Peroxidasin is required for full viability in development and for maintenance of tissue mechanics in adults.

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membranes that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved throughout the animal kingdom, indicating they are important, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a new CRISPR-generated catalytic null, and found that homozygotes were mostly lethal with 13 % viable escapers. Mouse mutants also show semi-lethality, with Mendelian analysis demonstrating ∼50 % lethality and ∼50 % escapers. Despite the strong mutations, the homozygous fly and mouse escapers had low but detectable levels of Col4 crosslinking, indicating the existence of inefficient alternative crosslinking mechanisms, probably responsible for the viable escapers. Fly mutant phenotypes are consistent with decreased basement membrane stiffness. Interestingly, we found that even after basement membranes are assembled and crosslinked in wild-type animals, continuing Peroxidasin activity is required in adults to maintain tissue stiffness over time. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.

Collagen IV of basement membranes: I. Origin and diversification of COL4 genes enabling animal evolution.

Collagen IV is a primordial component of basement membranes, a specialized form of extracellular matrix that enabled multi-cellular epithelial tissues. In mammals, collagen IV assembles from a family of six α-chains (α1 to α6), encoded by six genes (COL4A1 to COL4A6), into three distinct scaffolds: the α121, the α345 and a mixed scaffold containing both α121 and α565. The six mammalian COL4A genes occur in pairs that occur in a head-to-head arrangement on three distinct chromosomes. In Alport syndrome, variants in the COL4A3, 4 or 5 genes cause either loss or defective assembly of the collagen IV α345 scaffold which results in a dysfunctional glomerular basement membrane, proteinuria and progression to renal failure in millions of people worldwide. Here, we determine the evolutionary emergence and diversification of the COL4A genes using comparative genomics and biochemical analyses. Using syntenic relationships to genes closely linked to the COL4A genes, we determine that the COL4A3 and COL4A4 gene pair appeared in cyclostomes (hagfish and lampreys) while the COL4A5 and COL4A6 gene pair emerged in gnathostomes, jawed vertebrates. The more basal chordate species, lancelets and tunicates, do not have discrete kidneys and have a single COL4A gene pair, though often with single isolated COL4 genes similar to those found in C elegans . Remarkably, while the six COL4A genes are conserved in vertebrates, amphibians have lost the COL4A3 and COL4A4 genes. Our findings of the evolutionary emergence of these genes, together with the amphibian double-knockout, opens an experimental window to gain insights into functionality of the Col IV α345 scaffold.

Analysis of Drosophila and mouse mutants reveals that Peroxidasin is required for tissue mechanics and full viability.

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membrane that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved, indicating they are essential, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a newly generated catalytic null, and found that homozygotes were mostly lethal with 13% viable escapers. A Mendelian analysis of mouse mutants shows a similar pattern, with homozygotes displaying ~50% lethality and ~50% escapers. Despite the strong mutations, the homozygous escapers had low but detectable levels of Col4 crosslinking, indicating that inefficient alternative mechanisms exist and that are probably responsible for the viable escapers. Further, fly mutants have phenotypes consistent with a decrease in stiffness. Interestingly, we found that even after adult basement membranes are assembled and crosslinked, Peroxidasin is still required to maintain stiffness. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.

Cell-specific image-guided transcriptomics identifies complex injuries caused by ischemic acute kidney injury in mice.

The kidney's inherent complexity has made identifying cell-specific pathways challenging, particularly when temporally associating them with the dynamic pathophysiology of acute kidney injury (AKI). Here, we combine renal cell-specific luciferase reporter mice using a chemoselective luciferin to guide the acquisition of cell-specific transcriptional changes in C57BL/6 background mice. Hydrogen peroxide generation, a common mechanism of tissue damage, was tracked using a peroxy-caged-luciferin to identify optimum time points for immunoprecipitation of labeled ribosomes for RNA-sequencing. Together, these tools revealed a profound impact of AKI on mitochondrial pathways in the collecting duct. In fact, targeting the mitochondria with an antioxidant, ameliorated not only hydrogen peroxide generation, but also significantly reduced oxidative stress and the expression of the AKI biomarker, LCN2. This integrative approach of coupling physiological imaging with transcriptomics and drug testing revealed how the collecting duct responds to AKI and opens new venues for cell-specific predictive monitoring and treatment.

Zebrafish Genome Engineering Using the CRISPR-Cas9 System.

Geneticists have long sought the ability to manipulate vertebrate genomes by directly altering the information encoded in specific genes. The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease has the ability to bind single loci within vertebrate genomes and generate double-strand breaks (DSBs) at those sites. These DSBs induce an endogenous DSB repair response that results in small insertions or deletions at the targeted site. Alternatively, a template can be supplied, in which case homology-directed repair results in the generation of engineered alleles at the break site. These changes alter the function of the targeted gene facilitating the analysis of gene function. This tool has been widely adopted in the zebrafish model; we discuss the development of this system in the zebrafish and how it can be manipulated to facilitate genome engineering.

