A chick retinal proteome database and differential retinal protein expressions during early ocular development

Proteomics approach as a research tool has gained popularity in a growing number of basic and clinical researches. However, proteomic research has yet to gain significant momentum in eye research. Hence, we decided to build a retinal proteome database using postnatal retinal tissue from chick, a commonly used animal model in eye research. Employing 2-D gels with the coverage of 3-10 pH gradients, we were able to resolve hundreds of proteins from young chick retinae. Among them, 155 high abundant proteins were identified by Peptide Mass Fingerprinting (PMF) after the Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS). These proteins were then classified according to their functions. Making use of the retinal database, we were able to identify several differentially expressed proteins that might be involved in early retinal development by comparing the 2-DE maps of chick retinal tissues (3, 10, and 20 days after hatching). With the current proteomics approach, we not only documented the most abundant soluble proteins in the chick retinal tissue, but also demonstrated the dynamic protein expression changes during early ocular development. This represents one of the first steps in building a complete protein database in chick retinae which is applicable to the study of eye diseases from a few selected protein candidates to the whole proteome. Proteomic technology may provide a high throughput platform for advancing eye research in the feasible future.     

Cardiomyocytes re-enter the cell cycle and contribute to heart development after differentiation from cardiac progenitors expressing Isl1 in chick embryo

Cardiomyocytes are generated from the precardiac mesoderm and the size of the heart increases dramatically during embryogenesis. However, it is unclear how differentiation and proliferation correlate in the cardiac cell line during development. Here, we show that cardiomyocytes re-entered into a proliferative state after differentiation with a concomitant cell cycle arrest in chick embryo. The cells in the course of differentiation from Isl1-positive cardiac precursors to cardiomyocytes did not proliferate, but differentiated cardiomyocytes proliferated even after the acquisition of contractile function. After differentiation, cardiomyocytes developed a proliferative potential to contribute to the increase in cell numbers during heart development. Almost all differentiated cardiomyocytes (82.8%) incorporated bromodeoxyuridine (BrdU) in vitro, indicating the ability of DNA replication. Furthermore, mitotic chromosomes were observed in the cardiomyocytes in which a sarcomeric structure was sustained in the cytoplasm. We conclude that the sequential events of the differentiation to contractile myocytes and the re-entry into the cell cycle are strictly regulated during cardiac cell maturation. These results provide an insight into the maturation mechanism of the cardiac cell line.     

Red cell ferritin and iron storage during chick embryonic development

Ferritin, the iron storage protein, is at least 10 times as abundant in the circulating primitive red cells of the chick embryo as in the circulating definitive red cells of adult roosters. The decline in the ferritin content of the circulating red cells in the embryo corresponded to the replacement of primitive red cells by definitive red cells, monitored by the disappearance of primitive and embryonic hemoglobins. Iron concentrations in the yolk, the major nutrient storage site, changed little during the period when ferritin was lost from the circulating red cells. The storage of iron in the ferritin of the primitive red cells and the preferential loss of the stored red cell iron that was observed in chickens also occur in mice and bullfrogs, which suggests a special role for red cell ferritin in developing animals.     

The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest

For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months.     

Motoneurons and oligodendrocytes are sequentially generated from neural stem cells but do not appear to share common lineage-restricted progenitors in vivo

Olig gene expression is proposed to mark the common progenitors of motoneurons and oligodendrocytes. In an attempt to further dissect the in vivo lineage relationships between motoneurons and oligodendrocytes, we used a conditional cell-ablation approach to kill Olig-expressing cells. Although differentiated motoneurons and oligodendrocytes were eliminated, our ablation study revealed a continuous generation and subsequent death of their precursors. Most remarkably, a normal number of oligodendrocyte precursors are formed at day 12 of mouse development, after all motoneuron precursors have been killed. The data presented herein supports a sequential model in which motoneuron and oligodendrocyte precursors are sequentially generated in vivo from neuroepithelial stem cells, but do not share a common lineage-restricted progenitor.     

Antiglucocorticoid steroids have increased agonist activity in those hepatoma cell lines that are more sensitive to glucocorticoids

