Depletion Of Target Proteins Research Articles

Rapid depletion of target proteins in plants by an inducible protein degradation system.

Inducible protein knockdowns are excellent tools to test the function of essential proteins in short time scales and to capture the role of proteins in dynamic events. Current approaches destroy or sequester proteins by exploiting plant biological mechanisms such as the activity of photoreceptors for optogenetics or auxin-mediated ubiquitination in auxin degrons. It follows that these are not applicable for plants as light and auxin are strong signals for plant cells. We describe here an inducible protein degradation system in plants named E3-DART for E3-targeted Degradation of Plant Proteins. The E3-DART system is based on the specific and well-characterized interaction between the Salmonella-secreted protein H1 (SspH1) and its human target protein kinase N1 (PKN1). This system harnesses the E3 catalytic activity of SspH1 and the SspH1-binding activity of the homology region 1b (HR1b) domain from PKN1. Using Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana), we show that a chimeric protein containing the leucine-rich repeat and novel E3 ligase domains of SspH1 efficiently targets protein fusions of varying sizes containing HR1b for degradation. Target protein degradation was induced by transcriptional control of the chimeric E3 ligase using a glucocorticoid transactivation system, and target protein depletion was detected as early as 3 h after induction. This system could be used to study the loss of any plant protein with high-temporal resolution and may become an important tool in plant cell biology.

ATRT-14. ASSESSMENT OF MITOCHONDRIAL BIOENERGETICS AND FISSION-FUSION DYNAMICS IN ETMR

Abstract BACKGROUND Embryonal tumor with multilayered rosettes (ETMR) is a deadly pediatric brain tumor with limited available therapeutic options. Dysregulated mitochondrial bioenergetics and dynamics have been linked to the initiation and progression of diverse human cancers, however, their role in ETMR pathobiology remains unknown. We used the primary ETMR cell line, BT183, and an in-house developed human neural stem cell (NSC) line to evaluate mitochondrial morphology, bioenergetics and fission-fusion dynamics involved in ETMR growth and aggression. METHODS Mitochondrial morphology was determined by widefield immunofluorescence and dSTORM super-resolution microscopy in BT183 and NSC monolayers and by TEM in BT183 and NSCs grown as neurospheres. Relative expression of mitochondrial proteins were quantified in BT183 and NSC lysates by western blotting. RESULTS Widefield immunofluorescence and super-resolution imaging revealed profound differences in mitochondrial morphology in BT183 compared to NSCs. BT183 mitochondria appear as fragmented, rounded structures, with little interconnectivity, while NSC mitochondria present a typical elongated, filamentous morphology. These findings were corroborated by TEM image analysis of BT183 and NSC neurospheres, which revealed additional morphological alterations in BT183 mitochondria at the ultrastructural level, including aberrant cristae structure. Western blot analysis of BT183 and NSC lysates showed up to 5-fold differential expression of mitochondrial OXPHOS, glycolytic and fission-fusion proteins in the two cell types. Fission promoters phospho-Drp1 and MFF displayed significantly higher expression in BT183 relative to NSCs, whereas fusion-inducing proteins Opa1 and Miro2 showed significantly lower differential expression in BT183. CONCLUSIONS BT183 cells exhibited striking mitochondrial fragmentation and differential expression of proteins involved in fission-fusion, OXPHOS and glycolysis when compared to normal human NSCs. Ongoing studies include the analysis of mitochondrial structure and morphology in patient samples to validate our in-vitro findings, in addition to in-vitro metabolic flux analysis and targeted protein depletion studies to identify druggable pathways based on mitochondrial function and dynamics.

