Photo-regulation of rod precursor cell proliferation.

Teleosts are unique in their ability to undergo persistent neurogenesis and to regenerate damaged and lost retinal neurons in adults. This contrasts with the human retina, which is incapable of replacing lost retinal neurons causing vision loss/blindness in the affected individuals. Two cell populations within the adult teleost retina generate new retinal neurons throughout life. Stem cells within the ciliary marginal zone give rise to all retinal cell types except for rod photoreceptors, which are produced by the resident Müller glia that are located within the inner nuclear layer of the entire retina. Understanding the mechanisms that regulate the generation of photoreceptors in the adult teleost retina may ultimately aid developing strategies to overcome vision loss in diseases such as retinitis pigmentosa. Here, we investigated whether photic deprivation alters the proliferative capacity of rod precursor cells, which are generated from Müller glia. In dark-adapted retinas, rod precursor cell proliferation increased, while the number of proliferating Müller glia and their derived olig2:EGFP-positive neuronal progenitor cells was not significantly changed. Cell death of rod photoreceptors was excluded as the inducer of rod precursor cell proliferation, as the number of TUNEL-positive cells and l-plastin-positive microglia in both the outer (ONL) and inner nuclear layer (INL) remained at a similar level throughout the dark-adaptation timecourse. Rod precursor cell proliferation in response to dark-adaptation was characterized by an increased number of EdU-positive cells, i.e. cells that were undergoing DNA replication. These proliferating rod precursor cells in dark-adapted zebrafish differentiated into rod photoreceptors at a comparable percentage and in a similar time frame as those maintained under standard light conditions suggesting that the cell cycle did not stall in dark-adapted retinas. Inhibition of IGF1-receptor signaling reduced the dark-adaptation-mediated proliferation response; however, caloric restriction which has been suggested to be integrated by the IGF1/growth hormone signaling axis did not influence rod precursor cell proliferation in dark-adapted retinas, as similar numbers were observed in starved and normal fed zebrafish. In summary, photic deprivation induces cell cycle entry of rod precursor cells via IGF1-receptor signaling independent of Müller glia proliferation.

Repressing notch signaling and expressing TNFα are sufficient to mimic retinal regeneration by inducing Müller glial proliferation to generate committed progenitor cells.

Retinal damage in teleosts, unlike mammals, induces robust Müller glia-mediated regeneration of lost neurons. We examined whether Notch signaling regulates Müller glia proliferation in the adult zebrafish retina and demonstrated that Notch signaling maintains Müller glia in a quiescent state in the undamaged retina. Repressing Notch signaling, through injection of the γ-secretase inhibitor RO4929097, stimulates a subset of Müller glia to reenter the cell cycle without retinal damage. This RO4929097-induced Müller glia proliferation is mediated by repressing Notch signaling because inducible expression of the Notch Intracellular Domain (NICD) can reverse the effect. This RO4929097-induced proliferation requires Ascl1a expression and Jak1-mediated Stat3 phosphorylation/activation, analogous to the light-damaged retina. Moreover, coinjecting RO4929097 and TNFα, a previously identified damage signal, induced the majority of Müller glia to reenter the cell cycle and produced proliferating neuronal progenitor cells that committed to a neuronal lineage in the undamaged retina. This demonstrates that repressing Notch signaling and activating TNFα signaling are sufficient to induce Müller glia proliferation that generates neuronal progenitor cells that differentiate into retinal neurons, mimicking the responses observed in the regenerating retina.

Tumor necrosis factor-alpha is produced by dying retinal neurons and is required for Muller glia proliferation during zebrafish retinal regeneration.

Intense light exposure causes photoreceptor apoptosis in dark-adapted adult albino zebrafish (Danio rerio). Subsequently, Müller glia increase expression of the Achaete-scute complex-like 1a (Ascl1a) and Signal transducer and activator of transcription 3 (Stat3) transcription factors and re-enter the cell cycle to yield undifferentiated neuronal progenitors that continue to proliferate, migrate to the outer nuclear layer, and differentiate into photoreceptors. A proteomic analysis of light-damaged retinal homogenates, which induced Müller glia proliferation when injected into an undamaged eye, revealed increased expression of tumor necrosis factor α (TNFα) signaling proteins relative to undamaged retinal homogenates. TNFα expression initially increased in apoptotic photoreceptors and later in Müller glia. Morpholino-mediated knockdown of TNFα expression before light damage diminished the expression of both Ascl1a and Stat3 in Müller glia and significantly reduced the number of proliferating Müller glia without affecting photoreceptor cell death. Knockdown of TNFα expression in the Müller glia resulted in fewer proliferating Müller glia, suggesting that Müller glial-derived TNFα recruited additional Müller glia to re-enter the cell cycle. While TNFα is required for increased Ascl1a and Stat3 expression, Ascl1a and Stat3 are both necessary for TNFα expression in Müller glia. Apoptotic inner retinal neurons, resulting from intravitreal injection of ouabain, also exhibited increased TNFα expression that was required for Müller glia proliferation. Thus, TNFα is the first molecule identified that is produced by dying retinal neurons and is necessary to induce Müller glia to proliferate in the zebrafish retinal regeneration response.

Student performance on the Test of Scientific Literacy Skills (TOSLS) does not change with assignment of a low-stakes grade.

