X-ray crystallographic studies of RoAb13 bound to PIYDIN, a part of the N-terminal domain of C-C chemokine receptor 5.

C-C chemokine receptor 5 (CCR5) is a major co-receptor molecule used by HIV-1 to enter cells. This led to the hypothesis that stimulating an antibody response would block HIV with minimal toxicity. Here, X-ray crystallographic studies of the anti-CCR5 antibody RoAb13 together with two peptides were undertaken: one peptide is a 31-residue peptide containing the PIYDIN sequence and the other is the PIDYIN peptide alone, where PIYDIN is part of the N-terminal region of CCR5 previously shown to be important for HIV entry. In the presence of the longer peptide (the complete N-terminal domain), difference electron density was observed at a site within a hypervariable CDR3 binding region. In the presence of the shorter core peptide PIYDIN, difference electron density is again observed at this CDR3 site, confirming consistent binding for both peptides. This may be useful in the design of a new biomimetic to stimulate an antibody response to CCR5 in order to block HIV infection.

Hydrophobic Interface-Assisted Protein Crystallization: Theory and Experiment.

Macromolecular crystallization is crucial to a large number of scientific fields, including structural biology; drug design, formulation, and delivery; manufacture of biomaterials; and preparation of foodstuffs. The purpose of this study is to facilitate control of crystallization, by investigating hydrophobic interface-assisted protein crystallization both theoretically and experimentally. The application of hydrophobic liquids as nucleation promoters or suppressors has rarely been investigated, and provides an underused avenue to explore in protein crystallization. Theoretically, crystal nucleation is regarded as a two-step process, the first step being a local increase in protein concentration due to its adsorption on the hydrophobic surface. Subsequently, the protein is ordered in a crystal lattice. The energetic aspect of crystal nucleation on water/hydrophobic substance interfaces is approached by calculating the balance between the cohesive energy maintaining integrity of the two-dimensional crystal nucleus and the sum of destructive energies tending to tear up the crystal. This is achieved by comparing the number of bonds shared by the units forming the crystal and the number of unshared (dangling) bonds on the crystal surface pointing toward the solution. The same approach is extended to three-dimensional protein crystal nucleation at water/hydrophobic liquid interfaces. Experimentally, we studied protein crystallization over oils and other hydrophobic liquids (paraffin oil, FC-70 Fluorinert fluorinated oil, and three chlorinated hydrocarbons). Crystallizations of α-lactalbumin and lysozyme are compared, and additional information is acquired by studying α-crustacyanin, trypsin, an insulin analogue, and protein Lpg2936. Depending on the protein type, concentration, and the interface aging time, the proteins exhibit different crystallization propensities depending on the hydrophobic liquid used. Some hydrophobic liquids provoke an increase in the effective supersaturation, which translates to enhancement of crystal nucleation at their interface with the crystallization solution, leading to the formation of crystals.

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.

Pax6a and Pax6b are required at different points in neuronal progenitor cell proliferation during zebrafish photoreceptor regeneration.

The light-damaged zebrafish retina results in the death of photoreceptor cells and the subsequent regeneration of the missing rod and cone cells. Photoreceptor regeneration initiates with asymmetric Müller glial cell division to produce neuronal progenitor cells, which amplify, migrate to the outer nuclear layer (ONL), and differentiate into both classes of photoreceptor cells. In this study, we examined the role of the Pax6 protein in regeneration. In zebrafish, there are two Pax6 proteins, one encoded by the pax6a gene and the other encoded by the pax6b gene. We intravitreally injected and electroporated morpholinos that were complementary to either the pax6a or pax6b mRNA to knockdown the translation of the corresponding protein. Loss of Pax6b expression did not affect Müller glial cell division, but blocked the subsequent first cell division of the neuronal progenitors. In contrast, the paralogous Pax6a protein was required for later neuronal progenitor cell divisions, which maximized the number of neuronal progenitors. Without neuronal progenitor cell amplification, proliferation of resident ONL rod precursor cells, which can only regenerate rods, increased inversely proportional to the number of INL neuronal progenitor cells. This confirmed that Müller glial-derived neuronal progenitor cells are necessary to regenerate cones and that distinct mechanisms selectively regenerate rod and cone photoreceptors. This work also defines distinct roles for Pax6a and Pax6b in regulating neuronal progenitor cell proliferation in the adult zebrafish retina and increases our understanding of the molecular pathways required for photoreceptor cell regeneration.

