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During development, multipotent progenitors undergo temporally-restricted differentiation into post-mitotic retinal cells; however, the mechanisms of progenitor division that occurs during retinogenesis remain controversial. Using clonal analyses (lineage tracing and single cell cultures), we identify rod versus cone lineage-specific progenitors derived from both adult retinal stem cells and embryonic neural retinal precursors. Taurine and retinoic acid are shown to act in an instructive and lineage-restricted manner early in the progenitor lineage hierarchy to produce rod-restricted progenitors from stem cell progeny. We also identify an instructive, but lineage-independent, mechanism for the specification of cone-restricted progenitors through the suppression of multiple differentiation signaling pathways. These data indicate that exogenous signals play critical roles in directing lineage decisions and resulting in fate-restricted rod or cone photoreceptor progenitors in culture. Additional factors may be involved in governing photoreceptor fates in vivo., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

In mammals, a limited set of homeobox-containing transcription factors are expressed in the presumptive eye field and required to initiate eye development. How these factors interact together at the genetic and molecular level to coordinate this developmental process is poorly understood. We found that the Lhx2 and Pax6 transcription factors operate in a concerted manner during retinal development to promote transcriptional activation of the Six6 homeobox-gene in primitive and mature retinal progenitors. Lhx2 demarcates the presumptive retina field at the neural plate stage and Lhx2 inactivation delays initiation of Rx, Six3 and Pax6 expression in this domain. The later expressed Six6 is properly activated in the pituitary/hypothalamic axis of Lhx2(-/-) embryos, but expression fails to be initiated in the optic vesicle. Lhx2 and Pax6 associate with the chromatin at several regions of Six6 in vivo and cooperate for trans-activation of Six6 regulatory elements in vitro. In retinal progenitor/stem cells, both Lhx2 and Pax6 are genetically required for proper Six6 expression and forced co-expression of Lhx2 and Pax6 can synergistically trans-activate the Six6 locus. Our work reveals how two master regulators of eye development coordinate their action to sequentially promote tissue-specific transcriptional initiation and full activation of a retinal determinant gene.

In vertebrates, a limited number of homeobox-containing transcription factors are expressed in the optic vesicle primordium and are required and sufficient for eye formation. At present, little is known about the distinct functions of these factors in optic vesicle growth and on the nature of the main neuroepithelial (NE) progenitor population present in this organ. We have characterized a multipotent cell population present in the mouse optic vesicle that shows extensive proliferation potential and which expresses NE progenitor and retinal markers in vitro. In Pax6 mutant embryos, which form an optic vesicle, we found that the number of resident NE progenitors was greater than normal. In vitro, Pax6-null NE progenitors overproliferate and display reduced p16(Ink4a), p19(Arf), p27(kip1), p57(kip2), and p21(cip1) expression. Pax6 overexpression repressed cellular proliferation and secondary colonies formation, supporting the hypothesis that Pax6 acts cell-autonomously on NE progenitors cell cycle. Notably, these in vitro data correlated with aberrant numbers of mitosis observed in the optic vesicle of early stage Pax6 mutants, with Pax6 association with the chromatin upstream of p27(kip1) promoter region, and with reduced expression levels of p27(kip1), p57(kip2), and p21(cip1) in the primitive forebrain of Pax6 mutants. Taken together, our results suggest that, prior to retinal progenitor cell identity and neurogenesis, Pax6 is required to regulate the proliferation rate of NE progenitors present in the mouse optic vesicle.

