Collagen IV of basement membranes: II. Emergence of collagen IVα345 enabled the assembly of a compact GBM as an ultrafilter in mammalian kidneys.

The collagen IVα345 (Col-IVα345) scaffold, the major constituent of the glomerular basement membrane (GBM), is a critical component of the kidney glomerular filtration barrier. In Alport syndrome, affecting millions of people worldwide, over two thousand genetic variants occur in the COL4A3, COL4A4, and COL4A5 genes that encode the Col-IVα345 scaffold. Variants cause loss of scaffold, a suprastructure that tethers macromolecules, from the GBM or assembly of a defective scaffold, causing hematuria in nearly all cases, proteinuria, and often progressive kidney failure. How these variants cause proteinuria remains an enigma. In a companion paper, we found that the evolutionary emergence of the COL4A3, COL4A4, COL4A5, and COL4A6 genes coincided with kidney emergence in hagfish and shark and that the COL4A3 and COL4A4 were lost in amphibians. These findings opened an experimental window to gain insights into functionality of the Col-IVα345 scaffold. Here, using tissue staining, biochemical analysis and TEM, we characterized the scaffold chain arrangements and the morphology of the GBM of hagfish, shark, frog, and salamander. We found that α4 and α5 chains in shark GBM and α1 and α5 chains in amphibian GBM are spatially separated. Scaffolds are distinct from one another and from the mammalian Col-IVα345 scaffold, and the GBM morphologies are distinct. Our findings revealed that the evolutionary emergence of the Col-IVα345 scaffold enabled the genesis of a compact GBM that functions as an ultrafilter. Findings shed light on the conundrum, defined decades ago, whether the GBM or slit diaphragm is the primary filter.

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.

Overnutrition induces ß-cell differentiation through prolonged activation of ß-cells in zebrafish larvae.

Insulin from islet ß-cells maintains glucose homeostasis by stimulating peripheral tissues to remove glucose from circulation. Persistent elevation of insulin demand increases ß-cell number through self-replication or differentiation (neogenesis) as part of a compensatory response. However, it is not well understood how a persistent increase in insulin demand is detected. We have previously demonstrated that a persistent increase in insulin demand by overnutrition induces compensatory ß-cell differentiation in zebrafish. Here, we use a series of pharmacological and genetic analyses to show that prolonged stimulation of existing ß-cells is necessary and sufficient for this compensatory response. In the absence of feeding, tonic, but not intermittent, pharmacological activation of ß-cell secretion was sufficient to induce ß-cell differentiation. Conversely, drugs that block ß-cell secretion, including an ATP-sensitive potassium (K ATP) channel agonist and an L-type Ca(2+) channel blocker, suppressed overnutrition-induced ß-cell differentiation. Genetic experiments specifically targeting ß-cells confirm existing ß-cells as the overnutrition sensor. First, inducible expression of a constitutively active K ATP channel in ß-cells suppressed the overnutrition effect. Second, inducible expression of a dominant-negative K ATP mutant induced ß-cell differentiation independent of nutrients. Third, sensitizing ß-cell metabolism by transgenic expression of a hyperactive glucokinase potentiated differentiation. Finally, ablation of the existing ß-cells abolished the differentiation response. Taken together, these data establish that overnutrition induces ß-cell differentiation in larval zebrafish through prolonged activation of ß-cells. These findings demonstrate an essential role for existing ß-cells in sensing overnutrition and compensating for their own insufficiency by recruiting additional ß-cells.

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.

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.

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.

Macrophages and neutrophils are necessary for ER stress-induced ß cell loss.

Persistent endoplasmic reticulum (ER) stress induces islet inflammation and ß cell loss. How islet inflammation contributes to ß cell loss remains uncertain. We have reported previously that chronic overnutrition-induced ER stress in ß cells causes Ripk3-mediated islet inflammation, macrophage recruitment, and a reduction of ß cell numbers in a zebrafish model. We show here that ß cell loss results from the intricate communications among ß cells, macrophages, and neutrophils. Macrophage-derived Tnfa induces cxcl8a in ß cells. Cxcl8a, in turn, attracts neutrophils to macrophage-contacted "hotspots" where ß cell loss occurs. We also show potentiation of chemokine expression in stressed mammalian ß cells by macrophage-derived TNFA. In Akita and db/db mice, there is an increase in CXCL15-positive ß cells and intra-islet neutrophils. Blocking neutrophil recruitment in Akita mice preserves ß cell mass and slows diabetes progression. These results reveal an important role of neutrophils in persistent ER stress-induced ß cell loss.

Improved Growth and High Inheritance of Melanocortin-4 Receptor (mc4r) Mutation in CRISPR/Cas-9 Gene-Edited Channel Catfish, Ictalurus punctatus.

