Forkhead Foxe3 maps to the dysgenetic lens locus and is critical in lens development and differentiation

Here we report the isolation of a novel forkhead gene, Foxe3, that plays an important role in lens formation. During development Foxe3 is expressed in all undifferentiated lens tissues, and is turned off upon fiber cell differentiation. Foxe3 maps to a chromosomal region containing the dysgenetic lens (dyl) mutation. Mice homozygous for dyl display several defects in lens development. dyl mice also show altered patterns of crystallin expression suggesting a dysregulation of lens differentiation. We have identified mutations in Foxe3 that cosegregate with the dyl phenotype and are a likely cause of the mutant phenotype. Head ectoderm expression of Foxe3 is absent in Rx-/- and Small eye embryos indicating that Rx and Pax6 activity are necessary for Foxe3 expression.     

Foxe view of lens development and disease

The recent identification of a mutation in Foxe3 that causes congenital primary aphakia in humans marks an important milestone. Congenital primary aphakia is a rare developmental disease in which the lens does not form. Previously, Foxe3 had been shown to play a crucial role in vertebrate lens formation and this gene is one of the earliest integrators of several signaling pathways that cooperate to form a lens. In this review, we highlight recent advances that have led to a better understanding of the developmental processes and gene regulatory networks involved in lens development and disease.     

Severe developmental defects in Dictyostelium null mutants for actin-binding proteins

The actin cytoskeleton is implicated in many cellular processes, such as cell adhesion, locomotion, contraction and cytokinesis, which are central to any development. The extent of polymerization, cross-linking, and bundling of actin is regulated by several actin-binding proteins. Knock-out mutations in these proteins have revealed in many cases only subtle, if any, defects in development, suggesting that the actin system is redundant, with multiple proteins sharing overlapping functions. The apparent redundancy may, however, reflect limitations of available laboratory assays in assessing the developmental role of a given protein. By using a novel assay, which reproduces conditions closer to the natural ones, we have re-examined the effects of disruption of many actin-binding proteins, and show here that deletion of alpha-actinin, interaptin, synexin, 34-kDa actin-bundling protein, and gelation factor affect to varying degrees the efficiency of Dictyostelium cells to complete development and form viable spores. No phenotypic defects were found in hisactophilin or comitin null mutants.     

Mitochondrial respiration defects modulate differentiation but not proliferation of hematopoietic stem and progenitor cells

Mitochondrial energy production is involved in various cellular processes. Here we show that ATP content is significantly increased in lineage-restricted progenitor cells compared with hematopoietic stem and progenitor cells (HSPCs) or more differentiated cells. Transplantation analysis using a mouse model of mitochondrial disease revealed that mitochondrial respiration defects resulted in a significant decrease in the total number and repopulating activity of bone marrow cells, although the number of HSPCs increased. The proliferative activity of HSPCs and lineage-restricted progenitor cells was not impaired by reduction of ATP content and there seems to be no associated increase in reactive oxygen species levels and apoptosis. Our findings indicate that mitochondrial respiration defects modulate HSPC commitment/differentiation into lineage-restricted progenitor cells.     

A deletion in a cis element of Foxe3 causes cataracts and microphthalmia in rct mice

The Rinshoken cataract (rct) mutation, which causes congenital cataracts, is a recessive mutation found in SJL/J mice. All mutants present with opacity in the lens by 2?months of age. The rct locus was mapped to a 1.6-Mb region in Chr 4 that contains the Foxe3 gene. This gene is responsible for cataracts in humans and mice, and it plays a crucial role in the development of the lens. Furthermore, mutation of Foxe3 causes various ocular defects. We sequenced the genomic region of Foxe3, including the coding exons and UTRs; however, no mutations were discovered in these regions. Because there were no differences in Foxe3 sequences between the rct/rct and wild-type mice, we inferred that a mutation was located in the regulatory regions of the Foxe3 gene. To test this possibility, we sequenced a 5' noncoding region that is highly conserved among vertebrates and is predicted to be the major enhancer of Foxe3. This analysis revealed a deletion of 22-bp located approximately 3.2-kb upstream of the start codon of Foxe3 in rct mice. Moreover, we demonstrated by RT-PCR and in situ hybridization that the rct mutant has reduced expression of Foxe3 in the lens during development. We therefore suggest that cataracts in rct mice are caused by reduced Foxe3 expression in the lens and that this decreased expression is a result of a deletion in a cis-acting regulatory element.     