CNTF induces photoreceptor neuroprotection and Müller glial cell proliferation through two different signaling pathways in the adult zebrafish retina.

Ciliary neurotrophic factor (CNTF) acts in several processes in the vertebrate retina, including neuroprotection of photoreceptors in the stressed adult retina and regulation of neuronal progenitor cell proliferation during retinal development. However, the signaling pathway it utilizes (Jak/Stat, MAPK, or Akt) in these processes is ambiguous. Because dark-adapted albino zebrafish exhibit light-induced rod and cone cell death and subsequently regenerate the lost photoreceptor cells, zebrafish should be a useful model to study the role of CNTF in both neuroprotection and neuronal progenitor cell proliferation. We therefore investigated the potential roles of CNTF in both the undamaged and light-damaged adult zebrafish retinas. Intraocular injection of CNTF suppressed light-induced photoreceptor cell death, which then failed to exhibit the regeneration response that is marked by proliferating Müller glia and neuronal progenitor cells. Inhibiting the MAPK signaling pathway, but neither the Stat3 nor Akt pathways, significantly reduced the CNTF-mediated neuroprotection of light-induced photoreceptor cell death. Intraocular injection of CNTF into non-light-treated (undamaged) eyes mimicked constant intense light treatment by increasing Stat3 expression in Müller glia followed by increasing the number of proliferating Müller glia and neuronal progenitors. Knockdown of Stat3 expression in the CNTF-injected non-light-treated retinas significantly reduced the number of proliferating Müller glia, while coinjection of CNTF with either MAPK or Akt inhibitors did not inhibit the CNTF-induced Müller glia proliferation. Thus, CNTF utilizes a MAPK-dependant signaling pathway in neuroprotection of light-induced photoreceptor cell death and a Stat3-dependant signaling pathway to stimulate Müller glia proliferation.

Characterization of Müller glia and neuronal progenitors during adult zebrafish retinal regeneration.

The adult zebrafish retina exhibits a robust regenerative response following light-induced photoreceptor cell death. This response is initiated by the Müller glia proliferating in the inner nuclear layer (INL), which gives rise to neuronal progenitor cells that continue to divide and migrate to the outer nuclear layer (ONL), where they differentiate into rod and cone photoreceptors. We previously conducted a microarray analysis of retinal gene expression at 16, 31, 51, 68, and 96 h of constant intense-light treatment to identify genes and their corresponding proteins that may be involved in the generation and proliferation of the neuronal progenitor cells. We examined the expression of two candidate transcription factors, Pax6 and Ngn1, and one candidate transgene, olig2:EGFP, in the regenerating light-damaged retina. We compared the temporal and spatial expression patterns of these markers relative to PCNA (proliferating cell nuclear antigen), an established marker for proliferating cells in the zebrafish retina, and the Tg(gfap:EGFP) nt11 transgenic line that specifically labels Müller glial cells. We found that Müller glial cells dedifferentiate during regeneration, based on the loss of cell-specific markers such as GFAP (glial fibrillary acidic protein) and glutamine synthetase following their reentry into the cell cycle to produce neuronal progenitors. Pax6 expression was first detected in the proliferating neuronal progenitors by 51 h of constant light treatment, which is significantly after the Müller glia first reenter the cell cycle after 31h of light. This suggests that Pax6 expression increases in neuronal progenitors, rather than in the proliferating Müller glia. EGFP expression from the olig2 promoter was first detected by 68 h of constant light treatment in the dedifferentiated Müller glia, with Pax6 expressed in the closely associated proliferating neuronal progenitors migrating to the ONL. Both Pax6 and olig2 expression persisted until 3 days post-light treatment, when the neuronal progenitors begin differentiating into new rod and cone photoreceptors. Ngn1 protein expression was initially detected in proliferating neuronal progenitors at 68 h of light treatment. However, Ngn1 expression persisted in a subset of the INL nuclei until 17 days post-light treatment. Using the Tg(gfap:EGFP) nt11 transgenic line, Ngn1 was localized to the Müller glial nuclei that were reestablished following the regenerative response. These markers, therefore, can be used to identify different cell types at particular stages of retinal regeneration: neuronal progenitor formation, proliferation, and the reestablishment of the Müller glia cells. These markers will be important to further characterize the regeneration response in other retinal damage models and to elucidate the defects associated with mutants and morphants that disrupt the regeneration response.

The Tg(ccnb1:EGFP) transgenic zebrafish line labels proliferating cells during retinal development and regeneration.