Effects of CRISPR/Cas9 knockout of the melanocortin-4 receptor (mc4r) gene in channel catfish, Ictalurus punctatus, were investigated. Three sgRNAs targeting the channel catfish mc4r gene in conjunction with Cas9 protein were microinjected in embryos and mutation rate, inheritance, and growth were studied. Efficient mutagenesis was achieved as demonstrated by PCR, Surveyor® assay, and DNA sequencing. An overall mutation rate of 33% and 33% homozygosity/bi-allelism was achieved in 2017. Approximately 71% of progeny inherited the mutation. Growth was generally higher in MC4R mutants than controls (CNTRL) at all life stages and in both pond and tank environments. There was a positive relationship between zygosity and growth, with F1 homozygous/bi-allelic mutants reaching market size 30% faster than F1 heterozygotes in earthen ponds (p = 0.022). At the stocker stage (~ 50 g), MC4R × MC4R mutants generated in 2019 were 40% larger than the mean of combined CNTRL × CNTRL families (p = 0.005) and 54% larger than F1 MC4R × CNTRL mutants (p = 0.001) indicating mutation may be recessive. With a high mutation rate and inheritance of the mutation as well as improved growth, the use of gene-edited MC4R channel catfish appears to be beneficial for application on commercial farms.

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.

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.

Leptin system loss of function in the absence of obesity in zebrafish.

The leptin system plays a crucial role in the regulation of appetite and energy homeostasis in vertebrates. While the phenotype of morbid obesity due to leptin (Lep) or leptin receptor (LEPR) loss of function is well established in mammals, evidence in fish is controversial, questioning the role of leptin as the vertebrate adipostat. Here we report on three (Lepr) loss of function (LOF) and one leptin loss of function alleles in zebrafish. In order to demonstrate that the Lepr LOF alleles cannot transduce a leptin signal, we measured socs3a transcription after i.p. leptin which is abolished by Lepr LOF. None of the Lepr/Lepa LOF alleles leads to obesity/a body growth phenotype. We explore possible reasons leading to the difference in published results and find that even slight changes in background genetics such as inbreeding siblings and cousins can lead to significant variance in growth.

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.

Structural characterization of glycosaminoglycans from zebrafish in different ages.

The zebrafish (Danio rerio) is a popular model organism for the study of developmental biology, disease mechanisms, and drug discovery. Glycosaminoglycans (GAGs), located on animal cell membranes and in the extracellular matrix, are important molecules in cellular communication during development, in normal physiology and pathophysiology. Vertebrates commonly contain a variety of GAGs including chondroitin/dermatan sulfates, heparin/heparan sulfate, hyaluronan and keratan sulfate. Zebrafish might represent an excellent experimental organism to study the biological roles of GAGs. A recent study showing the absence of heparan sulfate in adult zebrafish, suggested a more detailed evaluation of the GAGs present in this important model organism needed to be undertaken. This report aimed at examining the structural alterations of different GAGs at the molecular level at different developmental stages. GAGs were isolated and purified from zebrafish in different stages in development ranging from 0.5 days to adult. The content and disaccharide composition of chondroitin sulfate and heparan sulfate were determined using chemical assays, liquid chromotography and mass spectrometry. The presence of HS in adult fish was also confirmed using (1)H-NMR.

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.

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.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing.

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Role of Hypohalous Acids in Basement Membrane Homeostasis.

SIGNIFICANCE: Basement membranes (BMs) are sheet-like structures of specialized extracellular matrix that underlie nearly all tissue cell layers including epithelial, endothelial, and muscle cells. BMs not only provide structural support but are also critical for the development, maintenance, and repair of organs. Animal heme peroxidases generate highly reactive hypohalous acids extracellularly and, therefore, target BMs for oxidative modification. Given the importance of BMs in tissue structure and function, hypohalous acid-mediated oxidative modifications of BM proteins represent a key mechanism in normal development and pathogenesis of disease. Recent Advances: Peroxidasin (PXDN), a BM-associated animal heme peroxidase, generates hypobromous acid (HOBr) to form sulfilimine cross-links within the collagen IV network of BM. These cross-links stabilize BM and are critical for animal tissue development. These findings highlight a paradoxical anabolic role for HOBr, which typically damages protein structure leading to dysfunction. CRITICAL ISSUES: The molecular mechanism whereby PXDN uses HOBr as a reactive intermediate to cross-link collagen IV, yet avoid collateral damage to nearby BM proteins, remains unclear. FUTURE DIRECTIONS: The exact identification and functional impact of specific hypohalous acid-mediated modifications of BM proteins need to be addressed to connect these modifications to tissue development and pathogenesis of disease. As seen with the sulfilimine cross-link of collagen IV, hypohalous acid oxidative events may be beneficial in select situations rather than uniformly deleterious. Antioxid. Redox Signal. 27, 839-854.