Spatiotemporal expression pattern of Myt/NZF family zinc finger transcription factors during mouse nervous system development.

BACKGROUND: Three members of the Myt/NZF family of transcription factors are involved in many processes of vertebrate development. Several studies have reported that Myt1/NZF-2 has a regulatory function in the development of cultured oligodendrocyte progenitors or in neuronal differentiation during Xenopus primary neurogenesis. However, little is known about the proper function of Myt/NZF family proteins during mammalian nervous system development. To assess the possible function of Myt/NZF transcription factors in mammalian neuronal differentiation, we determined the comparative spatial and temporal expression patterns of all three types of Myt/NZF family genes in the embryonic mouse nervous system using quantitative reverse transcriptase polymerase chain reaction and in situ hybridization. RESULTS: All three Myt/NZF family genes were extensively expressed in developing mouse nervous tissues, and their expression was transient. NZF-1 was expressed later in post-mitotic neurons. NZF-2 was initially expressed in neuronal cells a little earlier than NZF-3. NZF-3 was initially expressed in neuronal cells, just after proliferation was complete. CONCLUSION: These expression patterns suggest that the expression of NZF family genes is spatially and temporally regulated, and each Myt/NZF family gene may have a regulatory function in a specific phase during neuronal differentiation.

NADPH oxidase 4 function as a hydrogen peroxide sensor.

Nox4, a member of the NADPH- and oxygen-dependent oxidoreductases that generate reactive oxygen species (ROS), is widely expressed and constitutively active. To understand better its function and regulation, specific mutations in the Nox4 dehydrogenase (DH) domain were examined for effects on Nox4 oxidase activity. Transfection of His6-tagged Nox4 increased the amount of p22phox subunit in HEK293 cells, and a higher level of the heterodimer was observed in the nucleus-enriched fraction (NEF). NEF from Nox4-expressing HEK293 cells exhibited oxygen and H2O2 concentration-dependent NADPH oxidation rate. In Nox4-expressing cells, NEF and its partially purified form, the Nox4(P437H) mutant almost completely lost its oxidase activity, while Nox4(C546S), Nox4(C546L) or/and (C547L) had a significantly decreased rate of ROS production. The NADPH-dependent reduction of cytochrome c or cytochrome b5 by purified Nox4 DH domain was found regulated by the H2O2 concentration, and C546L and C547L mutants showed lower rates of the hemeprotein reduction. These conserved Cys residues in the DH domain respond to the cytosolic H2O2 concentration to regulate Nox4 activity.

Radial Columnar Patches in the Chimeric Cerebral Cortex Visualized by Use of Mouse Embryonic Stem Cells Expressing ß-Galactosidase: (mouse chimera/ES cell/cerebral cortex/ß-galactosidase).

Presumed radial migration of neuroblasts from the ventricular to pial surface during formation of the cerebral cortex predicts radial columnar patches in chimeric brains. Lack of adequate cell marker for neurons, however, has hindered such chimera analysis. We used a mouse embryonic stem cell line expressing ß-galactosidase gene to produce chimeric brains. Patches of the labeled cells were examined by whole mount staining and also by computer-assisted three-dimensional reconstruction from serial paraffin sections. Our study revealed presence of coherent radial columnar patches in the prenatal cerebral cortex, thus giving a direct evidence for the radial migration of neurons. These columnar patches were less clear in adult brains, suggesting cell mixing during later development and maturation.

Expression Pattern of E- and P-Cadherin in Mouse Embryos and Uteri during the Periimplantation Period: (implantation/mouse embryo/cell adhesion molecules E-cadherin/P-cadherin).

Periimplantation mouse embryos and uterine tissues were examined by means of immunohistochemistry for their expression of the Ca2+ dependent cell-cell adhesion molecules, E- and P-cadherin. E-cadherin was detected in all embryonic cells during periimplantation stages, and also detected in the uterine epithelium. When blastocysts attached to the uterine epithelium, E-cadherin was detected at implantation sites between the mural trophectoderm and the uterine epithelium on 5 day of pregnancy. P-cadherin was first detected in the mural trophectoderm on 4.5-day blastocysts, and then detected in the ectoplacental cone, giant cells and visceral endoderm from 5.5 day. P-cadherin was also detected in the maternal uterine decidual cells from 5.5 day. After degeneration of uterine epithelial cells, giant cells make direct contact with uterine decidual cells, and P-cadherin was detected at contact sites between these cells. Thus, the complicated process of implantation seems to be supported by temporal and spatial expression of the multiple classes of cadherins.