FU5-5 rat hepatoma (Reuber H35) cells are hypersensitive in that the same percentages of full induction of tyrosine aminotransferase (TAT) occur at much lower concentrations of glucocorticoids than in the related HTC rat hepatoma (Morris) cells. Unexpectedly, these hypersensitive FU5-5 cells also exhibited more agonist activity with the affinity labeling antiglucocorticoids cortisol 21-mesylate and dexamethasone 21-mesylate than did HTC cells (Mercier et al., Endocrinology 112, 601-609 [1983]). In the present study, several other antiglucocorticoids (11-desoxycortisone, progesterone, dexamethasone oxetanone, and RU 486 in addition to dexamethasone 21-mesylate) and the antiandrogen cyproterone acetate were examined to see if chemically unreactive, reversible antisteroids also would exhibit an altered activity (i.e. increased agonist activity) in FU5-5 cells. Each antiglucocorticoid examined did display a 2-fold increased amount of agonist activity in FU5-5 cells, as compared to HTC cells; only RU 486 was predominantly an antagonist in FU5-5 cells but the potency of RU 486 was about 9-fold less than in HTC cells. Dexamethasone, and especially progesterone, was metabolized in FU5-5 and HTC cells. However, differential metabolism in FU5-5 vs HTC cells cannot account for the increased induction of TAT in FU5-5 cells since the amount of agonist activity seen for dexamethasone mesylate (or its metabolites) depended not on the cell type used but rather on the glucocorticoid inducible enzyme monitored, i.e. TAT or glutamine synthetase. The combined data suggest that the hypersensitivity of FU5-5 cells towards glucocorticoid induction of TAT may be linked with the ability of both reversible and irreversible antiglucocorticoids to display increased TAT agonist activity in FU5-5 cells. This behavior was somewhat steroid specific since the antiandrogen cyproterone acetate did not display increased TAT agonist activity in FU5-5 cells compared to HTC cells and was only 2-fold less effective as an antiglucocorticoid in FU5-5.     

Posterior skeletal development and the segmentation clock period are sensitive to Lfng dosage during somitogenesis

The segmental structure of the axial skeleton is formed during somitogenesis. During this process, paired somites bud from the presomitic mesoderm (PSM), in a process regulated by a genetic clock called the segmentation clock. The Notch pathway and the Notch modulator Lunatic fringe (Lfng) play multiple roles during segmentation. Lfng oscillates in the posterior PSM as part of the segmentation clock, but is stably expressed in the anterior PSM during presomite patterning. We previously found that mice lacking overt oscillatory Lfng expression in the posterior PSM (Lfng^?FCE) exhibit abnormal anterior development but relatively normal posterior development. This suggests distinct requirements for segmentation clock activity during the formation of the anterior skeleton (primary body formation), compared to the posterior skeleton and tail (secondary body formation). To build on these findings, we created an allelic series that progressively lowers Lfng levels in the PSM. Interestingly, we find that further reduction of Lfng expression levels in the PSM does not increase disruption of anterior development. However tail development is increasingly compromised as Lfng levels are reduced, suggesting that primary body formation is more sensitive to Lfng dosage than is secondary body formation. Further, we find that while low levels of oscillatory Lfng in the posterior PSM are sufficient to support relatively normal posterior development, the period of the segmentation clock is increased when the amplitude of Lfng oscillations is low. These data support the hypothesis that there are differential requirements for oscillatory Lfng during primary and secondary body formation and that posterior development is less sensitive to overall Lfng levels. Further, they suggest that modulation of the Notch signaling by Lfng affects the clock period during development.     

Estrogen-mediated cell proliferation during chick oviduct development and its modulation by progesterone

The interactions of estrogen and progesterone on mitosis were examined in the surface epithelium of the developing chick oviduct. Both of these steroid hormones can stimulate cells to divide in the unstimulated oviduct. However, progesterone treatment results in a delayed suppression of cell division in both the presence and absence of estrogen. This progesterone induced depression of estrogen-mediated cell division is observed throughout oviduct development. During oviduct development estrogen is necessary for both cell division and the differentiation of specific cell types while progesterone appears to modify the action of estrogen by blocking the progression of cells through the cell cycle.     

Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs

Adult hematopoietic stem cells (HSCs) with serially transplantable activity comprise two subtypes. One shows a balanced output of mature lymphoid and myeloid cells; the other appears selectively lymphoid deficient. We now show that both of these HSC subtypes are present in the fetal liver (at a 1:10 ratio) with the rarer, lymphoid-deficient HSCs immediately gaining an increased representation in the fetal bone marrow, suggesting that the marrow niche plays a key role in regulating their ensuing preferential amplification. Clonal analysis of HSC expansion posttransplant showed that both subtypes display an extensive but variable self-renewal activity with occasional interconversion. Clonal analysis of their differentiation programs demonstrated functional and molecular as well as quantitative HSC subtype-specific differences in the lymphoid progenitors they generate but an indistinguishable production of multipotent and myeloid-restricted progenitors. These findings establish a level of heterogeneity in HSC differentiation and expansion control that may have relevance to stem cell populations in other hierarchically organized tissues.     