PURPOSE: To create the Tg(ccnb1:EGFP)(nt18) zebrafish line that spatially and temporally labels retinal progenitor cells with enhanced green fluorescent protein (EGFP) during zebrafish retinal development and regeneration. METHODS: We cloned the 1.5 kb promoter region of the zebrafish cyclin B1 (ccnb1) gene upstream of the EGFP gene in the Tol2 vector, which was used to generate the stable Tg(ccnb1:EGFP)(nt18) transgenic zebrafish line. Immunohistochemistry and in situ hybridization techniques verified that the ccnb1:EGFP transgene was expressed in retinal progenitor cells during retinal development, in the undamaged adult retina, and in the regenerating adult retina. RESULTS: At 36 h post-fertilization, both the enhanced green fluorescent protein (EGFP) and proliferating cell nuclear antigen (PCNA) expressions were observed throughout the developing transgenic retina, but they became restricted to the circumferential marginal zone by five days post-fertilization. In situ hybridization confirmed that this EGFP expression matched the cyclin B1 mRNA expression pattern. In comparison to the Tg(1016a1tubulin:EGFP) transgenic line that expresses EGFP in neuronal progenitor cells, the Tg(ccnb1:EGFP)(nt18) line more faithfully follows the rise and fall of PCNA expression through the developing retina and brain. In the adult retina, there are three cell types that continue to proliferate, the Müller glia in the inner nuclear layer, the rod precursor cells in the outer nuclear layer, and the stem cells in the circumferential marginal zone. In the Tg(ccnb1:EGFP)(nt18) retina, EGFP coexpressed with PCNA in all three of these proliferating cell types. Exposing the adult retina to constant intense light destroys the rod and cone photoreceptors and induces an increase in the number of proliferating Müller glia, which produces actively dividing neuronal progenitor cells that migrate to the outer nuclear layer (ONL) and replenish the lost photoreceptors. Following constant light damage, Tg(ccnb1:EGFP)(nt18) zebrafish expressed EGFP in both the proliferating Müller glia and the migrating neuronal progenitor cells. CONCLUSIONS: The spatial and temporal patterning of EGFP expression in the Tg(ccnb1:EGFP)(nt18) line directly reflects the known locations of proliferating cells in the zebrafish retina, making it a useful marker to study the transient nature of neuronal progenitor cells during the development and regeneration of the zebrafish retina.

Inhibition of Müller glial cell division blocks regeneration of the light-damaged zebrafish retina.

The adult zebrafish retina possesses a robust regenerative response. In the light-damaged retina, Müller glial cell divisions precede regeneration of rod and cone photoreceptors. Neuronal progenitors, which arise from the Müller glia, continue to divide and use the Müller glial cell processes to migrate to the outer nuclear layer and replace the lost photoreceptors. We tested the necessity of Müller glial cell division for photoreceptor regeneration. As knockdown tools were unavailable for use in the adult zebrafish retina, we developed a method to conditionally inhibit the expression of specific proteins by in vivo electroporation of morpholinos. We determined that two separate morpholinos targeted against the proliferating cell nuclear antigen (PCNA) mRNA reduced PCNA protein levels. Furthermore, injection and in vivo electroporation of PCNA morpholinos immediately prior to starting intense light exposure inhibited both Müller glial cell proliferation and neuronal progenitor marker Pax6 expression. PCNA knockdown additionally resulted in decreased expression of glutamine synthetase in Müller glia and Müller glial cell death, while amacrine and ganglion cells were unaffected. Finally, histological and immunological methods showed that long-term effects of PCNA knockdown resulted in decreased numbers of Müller glia and the failure to regenerate rod photoreceptors, short single cones, and long single cones. These data suggest that Müller glial cell division is necessary for proper photoreceptor regeneration in the light-damaged zebrafish retina and are consistent with the Müller glia serving as the source of neuronal progenitor cells in regenerating teleost retinas.

Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters.