ATRT-13. ASSESSMENT OF MITOCHONDRIAL BIOENERGETICS AND FISSION-FUSION DYNAMICS IN ETMR

Abstract BACKGROUND Embryonal tumor with multilayered rosettes (ETMR) is a deadly pediatric brain tumor with limited available therapeutic options. Dysregulated mitochondrial bioenergetics and dynamics have been linked to the initiation and progression of diverse human cancers, however, their role in ETMR pathobiology remains unknown. We used the primary ETMR cell line, BT183, and an in-house developed human neural stem cell (NSC) line to evaluate mitochondrial morphology, bioenergetics and fission-fusion dynamics involved in ETMR growth and aggression. METHODS Mitochondrial morphology was determined by widefield immunofluorescence and dSTORM super-resolution microscopy in BT183 and NSC monolayers and by TEM in BT183 and NSCs grown as neurospheres. Relative expression of mitochondrial proteins were quantified in BT183 and NSC lysates by western blotting. RESULTS Widefield immunofluorescence and super-resolution imaging revealed profound differences in mitochondrial morphology in BT183 compared to NSCs. BT183 mitochondria appear as fragmented, rounded structures, with little interconnectivity, while NSC mitochondria present a typical elongated, filamentous morphology. These findings were corroborated by TEM image analysis of BT183 and NSC neurospheres, which revealed additional morphological alterations in BT183 mitochondria at the ultrastructural level, including aberrant cristae structure. Western blot analysis of BT183 and NSC lysates showed up to 5-fold differential expression of mitochondrial OXPHOS, glycolytic and fission-fusion proteins in the two cell types. Fission promoters phospho-Drp1 and MFF displayed significantly higher expression in BT183 relative to NSCs, whereas fusion-inducing proteins Opa1 and Miro2 showed significantly lower differential expression in BT183. CONCLUSIONS BT183 cells exhibited striking mitochondrial fragmentation and differential expression of proteins involved in fission-fusion, OXPHOS and glycolysis when compared to normal human NSCs. Ongoing studies include the analysis of mitochondrial structure and morphology in patient samples to validate our in-vitro findings, in addition to in-vitro metabolic flux analysis and targeted protein depletion studies to identify druggable pathways based on mitochondrial function and dynamics.

Targeted Protein Depletion Using the Auxin-Inducible Degron 2 (AID2) System.

Targeted protein depletion using a conditional degron is a powerful method to probe the role of proteins in living cells because of the speed with which depletion can be induced and its reversibility. The auxin-inducible degron (AID) is one of the most common degron-based technologies used in cell biology. We recently established an improved system, called AID2, which involves expressing a mutant E3 ligase subunit, OsTIR1(F74G), and fusing a protein of interest to the mini-AID (mAID) tag, and that employs a new and more potent ligand, 5-phenyl-indole-3-acetic acid (5-Ph-IAA). The AID2 system overcomes some of the drawbacks associated with the original AID system, i.e., leaky degradation without auxin and the requirement of high auxin doses. With AID2 it is, therefore, now possible to control a degron-fused protein more precisely, enabling target proteins to be degraded with a half-life of 10 to 45 min via the addition of a low dose of 5-Ph-IAA. Importantly, in AID2, it is not necessary to control the expression of OsTIR1(F74G) for suppressing leaky degradation and a parental cell line constitutively expressing OsTIR1(F74G) can be used for the generation of multiple mAID-tagged proteins. Here, we describe a protocol for the tagging of endogenous proteins with mAID in diploid HCT116 cells. Our protocol can be applied to other mammalian cell lines and will enhance the utility of AID2 for studying protein functions in living cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Generation of a parental HCT116 cell line expressing OsTIR1(F74G) Basic Protocol 2: Construction of CRISPR and donor plasmids for tagging endogenous genes Basic Protocol 3: Generation of cell lines expressing a protein of interest fused with mAID.

Methods for Rapid Protein Depletion in C. elegans using Auxin-Inducible Degradation.

Numerous methods have been developed in model systems to deplete or inactivate proteins to elucidate their functional roles. In Caenorhabditis elegans, a common method for protein depletion is RNA interference (RNAi), in which mRNA is targeted for degradation. C. elegans is also a powerful genetic organism, amenable to large-scale genetic screens and CRISPR-mediated genome editing. However, these approaches largely lead to constitutive inhibition, which can make it difficult to study proteins essential for development or to dissect dynamic cellular processes. Thus, there have been recent efforts to develop methods to rapidly inactivate or deplete proteins to overcome these barriers. One such method that is proving to be exceptionally powerful is auxin-inducible degradation. In order to apply this approach in C. elegans, a 44-amino acid degron tag is added to the protein of interest, and the Arabidopsis ubiquitin ligase TIR1 is expressed in target tissues. When the plant hormone auxin is added, it mediates an interaction between TIR1 and the degron-tagged protein of interest, which triggers ubiquitination of the protein and its rapid degradation via the proteasome. Here, we have outlined multiple methods for inducing auxin-mediated depletion of target proteins in C. elegans, highlighting the versatility and power of this method. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Long-term auxin-mediated depletion on plates Support Protocol: Preparation of NGM and NGM-auxin plates Basic Protocol 2: Rapid auxin-mediated depletion via soaking Basic Protocol 3: Acute auxin-mediated depletion in isolated embryos Basic Protocol 4: Assessing auxin-mediated depletion.

A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair

The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.