Chondromodulin-I and tenomodulin are differentially expressed in the avascular mesenchyme during mouse and chick development

Chondromodulin-I (ChM-I) and tenomodulin (TeM) are homologous angiogenesis inhibitors. We have analyzed the spatial relationships between capillary networks and the localization of these molecules during mouse and chick development. ChM-I and TeM proteins have been localized to the PECAM-1-negative avascular region: ChM-I is expressed in the avascular cartilage, whereas TeM is detectable in dense connective tissues, including tendons and ligaments. We have also examined the vasculature of chick embryos by injection with India ink and have performed in situ hybridization of the ChM-I and TeM genes. The onset of ChM-I expression is associated with chondrogenesis during mouse embryonic development. ChM-I expression is also detectable in precartilaginous or noncartilaginous avascular mesenchyme in chick embryos, including the somite, sclerotome, and heart. Hence, the expression domains of ChM-I and TeM during vertebrate development incorporate the typical avascular regions of the mesenchymal tissues. This study was partly supported by Grants-in-Aid from the Ministry of Education, Culture, Sport, Science, and Technology of Japan and by the Tanabe Medical Frontier Conference.     

Sfrp-1 and sfrp-2 are expressed in overlapping and distinct domains during chick development

Secreted frizzled related proteins (Sfrps) are thought to bind and regulate Wnt activity through a cysteine rich domain that is highly similar to that of Frizzled receptors. To investigate possible roles for Sfrps in chick development, we have isolated partial cDNAs encoding Sfrp-1 and Sfrp-2 and have thoroughly characterized the expression patterns of both genes. Both sfrp-1 and sfrp-2 are expressed at all stages of development analyzed, ranging from Hamburger and Hamilton stage 4 through stage 32. Expression of both sfrp-1 and sfrp-2 is observed in mesodermal and ectodermal derivatives, while sfrp-1 is also found in endodermal lineages.     

Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development

The Wnt4 gene encodes a secreted signaling molecule controlling the development of several organs, such as the kidney, adrenal gland, ovary, mammary gland and pituitary gland. It is thought to act in the embryonic kidney as an auto-inducer of nephrogenesis controlling mesenchyme-to-epithelium transition, and Wnt4-deficient mice die soon after birth, probably of kidney failure. Given the requirement for Wnt4 signaling in the control of organogenesis, the targeting of Cre recombinase under the control of the Wnt4 promoter would provide a valuable tool for fate mapping and functional genomics. We report here on the generation and characterization of a Wnt4^EGFPCre knock-in allele where the EGFPCre fusion cDNA and Neo selection cassette were targeted into the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates of the E14.5 embryonic kidney that normally express Wnt4 mRNA. Characterization of the pattern of recombination of the floxed Rosa26^LacZ reporter with the Wnt4^EGFPCre allele revealed that in addition to the embryonic kidney, reporter-derived staining was observed in the embryonic gonad, spinal cord, lung and adrenal gland, i.e. the sites of Wnt4 gene expression. Time-lapse fate mapping of the Wnt4^EGFPCre-activated yellow fluorescent protein (YFP) from the Rosa26 locus in organ culture revealed that the cells that had expressed the Wnt4 gene contributed to the nephrons, some of the cells around the stalk of the developing ureter and also certain presumptive medullary stromal cells. Moreover, the time-lapse movies suggested that the first few pretubular cell aggregates may not mature into nephrons but instead appear to disintegrate. In association with this, Rosa26^YFP-positive stromal cells emerge around these disintegrating structures. Such cells may be transient, since their derivatives are neither detected later in the more mature kidney nor is there an overlap of the Wnt4^EGFPCre; Rosa26^LacZ-marked cells with those of the endothelial cells, the smooth muscle cells or the macrophages. The Wnt4^EGFPCre allele provides a useful new tool for conditional mutagenesis and provides the first time-lapse-based map of the fate of nephron precursor cells.     

Natural triterpenoids from Cecropia lyratiloba are cytotoxic to both sensitive and multidrug resistant leukemia cell lines

The cytotoxicity of four triterpenoids, euscaphic acid (1), tormentic acid (2), 2alpha-acetyl tormentic acid (3), and 3beta-acetyl tormentic acid (4), isolated from the roots of Cecropia lyratiloba (Moraceae) by countercurrent chromatography, was evaluated in vitro in sensitive and multidrug resistant leukemia cell lines. A structure/activity relationship analysis of the compounds was performed. Acetylation of compound 2 at C2 increased its activity by a factor of 2 while acetylation at C3 had a smaller effect. Compound 1 induces death by activation of caspase-3, dependent apoptotic pathway. Furthermore, the four triterpenoids were also active toward a multidrug resistant (MDR) leukemia cell line, overexpressing glycoprotein-P (P-gp). These results reveal the potential of the terpenoids as source for the development of new anti-neoplastic and anti-MDR drugs.