Myt/NZF family transcription factors regulate neuronal differentiation of P19 cells.

During mammalian central nervous system development, neural stem cells differentiate and then mature into various types of neurons. Myelin transcription factor (Myt)/neural zinc finger (NZF) family proteins were first identified as myelin proteolipid protein promoter binding factors and were shown to be involved in oligodendrocyte development. In this study, we found that Myt/NZF family molecules were expressed during neuronal differentiation in vivo and in vitro. Transient over-expression of Myt/NZF family genes could convert undifferentiated P19 cells into neurons without induction by retinoic acid (RA), and the ability of these genes to induce neuronal differentiation was comparable to that of Neurog1 and Neurod1. Additionally, we found that St18 (or NZF-3) was induced by several bHLH transcription factors. When NZF-3 and Neurog1 were co-expressed in P19 cells, the rate of neuronal differentiation was significantly increased. These data suggest not only that NZF-3 works downstream of Neurog1 but also that it plays a crucial role together with Neurog1 in neuronal differentiation.

CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes.

In the mammalian testis, spermatogenesis is initiated from a subset of stem cells belonging to undifferentiated type A spermatogonia. In spite of the biologic significance of undifferentiated type A spermatogonia, little is known about their behavior and properties because of a lack of specific cell surface markers. Here we show that CDH1 (previously known as E-cadherin) is expressed specifically in undifferentiated type A spermatogonia in the mouse testis. Histologic analysis showed that CDH1-positive cells had all the characteristics of undifferentiated type A spermatogonia. Whole-mount immunohistochemistry showed that CDH1-positive cells made clusters mainly comprising one, two, four, or eight cells. They survived after administration of the cytotoxic agent busulfan to mice, and then regenerated seminiferous epithelia. Transplantation experiments showed that only CDH1-positive cells had colonizing activity in the recipient testis. Our data clearly demonstrated that spermatogenic stem cells reside among undifferentiated type A spermatogonia, which express CDH1.

Chimeric analysis of Notch2 function: a role for Notch2 in the development of the roof plate of the mouse brain.

Notch proteins are transmembrane receptors involved in cell-fate determination throughout development. Targeted disruption of either the Notch1 or Notch2 gene in mice results in embryonic lethality around embryonic day (E) 10.5 with widespread cell death. Although Notch1-deficient mice show disorganized somitogenesis, Notch2 mutants did not show definitive abnormalities in any tissue expressing high levels of the Notch2 gene, including the central nervous system. To study Notch2 function in development beyond the embryonic lethal stage, we performed chimeric analysis between Notch2 mutant and wild-type mouse embryos. Chimeric embryos developed normally and homozygous Notch2 mutant-specific cell death was not observed. Although chimeric embryos showed normal mosaicism until E9.5 in all tissues studied to date, Notch2 homozygous mutant cells failed to contribute to formation of the roof plate of the diencephalon and mesencephalon at later developmental stages, when Notch2 is normally expressed at high levels at there. Furthermore, Notch2 heterozygous mutant cells were also excluded from the roof plate of the chimera, however, Notch2 heterozygous mutant mice developed normally. We also showed that Wnt-1 and Mash1 expression patterns at the roof plate were disorganized in Notch2 homozygous mutant embryos. These results indicate that Notch2 plays an important role in development of the roof plate of the diencephalon and mesencephalon, and suggest that cellular rearrangement is involved in this process.

Sequential activation of Notch family receptors during mouse spermatogenesis.

The expression pattern of Notch family receptors during mouse spermatogenesis was examined by immunohistochemistry. The entire cytoplasm of spermatogonia, spermatocytes and spermatids showed staining with antibodies against extracellular domains of Notch1, 2 and 4. In contrast, the nuclei of spermatogonia showed staining with an antibody against the intracellular domain of Notch3, and the nuclei of spermatocytes and spermatids showed staining with antibodies against the intracellular domains of Notch1 and 4. During regeneration of spermatogonia in busulfan-treated mice, the nuclei of all proliferating cells showed staining for the intracellular domain of Notch3. Western blot analysis showed that the molecular weights of the intracellular domains of Notch1 and 3 localizing in the nuclear fraction were smaller than those in the cytoplasmic fraction. This was consistent with the theory that the intracellular domain of Notch was cleaved in the cytoplasm and translocated to the nucleus. These results suggest that different Notch signals are sequentially activated during mouse spermatogenesis and control the proliferation and differentiation of spermatogenic stem cells.