Identification of Host Trafficking Genes Required for HIV-1 Virological Synapse Formation in Dendritic Cells.

Dendritic cells (DCs) are one of the earliest targets of HIV-1 infection acting as a "Trojan horse," concealing the virus from the innate immune system and delivering it to T cells via virological synapses (VS). To explicate how the virus is trafficked through the cell to the VS and evades degradation, a high-throughput small interfering RNA screen targeting membrane trafficking proteins was performed in monocyte-derived DCs. We identified several proteins including BIN-1 and RAB7L1 that share common roles in transport from endosomal compartments. Depletion of target proteins resulted in an accumulation of virus in intracellular compartments and significantly reduced viral trans-infection via the VS. By targeting endocytic trafficking and retromer recycling to the plasma membrane, we were able to reduce the virus's ability to accumulate at budding microdomains and the VS. Thus, we identify key genes involved in a pathway within DCs that is exploited by HIV-1 to traffic to the VS.IMPORTANCE The lentivirus human immunodeficiency virus (HIV) targets and destroys CD4+ T cells, leaving the host vulnerable to life-threatening opportunistic infections associated with AIDS. Dendritic cells (DCs) form a virological synapse (VS) with CD4+ T cells, enabling the efficient transfer of virus between the two cells. We have identified cellular factors that are critical in the induction of the VS. We show that ADP-ribosylation factor 1 (ARF1), bridging integrator 1 (BIN1), and Rab GTPases RAB7L1 and RAB8A are important regulators of HIV-1 trafficking to the VS and therefore the infection of CD4+ T cells. We found these cellular factors were essential for endosomal protein trafficking and formation of the VS and that depletion of target proteins prevented virus trafficking to the plasma membrane by retaining virus in intracellular vesicles. Identification of key regulators in HIV-1 trans-infection between DC and CD4+ T cells has the potential for the development of targeted therapy to reduce trans-infection of HIV-1 in vivo.

Tunable light and drug induced depletion of target proteins

Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development. These independent tools can be combined for spatial and temporal depletion of different sets of proteins, can help to distinguish immediate cellular responses from long-term adaptation effects and can facilitate the exploration of complex networks.

A Xylose-Inducible Expression System and a CRISPR Interference Plasmid for Targeted Knockdown of Gene Expression in Clostridioides difficile.

Here we introduce plasmids for xylose-regulated expression and repression of genes in Clostridioides difficile The xylose-inducible expression vector allows for ∼100-fold induction of an mCherryOpt reporter gene. Induction is titratable and uniform from cell to cell. The gene repression plasmid is a CRISPR interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of the Streptococcus pyogenes Cas9 protein (dCas9) that is targeted to a gene of interest by a constitutively expressed single guide RNA (sgRNA). Expression of dCas9 is induced by xylose, allowing investigators to control the timing and extent of gene silencing, as demonstrated here by dose-dependent repression of a chromosomal gene for a red fluorescent protein (maximum repression, ∼100-fold). To validate the utility of CRISPRi for deciphering gene function in C. difficile, we knocked down the expression of three genes involved in the biogenesis of the cell envelope: the cell division gene ftsZ, the S-layer protein gene slpA, and the peptidoglycan synthase gene pbp-0712 CRISPRi confirmed known or expected phenotypes associated with the loss of FtsZ and SlpA and revealed that the previously uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell division, and protection against lysis.IMPORTANCEClostridioides difficile has become the leading cause of hospital-acquired diarrhea in developed countries. A better understanding of the basic biology of this devastating pathogen might lead to novel approaches for preventing or treating C. difficile infections. Here we introduce new plasmid vectors that allow for titratable induction (P xyl ) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins in C. difficile Besides bypassing the lengthy process of mutant construction, CRISPRi can be used to study the function of essential genes, which are particularly important targets for antibiotic development.

A fast and tuneable auxin-inducible degron for depletion of target proteins in budding yeast.

The auxin‐inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in nonplant eukaryotes. Depletion is achieved by addition of the plant hormone auxin to the cell culture, which allows the auxin‐binding receptor, TIR1, to target the AID‐tagged protein for degradation by the proteasome. Fast depletion of the target protein requires good expression of TIR1 protein, but as we show here, high levels of TIR1 may cause uncontrolled depletion of the target protein in the absence of auxin. To enable conditional expression of TIR1 to a high level when required, we regulated the expression of TIR1 using the β‐estradiol expression system. This is a fast‐acting gene induction system that does not cause secondary effects on yeast cell metabolism. We demonstrate that combining the AID and β‐estradiol systems results in a tightly controlled and fast auxin‐induced depletion of nuclear target proteins. Moreover, we show that depletion rate can be tuned by modulating the duration of β‐estradiol preincubation. We conclude that TIR1 protein is a rate‐limiting factor for target protein depletion in yeast, and we provide new tools that allow tightly controlled, tuneable, and efficient depletion of essential proteins whereas minimising secondary effects.

