expression of the proneural gene neurogenin 2 (Ngn2) and prematurely depleted Bmp-expressing progenitor cells, leading to enhanced formation of Cajal-Retzius cells and complete loss of choroid plexus epithelial cells. Overexpression of Ngn2 had similar effects. These data indicate that Hes genes promote specification of the fate of choroid plexus epithelial cells rather than the fate of Cajal-Retzius cells by antagonizing Ngn2 in the dorsal telencephalic midline region, and thus this study has identified a novel role for bHLH genes in the process of deciding which cells will have a non-neural versus a neural fate.     

Synaptic relationships between proopiomelanocortin- and ghrelin-containing neurons in the rat arcuate nucleus

Both proopiomelanocortin (POMC) and ghrelin peptides are implicated in the feeding regulation. The synaptic relationships between POMC- and ghrelin-containing neurons in the hypothalamic arcuate nucleus were studied using double-immunostaining methods at the light and electron microscope levels. Many POMC-like immunoreactive axon terminals were found to be apposed to ghrelin-like immunoreactive neurons and also to make synapses with ghrelin-like immunoreactive neuronal perikarya and dendritic processes. Most of the synapses were symmetrical in shape. A small number of synapses made by ghrelin-like immunoreactive axon terminals on POMC-like immunoreactive neurons were also identified. Both the POMC- and ghrelin-like immunoreactive neurons were found to contain large dense granular vesicles. These data suggest that the POMC-producing neurons are modulated via synaptic communication with ghrelin-containing neurons. Moreover, ghrelin-containing neurons may also have a feedback effect on POMC-containing neurons through direct synaptic contacts.     

Transfection of Feminizing <Emphasis Type="Italic">Wolbachia</Emphasis> Endosymbionts of the Butterfly, <Emphasis Type="Italic">Eurema hecabe</Emphasis>, into the Cell Culture and Various Immature Stages of the Silkmoth, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Wolbachia are maternally inherited endosymbiotic bacteria of invertebrates that can manipulate the reproductive systems of their arthropod hosts in a variety of ways. To establish a useful model system for investigating the mechanism of Wolbachia-induced host feminization, we conducted the following series of experiments: (1) feminizing Wolbachia of the butterfly, Eurema hecabe, were transferred into cell cultures of the silkmoth, Bombyx mori, and (2) the transfected Wolbachia in cell cultures were inoculated into B. mori at four immature stages. Wolbachia were successfully transfected into the cell cultures and stably maintained for more than 1 year (>30 passages). However, none of the inoculated insects produced mature oocytes that were Wolbachia-positive. This finding was consistent with the fact that Wolbachia was not detected in individuals in subsequent generations. In contrast, Wolbachia were detected at relatively high frequencies (60–80% of individuals) in the somatic tissues of inoculated insects. Real-time quantitative polymerase chain reaction revealed that the Wolbachia densities in the cultured cells were approximately tenfold higher than those in the native host E. hecabe. Among B. mori individuals inoculated at various developmental stages, those inoculated at early stages exhibited higher Wolbachia densities at the adult stage. The Wolbachia densities in individuals inoculated at the second-instar stage were comparable to those in intact E. hecabe. These results suggest that infection and/or proliferation of Wolbachia in germline cells are actively hindered by regulation in B. mori but feasible in somatic cells and that the Wolbachia densities in somatic tissues are regulated by the living host insects.     

Roles of homeobox and bHLH genes in specification of a retinal cell type

Previous analysis of mutant mice has revealed that the bHLH genes Mash1 and Math3, and the homeobox gene Chx10 are essential for generation of bipolar cells, the interneurons present in the inner nuclear layer of the retina. Thus, a combination of the bHLH and homeobox genes should be important for bipolar cell genesis, but the exact functions of each gene remain largely unknown. We have found that in Mash1-Math3 double-mutant retina, which exhibits a complete loss of bipolar cells, Chx10 expression did not disappear but remained in Müller glial cells, suggesting that Chx10 expression per se is compatible with gliogenesis. In agreement with this, misexpression of Chx10 alone with retrovirus in the retinal explant cultures induced generation of the inner nuclear layer cells, including Müller glia, but few of them were mature bipolar cells. Misexpression of Mash1 or Math3 alone did not promote bipolar cell genesis either, but inhibited Müller gliogenesis. In contrast, misexpression of Mash1 or Math3 together with Chx10 increased the population of mature bipolar cells and decreased that of Müller glia. Thus, the homeobox gene provides the inner nuclear layer-specific identity while the bHLH genes regulate the neuronal versus glial fate determination, and these two classes of genes together specify the bipolar cell fate. Moreover, Mash1 and Math3 promoted the bipolar cell fate, but not the other inner nuclear layer-specific neuronal subtypes in the presence of Chx10, raising the possibility that the bHLH genes may be involved in neuronal subtype specification, in addition to simply making the neuronal versus glial fate choice.     

The Purple leaf (Pl) locus of rice: the Pl(w) allele has a complex organization and includes two genes encoding basic helix-loop-helix proteins involved in anthocyanin biosynthesis

The Purple leaf (Pl) locus of rice (Oryza sativa L.) affects regulation of anthocyanin biosynthesis in various plant tissues. The tissue-specific patterns of anthocyanin pigmentation, together with the syntenic relationship, indicate that the rice Pl locus may play a role in the anthocyanin pathway similar to the maize R/B loci. We isolated two cDNAs showing significant identity to the basic helix-loop-helix (bHLH) proteins found in the maize R gene family. OSB1 appeared to be allelic to the previously isolated R homologue, Ra1, but showed a striking difference at the C-terminus because of a 2-bp deletion. Characterization of the corresponding genomic region revealed that the sequence identical to a 5'-portion of OSB2 existed approximately 10-kb downstream of the OSB1 coding region. OSB2 lacks a conserved C-terminal domain. Restriction fragment length polymorphism analyses using an F(2) population indicate that both genes co-segregate with the purple leaf phenotype. A transient complementation assay showed that the anthocyanin pathway is inducible by OSB1 or OSB2. These results suggest that the Pl(w) allele may be complex and composed of at least two genes encoding bHLH proteins.