Leptin signaling regulates glucose homeostasis, but not adipostasis, in the zebrafish.

Leptin is the primary adipostatic factor in mammals. Produced largely by adipocytes in proportion to total adipose mass, the hormone informs the brain regarding total energy stored as triglycerides in fat cells. The hormone acts on multiple circuits in the brain to regulate food intake, autonomic outflow, and endocrine function to maintain energy balance. In addition to regulating adipose mass, mammalian leptin also plays a role in the regulation of glucose homeostasis and as a gating factor in reproductive competence. Leptin-deficient mice and people exhibit early onset profound hyperphagia and obesity, diabetes, and infertility. Although leptin and the leptin receptor are found in fish, the hormone is not expressed in adipose tissue, but is found in liver and other tissues. Here, we show that adult zebrafish lacking a functional leptin receptor do not exhibit hyperphagia or increased adiposity, and exhibit normal fertility. However, leptin receptor-deficient larvae have increased numbers of ß-cells and increased levels of insulin mRNA. Furthermore, larval zebrafish have been shown to exhibit ß-cell hyperplasia in response to high fat feeding or peripheral insulin resistance, and we show here that leptin receptor is required for this response. Adult zebrafish also have increased levels of insulin mRNA and other alterations in glucose homeostasis. Thus, a role for leptin in the regulation of ß-cell mass and glucose homeostasis appears to be conserved across vertebrates, whereas its role as an adipostatic factor is likely to be a secondary role acquired during the evolution of mammals.

Conditional control of gene function by an invertible gene trap in zebrafish.

Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.

Retinal degeneration and failure of photoreceptor outer segment formation in mice with targeted deletion of the Joubert syndrome gene, Ahi1.

Vertebrate photoreceptors have a modified cilium composed of a basal body, axoneme and outer segment. The outer segment includes stacked membrane discs, containing opsin and the signal transduction apparatus mediating phototransduction. In photoreceptors, two distinct classes of vesicles are trafficked. Synaptic vesicles are transported down the axon to the synapse, whereas opsin-containing vesicles are transported to the outer segment. The continuous replacement of the outer segments imposes a significant biosynthetic and trafficking burden on the photoreceptors. Here, we show that Ahi1, a gene that when mutated results in the neurodevelopmental disorder, Joubert syndrome (JBTS), is required for photoreceptor sensory cilia formation and the development of photoreceptor outer segments. In mice with a targeted deletion of Ahi1, photoreceptors undergo early degeneration. Whereas synaptic proteins are correctly trafficked, photoreceptor outer segment proteins fail to be transported appropriately or are significantly reduced in their expression levels (i.e., transducin and Rom1) in Ahi1(-/-) mice. We show that vesicular targeting defects in Ahi1(-/-) mice are cilium specific, and our evidence suggests that the defects are caused by a decrease in expression of the small GTPase Rab8a, a protein required for accurate polarized vesicular trafficking. Thus, our results suggest that Ahi1 plays a role in stabilizing the outer segment proteins, transducin and Rom1, and that Ahi1 is an important component of Rab8a-mediated vesicular trafficking in photoreceptors. The retinal degeneration observed in Ahi1(-/-) mice recapitulates aspects of the retinal phenotype observed in patients with JBTS and suggests the importance of Ahi1 in photoreceptor function.

Cardiac response to startle stimuli in larval zebrafish: sympathetic and parasympathetic components.

Central regulation of cardiac output via the sympathetic and parasympathetic branches of the autonomic nervous system allows the organism to respond to environmental changes. Sudden onset stimuli, startle stimuli, are useful probes to study central regulatory responses to the environment. In mammals, startle stimuli induce a transient bradycardia that habituates with repeated stimulation. Repeated presentation of the stimulus results in tachycardia. In this study, we investigate the behavioral regulation of heart rate in response to sudden stimuli in the zebrafish. Larval zebrafish show a stereotyped heart rate response to mild electrical shock. Naïve fish show a significant increase in interbeat interval that resolves in the 2 s following stimulation. This transient bradycardia decreases on repeated exposure to the stimulus. Following repeated stimulation, the fish become tachycardic within 1 min of stimulation. Both the transient bradycardia and following tachycardia responses are blocked with administration of the ganglionic blocker hexamethonium, demonstrating that these responses are mediated centrally. The transient bradycardia is blocked by the muscarinic antagonist atropine, suggesting that this response is mediated by the parasympathetic system, while the following tachycardia is specifically blocked by the beta-adrenergic antagonist propranolol, suggesting that this response is mediated by the sympathetic nervous system. Together, these results demonstrate that at the larval stage, zebrafish actively regulate cardiac output to changes in their environment using both the parasympathetic and sympathetic branches of the autonomic nervous system, a behavioral response that is markedly similar to that observed in mammals to similar sudden onset stimuli.