The auxin-inducible degradation system enables conditional PERIOD protein depletion in the nervous system of Drosophila melanogaster.

Tools that allow inducible and reversible depletion of target proteins are critical for biological studies. The plant-derived auxin-inducible degradation system (AID) enables the degradation of target proteins tagged with the AID motif. This system has been recently employed in mammalian cells as well as in Caenorhabditis elegans and Drosophila. To test the utility of the AID approach in the nervous system, we used circadian locomotor rhythms as a model and applied the AID method to temporally and spatially degrade PERIOD (PER), a critical pacemaker protein in Drosophila. We found that the period locus can be efficiently tagged with the AID motif by CRISPR/Cas9-based genome editing without disrupting PER function. Moreover, we demonstrated that the AID system could be used to induce rapid and efficient protein degradation in the nervous system as shown by effects on circadian and sleep behaviors. Furthermore, the protein degradation by AID was rapidly reversible after auxin removal. Together, our results show that the AID system provides a powerful tool for behavior studies in Drosophila.

Design and Functional Characterization of Synthetic E3 Ubiquitin Ligases for Targeted Protein Depletion.

A number of techniques now exist for decreasing the expression of cellular proteins without the need for genomic modification. One such technique involves engineered protein chimeras that combine the ubiquitination activity of E3 ubiquitin ligases with the binding affinity and substrate specificity of designer binding proteins (DBPs). These chimeras, called "ubiquibodies," are capable of selectively and controllably steering virtually any protein to the ubiquitin proteasome pathway (UPP) for degradation, making ubiquibodies a powerful addition to the protein knockout toolbox. A distinguishing feature of ubiquibodies is their modularity-simply swapping DBPs can generate a new ubiquibody with specificity for a different substrate protein. Moreover, by employing DBPs that recognize particular protein states (e.g., active versus inactive conformation, mutant versus wild-type, post-translational modification), it becomes possible to deplete certain protein subpopulations while sparing others. This protocol outlines the steps necessary to design and functionally evaluate ubiquibodies for customizable silencing of cellular proteins. © 2018 by John Wiley & Sons, Inc.

Simultaneous depletion of albumin and immunoglobulin G by using twin affinity magnetic nanotraps

ABSTRACTFor biomarker discovery, simultaneous removal of albumin and immunoglobulin G (IgG) become more important for both concentrating other species including potential biomarkers and getting rid of their masking effect. In this study, we have proposed a covalent and photosensitive cross-linking conjugation of the biomolecules on nanostructures for the depletion of target proteins from aqueous solution and serum. The effect of concentration, pH, temperature and ionic strength on the adsorption of twin affinity nanotraps based on Cibacron Blue F3GA and Protein A were investigated. The efficiency of albumin and IgG depletion from human serum was performed using sodium dodecylsulfate–polyacrylamide gel electrophoresis.

An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

Sec62 as a novel gating modulator for protein translocation and ion permeability of the mammalian Sec61 complex (LB196)

In mammalian cells the endoplasmic reticulum (ER) plays a key role during the biogenesis of secretory and membrane proteins and acts as a major regulator for intracellular Ca2+ signaling. Sec62, a component of the ER‐protein translocase, acts during post‐translational translocation of small precursor proteins and in ER‐Ca2+ leakage‐control. Our project aims to elucidate the molecular mechanisms how it regulates the translocation machinery concerning these two fundamental and seemingly opposing functions. We used siRNA mediated depletion of target proteins from human cells followed by functional assays for protein transport and live cell calcium imaging.Depletion of Sec62 in combination with proteasomal inhibition led to accumulation of the precursor of the ER resident protein ERj3 at the cytosolic face of the ER membrane. Control experiments using different siRNA as well as a plasmid rescue approach confirmed the specificity for Sec62. Follow‐up experiments revealed a pattern of dependencies upon translocon components which is characteristic for a post‐translational translocation pathway.Decrease of Sec62 level led to an increased ER calcium leakage that could be attributed to Sec61α. We observed that Sec62 binds the N‐terminus of Sec61α in a Ca2+ sensitive manner and identified a crucial putative EF‐hand in the C‐terminal part of Sec62.These experiments demonstrated for the first time that human Sec62 is important for the early steps of translocation of a large precursor protein indicating that Sec62 participation is not restricted to small proteins as suggested in previous studies.Additionally, Sec62 acts as a modulator, shifting the translocon from the open to the closed state after translocation is completed in order to maintain the ion impermeability of the ER membrane.Further experiments will allow us to determine the recently proposed role of Sec62 for various cancer entities.

