A novel gene coding for the pre-B-cell colony-enhancing factor (PBEF)

Photoreceptor degeneration is a common cause of inherited blindness worldwide. We have identified a blind zebrafish mutant with rapid degeneration of cone photoreceptors caused by a mutation in the cone phosphodiesterase c ( pde6c) gene, a key regulatory component in cone phototransduction. Some rods also degenerate, primarily in areas with a low density of rods. Rod photoreceptors in areas of the retina that always have a high density of rods are protected from degeneration. Our findings demonstrate that, analogous to what happens to rod photoreceptors in the rd1 mouse model, loss of cone phosphodiesterase leads to rapid degeneration of cone photoreceptors. Furthermore, we propose that cell density plays a key role in determining whether rod photoreceptors degenerate as a secondary consequence to cone degeneration. Our zebrafish mutant serves as a model for developing therapeutic treatments for photoreceptor degeneration in humans.

Ciliopathies are a genetically and phenotypically heterogeneous group of human developmental disorders whose root cause is the absence or dysfunction of primary cilia. Joubert syndrome is characterized by a distinctive hindbrain malformation variably associated with retinal dystrophy and cystic kidney disease. Mutations in CC2D2A are found in ∼10% of patients with Joubert syndrome. Here we describe the retinal phenotype of cc2d2a mutant zebrafish consisting of disorganized rod and cone photoreceptor outer segments resulting in abnormal visual function as measured by electroretinogram. Our analysis reveals trafficking defects in mutant photoreceptors affecting transmembrane outer segment proteins (opsins) and striking accumulation of vesicles, suggesting a role for Cc2d2a in vesicle trafficking and fusion. This is further supported by mislocalization of Rab8, a key regulator of opsin carrier vesicle trafficking, in cc2d2a mutant photoreceptors and by enhancement of the cc2d2a retinal and kidney phenotypes with partial knockdown of rab8. We demonstrate that Cc2d2a localizes to the connecting cilium in photoreceptors and to the transition zone in other ciliated cell types and that cilia are present in these cells in cc2d2a mutants, arguing against a primary function for Cc2d2a in ciliogenesis. Our data support a model where Cc2d2a, localized at the photoreceptor connecting cilium/transition zone, facilitates protein transport through a role in Rab8-dependent vesicle trafficking and fusion.

Visual, vestibular, and auditory neurons rely on ribbon synapses for rapid continuous release and recycling of synaptic vesicles. Molecular mechanisms responsible for the properties of ribbon synapses are mostly unknown. The zebrafish vision mutant nrc has unanchored ribbons and abnormal synaptic transmission at cone photoreceptor synapses. We used positional cloning to identify the nrc mutation as a premature stop codon in the synaptojanin1 (synj1) gene. Synaptojanin 1 (Synj1) is undetectable in nrc extracts, and biochemical activities associated with it are reduced. Furthermore, morpholinos directed against synj1 phenocopy the nrc mutation. Synj1 is a polyphosphoinositide phosphatase important at conventional synapses for clathrin-mediated endocytosis and actin cytoskeletal rearrangement. In the nrc cone photoreceptor pedicle, not only are ribbons unanchored, but synaptic vesicles are reduced in number, abnormally distributed, and interspersed within a dense cytoskeletal matrix. Our findings reveal a new role for Synj1 and link phosphoinositide metabolism to ribbon architecture and function at the cone photoreceptor synapse.