OBJECTIVE: Response-validated multiple-choice assessments are used in college courses to assess student learning gains. The ability of a test to accurately reflect student learning gains is highly dependent on the students' effort. Within our institution, lackluster student effort is common on response-validated multiple-choice concept assessments that are not included as a portion of the semester grade but are used to inform curricular changes. Thus, we set out to determine whether increasing testing stakes by assigning a grade on student performance had an effect on student score and self-reported effort. The Test of Scientific Literacy Skills (TOSLS) is a response-validated multiple-choice assessment used to measure scientific literacy in undergraduates. We administered the TOSLS to students enrolled in a general education Biology course, both during the first 2 weeks (pretest) and the last 2 weeks (posttest) of the course. RESULTS: Self-reported effort and TOSLS performance were significantly correlated in the ungraded cohort. This relationship did not exist in the graded sections. Our data indicate that assigning a low-stakes grade has no significant effect on mean student performance or self-reported effort on the TOSLS within our general education course.

Stat3 defines three populations of Müller glia and is required for initiating maximal müller glia proliferation in the regenerating zebrafish retina.

We analyzed the role of Stat3, Ascl1a, and Lin28a in Müller glia reentry into the cell cycle following damage to the zebrafish retina. Immunohistochemical analysis was employed to determine the temporal and spatial expression of Stat3 and Ascl1a proteins following rod and cone photoreceptor cell apoptosis. Stat3 expression was observed in all Müller glia, whereas Ascl1a expression was restricted to only the mitotic Müller glia. Knockdown of Stat3 protein expression did not affect photoreceptor apoptosis, but significantly reduced, without abolishing, the number of proliferating Ascl1a-positive Müller glia. Knockdown of Ascl1a protein also did not change the extent of photoreceptor apoptosis, but did yield significantly fewer Müller glia that reentered the cell cycle relative to the stat3 morphant and significantly decreased the number and intensity of Stat3-expressing Müller glia. Finally, introduction of lin28a morpholinos resulted in decreased Müller glia expression of Stat3 and Ascl1a, significantly reducing the number of proliferating Müller glia. Thus, there are three populations of Müller glia in the light-damaged zebrafish retina: 1) Stat3-expressing Ascl1a-nonexpressing nonproliferating (quiescent) Müller glia; 2) Stat3-dependent Ascl1a-dependent proliferating Müller glia; and 3) Stat3-independent Ascl1a-dependent proliferating Müller glia. Whereas Ascl1a and Lin28a are required for Müller glia proliferation, Stat3 is necessary for the maximal number of Müller glia to proliferate during regeneration of the damaged zebrafish retina.

Inhibition of the Pim1 oncogene results in diminished visual function.

Our objective was to profile genetic pathways whose differential expression correlates with maturation of visual function in zebrafish. Bioinformatic analysis of transcriptomic data revealed Jak-Stat signalling as the pathway most enriched in the eye, as visual function develops. Real-time PCR, western blotting, immunohistochemistry and in situ hybridization data confirm that multiple Jak-Stat pathway genes are up-regulated in the zebrafish eye between 3-5 days post-fertilisation, times associated with significant maturation of vision. One of the most up-regulated Jak-Stat genes is the proto-oncogene Pim1 kinase, previously associated with haematological malignancies and cancer. Loss of function experiments using Pim1 morpholinos or Pim1 inhibitors result in significant diminishment of visual behaviour and function. In summary, we have identified that enhanced expression of Jak-Stat pathway genes correlates with maturation of visual function and that the Pim1 oncogene is required for normal visual function.

Cloning and spatiotemporal expression of zebrafish neuronal nicotinic acetylcholine receptor alpha 6 and alpha 4 subunit RNAs.

Acetylcholine plays an important role in regulation of nervous system development and function. We are developing zebrafish (Danio rerio) as a model system to study the role of specific neuronal nicotinic acetylcholine receptor (nAChR) subtypes in development and the effects of nicotine on the developing vertebrate nervous system. We previously characterized the expression of several zebrafish nAChR subunits. To further develop the zebrafish model, here we report a study on the molecular characterization of two additional nAChR subunit genes, designated chrna6 and chrna4. Both zebrafish nAChRs have a high degree of sequence identity to nAChRs expressed in a variety of mammalian species. Reverse transcription polymerase chain reaction was used to show that both nAChR subunit RNAs were expressed early in zebrafish development, with the chrna4 transcript present at 3 hours postfertilization (hpf) and the chrna6 RNA present at 10 hpf. In situ hybridization was used to localize chrna6 and chrna4 RNA expression in 24, 48, 72, and 96 hpf zebrafish. The chrna6 and chrna4 RNAs were each expressed in a unique pattern, which changed during development. At various ages, chrna6 was expressed in Rohon-Beard sensory neurons, trigeminal ganglion, retina, and the pineal gland. Most notably, chrna6 was expressed in catecholaminergic neurons in the midbrain, but was also present in noncatecholaminergic cells in both midbrain and hindbrain. The expression of chrna6 RNA in catecholaminergic cells supports the use of zebrafish as a valid model system to better understand the molecular basis of cholinergic regulation of dopaminergic signaling and the role of alpha6-containing nAChRs in Parkinson's disease. The most notable chrna4 expression was in neural crest cells at 24 hpf and reticulospinal neurons in hindbrain at 48 hpf. chrna4 RNA exhibited a widespread and robust expression pattern in the midbrain in 72 hpf and 96 hpf zebrafish.