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Defects in the retina or the anterior segment of the eye lead to compromised vision and affect millions of people. Understanding how these ocular structures develop and are maintained is therefore of paramount importance. The maintenance of proper vision depends, among other factors, on the function of genes controlling apico-basal polarity. In fact, mutations in polarity genes are linked to retinal degeneration in several species, including human. Here we describe a novel zebrafish crb2b allele (crb2be40 ), which specifically affects the crb2b long isoform. crb2be40 mutants are viable and display normal ocular development. However, old crb2be40 mutant fish develop multiple defects in structures of the anterior segment, which includes the cornea, the iris and the lens. Phenotypes are characterised by smaller pupils due to expansion of the iris and tissues of the iridocorneal angle, an increased number of corneal stromal keratocytes, an abnormal corneal endothelium and an expanded lens capsule. These findings illustrate a novel role for crb2b in the maintenance of the anterior segment and hence add an important function to this polarity regulator, which may be conserved in other vertebrates including humans.

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The vertebrate retina is a complex tissue built from multiple neuronal cell types, which develop from a pseudostratified neuroepithelium. These cells are arranged into a highly organized and stereotypic pattern formed by nuclear and plexiform layers. The process of lamination as well as the maturation and differentiation of photoreceptor cells rely on the establishment and maintenance of apico-basal cell polarity and formation of adhesive junctions. Defects in any of these processes can result in impaired vision and are causally related to a variety of human diseases leading to blindness. While the importance of apical polarity regulators in retinal stratification and disease is well established, little is known about the function of basal regulators in retinal development. Here, we analyzed the role of Lgl2, a basolateral polarity factor, in the zebrafish retina. Lgl2 is upregulated in photoreceptor cells and in the retinal pigment epithelium by 72 h post fertilization. In both cell types, Lgl2 is localized basolaterally. Loss of zygotic Lgl2 does not interfere with retinal lamination or photoreceptor cell polarity or maturation. However, knockdown of both maternal and zygotic Lgl2 leads to impaired cell adhesion. As a consequence, severe layering defects occur in the distal retina, manifested by a breakdown of the outer plexiform layer and the outer limiting membrane. These results define zebrafish Lgl2 as an important regulator of retinal lamination, which, given the high degree of evolutionary conservation, may be preserved in other vertebrates, including human.

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Vertebrates develop organs and appendages in a proportionally coordinated manner, and animals that regenerate them do so to the same dimensions as the original structures. Coordinated proportional growth involves controlled regulation between allometric and isometric growth programs, but it is unclear what executes this control. We show that calcineurin inhibition results in continued allometric outgrowth of regenerating fins beyond their original dimensions. Calcineurin inhibition also maintains allometric growth of juvenile fins and induces it in adult fins. Furthermore, calcineurin activity is low when the regeneration rate is highest, and its activity increases as the rate decreases. Growth measurements and morphometric analysis of proximodistal asymmetry indicate that calcineurin inhibition shifts fin regeneration from a distal growth program to a proximal program. This shift is associated with the promotion of retinoic acid signaling. Thus, we identified a calcineurin-mediated mechanism that operates as a molecular switch between position-associated isometric and allometric growth programs.

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Signal transduction pathways in megakaryocytes, a rare population of bone marrow cells, are poorly understood. We have previously shown that the calcineurin-dependent transcription factor Nuclear Factor of Activated T cells (NFAT) is expressed in megakaryocytes and is required for the transcription of specific megakaryocytic genes. The biological role of NFAT in megakaryocytes, however, is unknown. Here we show that activation of the calcineurin/NFAT pathway in megakaryocytes forces the cells to go into apoptosis. Calcineurin/NFAT activation in megakaryocytes leads to membrane expression of Fas ligand (FASLG), a pro-apoptotic member of the tumour necrosis factor superfamily. Expression of FASLG was augmented in cells stably overexpressing NFATC2 and suppressed in cells either pretreated with the calcineurin inhibitor ciclosporin A (CsA) or expressing the specific peptide inhibitor of NFAT, VIVIT. In cocultures with Fas-expressing Jurkat T cells, the presence of activated megakaryocytic cells, but not of unstimulated cells or cells stimulated in the presence of CsA, significantly induced apoptosis in Jurkat cells in a Fas/FASLG- and NFAT-dependent manner. These results represent the first evidence for a biological function of the calcineurin/NFAT pathway in megakaryocytes, and suggest that the biological role of megakaryocytes may include the induction of apoptosis in bystander cells.

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BACKGROUND: Nuclear factors of activated T cells (NFAT) are transcription factors that are central to cytokine production in activated T cells and regulate the development and differentiation of various tissues. NFATc2 is expressed in hematopoietic stem cells and regulated during myeloid commitment in a lineage-specific manner. The biological role of NFATc2 in hematopoiesis is, however, unclear. DESIGN AND METHODS: In the present study, we analyzed steady-state hematopoiesis in young (<3 months) and old (>12 months) mice lacking NFATc2. Complete blood counts were performed in the peripheral blood, bone marrow and spleen. Using cytological and histological analyses, the blood cell differential was determined. Colony-formation assays were used to determine the differentiation potential of hematopoietic cells. Bone cell cultures were derived from the bone marrow, and bone remodeling markers were determined in the serum. RESULTS: NFATc2(-/-) mice older than 12 months were anemic and thrombocytopenic. The bone marrows of these mice showed a markedly reduced number of hematopoietic cells, of which megakaryocytic and erythroid lineages were most affected. While the number of hematopoietic progenitor cells in NFATc2-deficent bone marrow was reduced, the myeloid differentiation potential of these cells remained intact. Aged NFATc2(-/-) mice showed ossification of their bone marrow space and developed extramedullary hematopoiesis in the spleen. Ex vivo differentiation assays revealed an intrinsic defect of NFATc2-deficient stromal cells, in which NFATc2(-/-) osteoblasts differentiated more efficiently than wild-type cells, whereas osteoclast differentiation was impaired. CONCLUSIONS: Our data suggest that NFATc2 may play a role in the maintenance of steady-state hematopoiesis and bone remodeling in adult organisms.

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