The transcription factor Pax6 regulates multiple aspects of central nervous system (CNS) development. At the cellular level, the Pax6 mutation was reported to affect homophilic and heterophilic cellular adhesion, neuron polarity and neurite outgrowth. These abnormalities were observed in multiple regions of Pax6-mutant CNS, suggesting a common function for Pax6 in regulating cytoskeletal dynamics. However, target genes mediating Pax6 function in cytoskeletal dynamics remain largely unknown. Using DNA microarrays, we identified delta-catenin (delta-catenin /neurojugin) as a potential direct target of Pax6 in the CNS. delta-catenin encodes a large cytoskeletal protein that localizes at adherens junction in the CNS and that can modulate neurite outgrowth and N-cadherin turnover. delta-catenin was found to be co-expressed with Pax6 in several regions of the developing CNS. In Pax6 mutant embryos, delta-catenin expression was severely reduced in the optic vesicle neural ectoderm, in the ventricular zone of the neocortex and in the external granule layer of the cerebellum. We identified a Pax6 binding site in delta-catenin promoter that is conserved between mice and humans and which is effectively bound by Pax6 in vitro. Our results suggest that Pax6 regulates delta-catenin expression during CNS development in mice.

Rare pigmented cells showing retinal stem cell characteristics have been identified in the ocular ciliary body (CB) of adult mammals. In vitro, these cells were reported to clonally proliferate and generate pigmented sphere colonies (PSC) containing multipotent retinal progenitor-like cells. Because these cells may have important clinical applications and because their embryonic origin is unclear, we have analyzed their local environment and gene expression profile. We found that transcription factors Pax6, Six3, and Rx, all involved in early eye morphogenesis, were expressed in the CB of adult mice. By sequencing a PSC cDNA library, we found that PSC expressed at high levels transcripts involved in the control of redox metabolism and cellular proliferation. PSC also expressed the retinal transcription factor Six6, which expression was not detected in the CB epithelium. By in situ hybridization screen, we found that Palmdelphin (Palm), Hmga2, and a novel transcript were expressed in the central nervous system of early embryos. Palm expression delineated the pigmented epithelium of the future CB and the developing myotome. Hmga2 was expressed in the ventricular zone of the telencephalon, the developing retinal ciliary margin and lens. Several genes expressed in PSC were also expressed in the nasal anlagen. Taken together, our study reveals that PSC isolated from the ocular CB express genes involved in the control of embryonic development, retinal identity, redox metabolism, and cellular proliferation.

During vertebrate eye development, the expression of the homeobox gene Six6 is restricted to the neural retina and is initiated later than Rx and Pax6 in the presumptive retina field. We show here that overexpression of mouse Six6 in Xenopus embryos can induce transformation of competent tissue of the anterior neural plate into retinal tissue. In Six6 injected embryos, the molecular identity of the presumptive midbrain and rostral hindbrain regions was lost, as shown by the absence of XEn-2 and Xpax2 expression, being replaced by the ectopic expression of the retinal markers Xpax6 and Xrx. When allowed to grow further, Six6 injected embryos developed ectopic eye-like structures in the rostral brain and showed a transformation of the midbrain into retina. Similar results were obtained upon overexpression of Six3 or Xsix3, revealing a possible redundance of Six3 and Six6 activities. Taken together, results obtained suggest that during normal retina development, the relatively late expressed Six6 gene becomes part of a network of retinal homeobox genes that are linked together by positive feedback loops. Furthermore, our results demonstrate that the primitive neural ectoderm of the future midbrain and rostral hindbrain is competent to form retinal tissue.

We report on the isolation of a murine homeobox-containing gene, Six6 (Optx2), that shows extended identity in its coding region with Six3, the only member of the mammalian Six gene family known to be expressed in the optic primordium. Phylogenetic analysis demonstrates that Six6 and Six3 belong to a separate group of homeobox-genes that are closely related to the recently identified Drosophila optix. Earliest Six6 expression was detected in the floor of the diencephalic portion of the primitive forebrain, a region predicted to give rise to the neurohypophysis and to the hypothalamus. Later on, Six6 mRNA was found in the primordial tissues giving rise to the mature pituitary: the Rathke's pouch and the infundibular recess. In the optic primordium, Six6 demarcates the presumptive ventral optic stalk and the ventral portion of the future neural retina. In the developing eye. Six6 expression was detected in the neural retina, the optic chiasma and optic stalk, but not in the lens. When compared to Six6, Six3 expression pattern was highly similar, but with a generally broader transcripts distribution in the brain and in the visual system. We finally show that Six6 does not require Pax6 for its expression in the optic primordium, suggesting that Six6 acts on a parallel and/or independent pathway with Pax6 in the genetic cascade governing early development of the eye.