Ahi1, whose human ortholog is mutated in Joubert syndrome, is required for Rab8a localization, ciliogenesis and vesicle trafficking.

The primary non-motile cilium, a membrane-ensheathed, microtubule-bundled organelle, extends from virtually all cells and is important for development. Normal functioning of the cilium requires proper axoneme assembly, membrane biogenesis and ciliary protein localization, in tight coordination with the intraflagellar transport system and vesicular trafficking. Disruptions at any level can induce severe alterations in cell function, giving rise to a myriad of human genetic diseases known as ciliopathies. Here we show that the Abelson helper integration site 1 (Ahi1) gene, whose human ortholog is mutated in Joubert syndrome, regulates cilium formation via its interaction with Rab8a, a small GTPase critical for polarized membrane trafficking. We find that the Ahi1 protein localizes to a single centriole, the mother centriole, which becomes the basal body of the primary cilium. In order to determine whether Ahi1 functions in ciliogenesis, loss of function analysis of Ahi1 was performed in cell culture models of ciliogenesis. Knockdown of Ahi1 expression by shRNAi in cells or targeted deletion of Ahi1 (Ahi1 knockout mouse) leads to impairments in ciliogenesis. In Ahi1-knockdown cells, Rab8a is destabilized and does not properly localize to the basal body. Since Rab8a is implicated in vesicular trafficking, we next examined this process in Ahi1-knockdown cells. Defects in the trafficking of endocytic vesicles from the plasma membrane to the Golgi and back to the plasma membrane were observed in Ahi1-knockdown cells. Overall, our data indicate that the distribution and functioning of Rab8a is regulated by Ahi1, not only affecting cilium formation, but also vesicle transport.

Species differences in the expression of Ahi1, a protein implicated in the neurodevelopmental disorder Joubert syndrome, with preferential accumulation to stigmoid bodies.

Joubert syndrome (JBTS) is an autosomal recessive disorder characterized by cerebellum and brainstem malformations. Individuals with JBTS have abnormal breathing and eye movements, ataxia, hypotonia, and cognitive difficulty, and they display mirror movements. Mutations in the Abelson-helper integration site-1 gene (AHI1) cause JBTS in humans, suggesting that AHI1 is required for hindbrain development; however AHI1 may also be required for neuronal function. Support for this idea comes from studies demonstrating that the AHI1 locus is associated with schizophrenia. To gain further insight into the function of AHI1 in both the developing and mature central nervous system, we determined the spatial and temporal expression patterns of the gene products of AHI1 orthologs throughout development, in human, mouse, and zebrafish. Murine Ahi1 was distributed throughout the cytoplasm, dendrites, and axons of neurons, but was absent in glial cells. Ahi1 expression in the mouse brain was observed as early as embryonic day 10.5 and persisted into adulthood, with peak expression during the first postnatal week. Murine Ahi1 was observed in neurons of the hindbrain, midbrain, and ventral forebrain. Generally, the AHI1/Ahi1/ahi1 orthologs had a conserved distribution pattern in human, mouse, and zebrafish, but mouse Ahi1 was not present in the developing and mature cerebellum. Ahi1 was also observed consistently in the stigmoid body, a poorly characterized cytoplasmic organelle found in neurons. Overall, these results suggest roles for AHI1 in neurodevelopmental processes that underlie most of the neuroanatomical defects in JBTS, and perhaps in neuronal functions that contribute to schizophrenia.

Forward genetic analysis of visual behavior in zebrafish.

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders.

Retinal network adaptation to bright light requires tyrosinase.

The visual system adjusts its sensitivity to a wide range of light intensities. We report here that mutation of the zebrafish sdy gene, which encodes tyrosinase, slows down the onset of adaptation to bright light. When fish larvae were challenged with periods of darkness during the day, the sdy mutants required nearly an hour to recover optokinetic behavior after return to bright light, whereas wild types recovered within minutes. This behavioral deficit was phenocopied in fully pigmented fish by inhibiting tyrosinase and thus does not depend on the absence of melanin pigment in sdy. Electroretinograms showed that the dark-adapted retinal network recovers sensitivity to a pulse of light more slowly in sdy mutants than in wild types. This failure is localized in the retinal neural network, postsynaptic to photoreceptors. We propose that retinal pigment epithelium (which normally expresses tyrosinase) secretes a modulatory factor, possibly L-DOPA, which regulates light adaptation in the retinal circuitry.