Cultured myf5 null and myoD null muscle precursor cells display distinct growth defects

Myf-5 and MyoD are the two muscle regulatory factors expressed from the myoblast stage to maintain the identity and to promote the subsequent differentiation of muscle precursor cells. To get insight into their role we have studied the capacity to proliferate and to differentiate of myf-5 and myoD null myoblasts in primary cultures and in the subsequent passages. Our results indicate that myf-5 null myoblasts differ from wild type (wt) myoblasts in that they undergo precocious differentiation: they become myogenin- and troponin T-positive and fail to incorporate bromodeoxyuridine (BrdU) under culture conditions and at a time when wt cells are not yet differentiated and continue to proliferate. In primary cultures of myoD null cells, up to 60% of the cells were scored as myoblasts on the basis of the expression of myf-5. These myoD-deficient myoblasts, unlike myoD-expressing cells, were poorly differentiating and displayed a severe growth defect that led to their elimination from the cultures: within a few passages myoblasts were absent from myoD-deficient cultures, which mostly consisted of senescent cells. That a null mutation in either gene reduces the proliferative potential of cultured myoblasts raises the possibility that Myf-5 and MyoD serve proliferation of muscle precursor cells.     

DISP3 promotes proliferation and delays differentiation of neural progenitor cells

DISP3 (PTCHD2), a sterol-sensing domain-containing protein, is highly expressed in neural tissue but its role in neural differentiation is unknown. In the present study we used a multipotent cerebellar progenitor cell line, C17.2, to investigate the impact of DISP3 on the proliferation and differentiation of neural precursors. We found that ectopically expressed DISP3 promotes cell proliferation and alters expression of genes that are involved in tumorigenesis. Finally, the differentiation profile of DISP3-expressing cells was altered, as evidenced by delayed expression of neural specific markers and a reduced capacity to undergo neural differentiation.     

Pax6 is misexpressed in Sox1 null lens fiber cells

Sox1 null lens fiber cells fail to elongate and have disrupted expression of gamma-crystallin. We have evaluated the expression of Sox1 and Pax6 proteins during critical stages of lens morphogenesis, with particular focus on fiber cell differentiation. While Pax6 and Sox1 are co-expressed during early stages of fiber cell differentiation, Sox1 up-regulation coincides temporally with the down-regulation of Pax6, and these proteins therefore display a striking inverse expression pattern in the lens fiber cell compartment. Furthermore, Pax6 is inappropriately expressed in the fiber cells of Sox1 null mice and the Pax6 target, alpha5 integrin, is simultaneously misexpressed. Finally, we demonstrate a genetic interaction between Sox1 and Pax6, as Sox1 heterozygosity partially rescues the diameter of Pax6(Sey) lenses by increasing the number of cells in the fiber cell compartment.     

Severe defect in proglucagon processing in islet A-cells of prohormone convertase 2 null mice

Mice homozygous for a deletion in the gene encoding prohormone convertase 2 (PC2) are generally healthy but have mild hypoglycemia and flat glucose-tolerance curves. Their islets show marked alpha (A)-cell hyperplasia, suggesting a possible defect in glucagon processing (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H., and Steiner, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646-6651). In this report we have examined the biosynthesis and processing of proglucagon in isolated islets from these mice via pulse-chase labeling and find that proglucagon undergoes essentially no processing in chase periods up to 8 h in duration. Only a small percent of cleavage at the sensitive interdomain site (residues 71 and 72) appears to occur. These observations thus conclusively demonstrate the essentiality of PC2 for the production of glucagon in the islet A-cells. Ultrastructural and immunocytochemical studies indicate the presence of large amounts of proglucagon in atypical appearing secretory granules in the hyperplastic and hypertrophic A-cells, along with morphological evidence of high rates of proglucagon secretion in PC2 null islets. These findings provide strong evidence that active glucagon is required to maintain normal blood glucose levels, counterbalancing the action of insulin at all times.     

Identification and differentiation of lens cells in tissue culture]

The cells of S-phase labelled prior to cultivation with H3-thymidine and other neighbouring cambial cells of the lens of the pig, cattle and sheep were found to form morphologically underdifferentiated zones of growth. The zones of growth were formed in the culture from differentiating in vivo cells of the lens. The cells of these zones occasionally resembled abortively differentiated lens fibres in vivo. The growth zones of the lens cells in vitro are comparable by its growings in trauma or cataract in vivo. In lens cultures under routine conditions of cultivation there occurs disturbance of normal embryonic histogenesis and abortive differentiation of the already differentiated in vivo cells.     

Lunatic fringe null female mice are infertile due to defects in meiotic maturation

We have demonstrated that Notch genes are expressed in developing mammalian ovarian follicles. Lunatic fringe is an important regulator of Notch signaling. In this study, data are presented that demonstrate that radical fringe and lunatic fringe are expressed in the granulosa cells of developing follicles. Lunatic fringe null female mice were found to be infertile. Histological analysis of the lunatic fringe-deficient ovary demonstrated aberrant folliculogenesis. Furthermore, oocytes from these mutants did not complete meiotic maturation. This is a novel observation because this is the first report describing a meiotic defect that results from mutations in genes that are expressed in the somatic granulosa cells and not the oocytes. This represents a new role for the Notch signaling pathway and lunatic fringe in mammalian folliculogenesis.     