The apparent simultaneous presence of surface markers characteristic of both B and T cells is a phenomenon being described with increasing frequency in patients with chronic lymphocytic leukemia (CLL). We describe a patient with CLL whose B lymphocytes possessed surface immunoglobulin reactive with neuraminidase-treated sheep erythrocytes (SRBCs) and produced E rosette formation. Cytofluorography using monoclonal antibodies demonstrated the B cell nature of these cells and the absence of the SRBC receptor. Further documentation that the binding of SRBCs was mediated through immunologic reaction included E rosette formation inhibition by monospecific antisera and hemagglutination of SRBCs by a paraprotein isolated from the patient's serum. Fusion of the CLL cells with a human hypoxanthine-aminopterin-thymidine-sensitive plasma cell line resulted in the production of human hybridomas that secreted the SRBC-reactive IgM antibody. An analysis of clinical histories of CLL patients whose cells exhibited this phenomenon from both immunologic and clinical perspectives is presented.

Three patients with acute myelogenous leukemia (AML) in relapse were treated with intravenous infusions of one or more purified murine monoclonal antibodies (MoAbs) specific for differentiation antigens on normal and malignant myeloid cells. Three of the MoAbs used were IgM immunoglobulins that react with glycolipids, while the fourth, an IgG2b, reacts with a protein antigen. Peripheral blood leukemia cell counts decreased significantly, but transiently, during treatment. Evidence of in vivo binding of each MoAb to leukemia cells was obtained, although two of the four MoAbs could not be detected in the plasma following infusion, perhaps due to circulating blocking factors. Antigenic modulation was not encountered in these studies. However, the induction of human antibody to murine MoAb was observed in one patient who was treated over a 70-day period. Toxicities encountered were minimal and included fever (3 patients), back pain (1 patient), and arthralgias and myalgias (1 patient). This is the first reported clinical trial of (1) IgM MoAbs, (2) MoAb therapy in patients with AML, (3) combinations of MoAbs directed toward different myeloid differentiation antigens, and (4) MoAbs directed to glycolipids. The relative lack of toxicity and the positive effects of MoAb treatment in the reduction of leukemia cell counts permit the continued study of more innovative approaches to the treatment of AML with MoAbs.

The bone marrow of a patient with light chain myeloma and amyloidosis was substantially infiltrated with basophilic globular particles. The globules, which were confined to the bone marrow, ranged in size from tiny intracytoplasmic inclusions to large (100 mu) extracellular particles. Both the intra- and extracellular globules stained with fluorescent antibody directed against the light chain (kappa) produced by the patient's abnormal clone, and not with other fluorescent antiserums. By electron microscopy, even the largest extracytophasmic globules were bounded at least in part by rough membranes, suggesting that their extreme size was the result of cellular synthesis rather than extracellular coalescence.

The heterogeneity of human circulating anticoagulants against antihemophilic factor (AHF, factor VIII) observed in seven patients, both with and without classic hemophilia, was investigated by neutralization of their activity with antiserums directed to whole IgG and to lambda and kappa light chains. All seven anticoagulants were immunoglobulins. Six appeared to contain both kinds of light chains, although the dual light chain composition of two of these could be demonstrated only at high concentration of antiserum. In one circulating anticoagulant, light chain specificity could not be demonstrated with small amounts of antiserum, and with larger amounts, only lambda light chain specificity was revealed. Whether or not this circulating anticoagulant really contained a single light chain type could not be ascertained with our technique. The evidence presented suggested that circulating anticoagulant antibodies against AHF are polyclonal in nature.