Oxidized halogen antimicrobials, such as hypochlorous and hypobromous acids, have been used extensively for microbial control in industrial systems. Recent discoveries have shown that acylated homoserine lactone cell-to-cell signaling molecules are important for biofilm formation in Pseudomonas aeruginosa, suggesting that biofouling can be controlled by interfering with bacterial cell-to-cell communication. This study was conducted to investigate the potential for oxidized halogens to react with acylated homoserine lactone-based signaling molecules. Acylated homoserine lactones containing a 3-oxo group were found to rapidly react with oxidized halogens, while acylated homoserine lactones lacking the 3-oxo functionality did not react. The Chromobacterium violaceum CV026 bioassay was used to determine the effects of such reactions on acylated homoserine lactone activity. The results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity. Experiments with the marine alga Laminaria digitata demonstrated that natural haloperoxidase systems are capable of mediating the deactivation of acylated homoserine lactones. This may illustrate a natural defense mechanism to prevent biofouling on the surface of this marine alga. The Chromobacterium violaceum activity assay illustrates that reactions between 3-oxo acylated homoserine lactone molecules and oxidized halogens do occur despite the presence of biofilm components at much greater concentrations. This work suggests that oxidized halogens may control biofilm not only via a cidal mechanism, but also by possibly interfering with 3-oxo acylated homoserine lactone-based cell signaling.

Two alleles of an eyeless mutant, chokh (chk), were identified in ongoing zebrafish F(3) mutagenesis screens. Morphologically, chk mutants can be identified at 15 h post-fertilization by the failure of optic primordia to evaginate from the forebrain. The chk phenotype appears specific, as marker genes in the forebrain, midbrain, and pineal are expressed in normal temporal, spatial, and circadian patterns. Sequence analysis of the chk alleles revealed nonsense or missense mutations in the rx3 homeobox. Rx genes encode paired-type homeodomain transcription factors known to be key regulators of eye development in mouse, medaka, Xenopus, and zebrafish. To uncover novel Rx targets, we analyzed the expression of multiple eye development genes in chk. We find that expression of mab21l2, mab21l1 and rx2 are specifically absent in the eye field of chk embryos. Knockdown of Mab21l2 by antisense morpholino microinjections partially phenocopies the rx3 mutation, leading to microphthalmia, incomplete eye maturation, and dramatic increases in apoptotic eye progenitors. We propose that mab21l2 is an early downstream effector of rx3 and is critical for survival of eye progenitors.

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor's synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.energy metabolism | phototransduction A significant energy distribution problem can arise from the relative locations of mitochondria, ion pumps, and synapses in neurons. In photoreceptors, ion pumps occupy the intervening space between the centrally located mitochondria and the synaptic terminal. Ion pumping in dark-adapted photoreceptors consumes ∼20× more energy than neurotransmission (1). Therefore, the pumps could intercept all the metabolic energy made by the mitochondria before it can reach the synaptic terminal. In the vascularized retinas of mice, rats, and humans (2-4) this problem is solved by the presence of additional mitochondria in the terminal. However, in the avascular retinas of zebrafish, salamanders, rabbits, and guinea pigs there are no mitochondria in the terminals (2, 4, 5), which creates a need to partition some of the energy made by the central mitochondria into a protected form that can bypass the ion pumps to support the essential energy demands of the synaptic terminal.Energy consumption within retinal photoreceptors is compartmentalized and light-dependent. During illumination, phototransduction and light adaptation consume energy in the outer segment (OS). In darkness, energy is consumed by ion pumps in the inner segment and by glutamate release at the synaptic terminal (1). Energy demands and O 2 consumption are far greater in darkness than in light (1, 6-8).Metabolic energy is distributed in most cells as either ATP or phosphocreatine (PCr). There are 2 isoforms of creatine kinase (CK) in neurons, ubiquitous mitochondrial creatine kinase (uMtCK), and brain-type cytoplasmic creatine kinase (CK-B). uMtCK creates PCr from ATP at mitochondria (9), and CK-B can recreate ATP from PCr at sites of energy demand. In this way uMtCK and CK-B can collaborate to transfer metabolic energy between neuronal compartments (10, 11). This paper descr...

A 3.2-kb TalphaC promoter fragment replicates the temporal and spatial pattern of endogenous TalphaC expression. The integrity of cones can be readily assessed in an EGFP transgenic line generated with this promoter, enabling downstream genetic and chemical screens for cone determinants.