Sef and Sprouty expression in the developing ocular lens: implications for regulating lens cell proliferation and differentiation

In many developmental systems, growth factor signalling must be temporally and spatially regulated, and this is commonly achieved by growth factor antagonists. Here, we describe the expression patterns of newly identified growth factor inhibitors, Sprouty and Sef, in the developing ocular lens. Sprouty and Sef are both expressed in the lens throughout embryogenesis, and become restricted to the lens epithelium, indicating that lens cell proliferation and fibre differentiation may be tightly regulated by such antagonists. Future studies will be aimed at understanding how these negative regulatory molecules modulate growth factor-induced signalling pathways and cellular processes in the lens.     

Roles for Kisspeptin in proliferation and differentiation of spermatogonial cells isolated from mice offspring when the cells are cocultured with somatic cells

Kisspeptin (Kp) expression in testis has caused most of the recent research surveying its functional role in this organ. This peptide influences spermatogenesis and sperm capacitation, so it is considered as a regulator of reproduction. Kp roles exert through hypothalamic/pituitary/gonadal axis. We aimed to evaluate direct roles for Kp on proliferation and differentiation of spermatogonial cells (SCs) when the cells are cocultured with somatic cells. Somatic cells and SCs were isolated from adult azoospermic and newborn mice and then enriched using a differential attachment technique. After the evaluation of identity and colonization for SCs, the cells were cocultured with somatic cells, and three doses of Kp (10⁻⁸-10⁻⁶ M) was assessed on proliferation (through evaluation of MVH and ID4 markers) and differentiation (via evaluation of c-Kit and SCP₃, TP_1, TP₂, and, Prm₁ markers) of the coculture system. Investigations were continued for four succeeding weeks. At the end of each level of testosterone in the culture media was also evaluated. We found positive influence from Kp on proliferative and differentiative markers in SCs cocultured with somatic cells. These effects were dose-dependent. There was no effect for Kp on testosterone level. From our findings, we simply conclude that Kp as a neuropeptide for influencing central part of reproductive axis could also positively affect peripheral processes related to spermatogenesis without having an effect on steroidogenesis.     

Redox regulation of proliferation of lens epithelial cells in culture

Both oxidants and antioxidants have been shown to modulate cell proliferation. We studied the effects of hydrogen peroxide and two antioxidants on the rate of proliferation of lens epithelial cells in culture. Hydrogen peroxide at concentrations higher than 32 microM caused a significant inhibition of proliferation. However, in the concentration range of 0.01-0.5 microM, hydrogen peroxide stimulated the rate of proliferation. The effect of hydrogen peroxide was dependent on the amount of cells in an individual culture well, indicating decomposition of hydrogen peroxide by cellular enzymes. In order to eliminate the possibility of decomposition of the dose of hydrogen peroxide given as a bolus, we induced continual production of hydrogen peroxide by adding glucose oxidase to the incubation medium. We found that hydrogen peroxide, generated by 1-50 microU x ml(-1) of glucose oxidase significantly increased the rate of cell proliferation. This effect was most apparent at the beginning of the exponential phase of cellular growth. Glucose oxidase alone (100-500 microU x ml(-1)) did not produce any effect. The effects of pro-oxidative hydrogen peroxide were compared with the effects of two biologically important antioxidants, alpha-tocopherol and retinol. Both antioxidants completely inhibited proliferation at concentrations of 30 microM and higher. In contrast to retinol, the effect of alpha-tocopherol was dependent on the amount of cells, indicating cellular decomposition of alpha-tocopherol. The results document the possibility of redox regulation of cellular proliferation at physiologically relevant reactant concentrations.     

Enhancement of proliferation and differentiation in bone marrow hematopoietic cells by Spirulina (Arthrospira) platensis in mice

This study evaluates whether Spirulina, including its components such as phycocyanin, enhances or sustains immune functions by promoting immune competent-cell proliferation or differentiation. The effects of Spirulina of a hot-water extract (SpHW), phycocyanin (Phyc), and cell-wall component extract (SpCW) on proliferation of bone marrow cells and induction of colony-forming activity in mice were investigated. The Spirulina extracts, SpHW, Phyc, and SpCW, enhanced proliferation of bone-marrow cells and induced colony-forming activity in the spleen-cell culture supernatant. Granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were detected in the culture supernatant of the spleen cells stimulated with the Spirulina extracts. Bone marrow-cell colony formation in soft-agar assay was also significantly induced by the blood samples and the culture supernatants of the spleen and Peyer's patch cells of the mice which ingested Spirulina extracts orally for 5 weeks in in vivo study. Ratios of neutrophils and lymphocytes in the peripheral blood and bone marrow, consequently, increased in the mice. Spirulina may have potential therapeutic benefits for improvement of weakened immune functions caused by, for example, the use of anticancer drugs.