Two commonly used promoters to ubiquitously express transgenes in zebrafish are the Xenopus laevis elongation factor 1 alpha promoter (XlEef1a1) and the zebrafish histone variant H2A.F/Z (h2afv) promoter. Recently, transgenes utilizing these promoters were shown to be silenced in certain adult tissues, particularly the central nervous system. To overcome this limitation, we cloned the promoters of four zebrafish genes that likely are transcribed ubiquitously throughout development and into the adult. These four genes are the TATA box binding protein gene, the taube nuss-like gene, the eukaryotic elongation factor 1-gamma gene, and the beta-actin-1 gene. We PCR amplified approximately 2.5 kb upstream of the putative translational start site of each gene and cloned each into a Tol2 expression vector that contains the EGFP reporter transgene. We used these four Tol2 vectors to independently generate stable transgenic fish lines for analysis of transgene expression during development and in the adult. We demonstrated that all four promoters drive a very broad pattern of EGFP expression throughout development and the adult. Using the retina as a well-characterized component of the CNS, all four promoters appeared to drive EGFP expression in all neuronal and non-neuronal cells of the adult retina. In contrast, the h2afv promoter failed to express EGFP in the adult retina. When we examined EGFP expression in the various cells of the blood cell lineage, we observed that all four promoters exhibited a more heterogenous expression pattern than either the XlEef1a1 or h2afv promoters. While these four ubiquitous promoters did not express EGFP in all the adult blood cells, they did express EGFP throughout the CNS and in broader expression patterns in the adult than either the XlEef1a1 or h2afv promoters. For these reasons, these four promoters will be valuable tools for expressing transgenes in adult zebrafish.

Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish.

Constant intense light causes apoptosis of rod and cone photoreceptors in adult albino zebrafish. The photoreceptors subsequently regenerate from proliferating inner nuclear layer (INL) progenitor cells that migrate to the outer nuclear layer (ONL) and differentiate into rods and cones. To identify gene expression changes during this photoreceptor regeneration response, a microarray analysis was performed at five time points during the light treatment. The time course included an early time point during photoreceptor death (16 h), later time points during progenitor cell proliferation and migration (31, 51, and 68 h) and a 96 h time point, which likely corresponds to the initial photoreceptor differentiation. Mean expression values for each gene were calculated at each time point relative to the control (0 h light exposure) and statistical analysis by one-way ANOVA identified 4567 genes exhibiting significant changes in gene expression along the time course. The genes within this data set were clustered based on their temporal expression patterns and proposed functions. Quantitative real-time PCR validated the microarray expression profiles for selected genes, including stat3 whose expression increased markedly during the light exposure. Based on immunoblots, both total and activated Stat3 protein expression also increased during the light treatment. Immunolocalization of Stat3 on retinal tissue sections demonstrated increased expression in photoreceptors and Müller glia by 16 h of light exposure. Some of the Stat3-positive Müller cells expressed PCNA at 31 h, suggesting that Stat3 may play a role in signaling a subset of Müller cells to proliferate during the regeneration response.

Retinal regional differences in photoreceptor cell death and regeneration in light-lesioned albino zebrafish.

Teleost fish regenerate retinal cells from a population of inner nuclear layer (INL) stem cells. To characterize photoreceptor regeneration in zebrafish (Danio rerio), adult albino fish were subjected to constant intense light to cause photoreceptor cell death. Retinal morphometry was performed on histological sections of control and light-lesioned albino retinas to compare the extent of light damage in the ventral, central and dorsal retinal regions. In addition, opsin immunohistochemistry and TUNEL were used to compare photoreceptor cell death in these different retinal areas, while PCNA immunolabeling quantified the cell proliferation that precedes the photoreceptor regeneration. Transgenic albino; Tg(alpha1-tubulin:egfp) zebrafish were also exposed to the intense light in order to examine regeneration-related gene expression changes. The light-lesioned retinas are characterized by extensive rod and cone photoreceptor cell death in the central and dorsal regions. In contrast, many of the rods and cones survive in the ventral retina. The highest levels of INL cell proliferation, which occurs subsequent to photoreceptor death, correspond to the retinal regions that suffer the greatest levels of photoreceptor damage. In the ventral retina, where photoreceptor cell death is minimal, cell proliferation is confined to the ONL. In addition, EGFP expression from the alpha1-tubulin promoter is increased in Müller glial cells in the light-damaged central and dorsal retina, while transgene expression in the ventral retina is restricted to small, round INL cells. Furthermore, expression of the HuC/D neuronal antigen is detected in a subpopulation of the Müller cells in the light-damaged superior retinal region. These data demonstrate that adult albino zebrafish display retinal regional differences in photoreceptor cell death and in the regeneration-related INL cell proliferation response. The high levels of INL cell proliferation and alpha1-tubulin:egfp transgene expression in the Müller cells may be graded in response to the degree of photoreceptor cell death. This suggests that the levels of photoreceptor damage may directly influence cell responses in the underlying retinal layers.