Rapid Depletion of Target Proteins Allows Identification of Coincident Physiological Responses

Targeted protein degradation is a powerful tool that can be used to create unique physiologies depleted of important factors. Current strategies involve modifying a gene of interest such that a degradation peptide is added to an expressed target protein and then conditionally activating proteolysis, either by expressing adapters, unmasking cryptic recognition determinants, or regulating protease affinities using small molecules. For each target, substantial optimization may be required to achieve a practical depletion, in that the target remains present at a normal level prior to induction and is then rapidly depleted to levels low enough to manifest a physiological response. Here, we describe a simplified targeted degradation system that rapidly depletes targets and that can be applied to a wide variety of proteins without optimizing target protease affinities. The depletion of the target is rapid enough that a primary physiological response manifests that is related to the function of the target. Using ribosomal protein S1 as an example, we show that the rapid depletion of this essential translation factor invokes concomitant changes to the levels of several mRNAs, even before appreciable cell division has occurred.

Quantitative Analysis of Survivin Protein Expression and Its Therapeutic Depletion by an Antisense Oligonucleotide in Human Lung Tumors

RNA-directed antisense and interference therapeutics are a promising treatment option for cancer. The demonstration of depletion of target proteins within human tumors in vivo using validated methodology will be a key to the application of this technology. Here, we present a flow cytometric-based approach to quantitatively determine protein levels in solid tumor material derived by fiber optic brushing (FOB) of non-small cell lung cancer (NSCLC) patients. Focusing upon the survivin protein, and its depletion by an antisense oligonucleotide (ASO) (LY2181308), we show that we can robustly identify a subpopulation of survivin positive tumor cells in FOB samples, and, moreover, detect survivin depletion in tumor samples from a patient treated with LY2181308. Survivin depletion appears to be a result of treatment with this ASO, because a tumor treated with conventional cytotoxic chemotherapy did not exhibit a decreased percentage of survivin positive cells. Our approach is likely to be broadly applicable to, and useful for, the quantification of protein levels in tumor samples obtained as part of clinical trials and studies, facilitating the proof-of-principle testing of novel targeted therapies.

Targeted protein depletion in Saccharomyces cerevisiae by activation of a bidirectional degron

BackgroundTools for in vivo manipulation of protein abundance or activity are highly beneficial for life science research. Protein stability can be efficiently controlled by conditional degrons, which induce target protein degradation at restrictive conditions.ResultsWe used the yeast Saccharomyces cerevisiae for development of a conditional, bidirectional degron to control protein stability, which can be fused to the target protein N-terminally, C-terminally or placed internally. Activation of the degron is achieved by cleavage with the tobacco etch virus (TEV) protease, resulting in quick proteolysis of the target protein. We found similar degradation rates of soluble substrates using destabilization by the N- or C-degron. C-terminal tagging of essential yeast proteins with the bidirectional degron resulted in deletion-like phenotypes at non-permissive conditions. Developmental process-specific mutants were created by N- or C-terminal tagging of essential proteins with the bidirectional degron in combination with sporulation-specific production of the TEV protease.ConclusionsWe developed a system to influence protein abundance and activity genetically, which can be used to create conditional mutants, to regulate the fate of single protein domains or to design artificial regulatory circuits. Thus, this method enhances the toolbox to manipulate proteins in systems biology approaches considerably.

A central function for perlecan in skeletal muscle and cardiovascular development

Perlecan's developmental functions are difficult to dissect in placental animals because perlecan disruption is embryonic lethal. In contrast to mammals, cardiovascular function is not essential for early zebrafish development because the embryos obtain adequate oxygen by diffusion. In this study, we use targeted protein depletion coupled with protein-based rescue experiments to investigate the involvement of perlecan and its C-terminal domain V/endorepellin in zebrafish development. The perlecan morphants show a severe myopathy characterized by abnormal actin filament orientation and disorganized sarcomeres, suggesting an involvement of perlecan in myopathies. In the perlecan morphants, primary intersegmental vessel sprouts, which develop through angiogenesis, fail to extend and show reduced protrusive activity. Live videomicroscopy confirms the abnormal swimming pattern caused by the myopathy and anomalous head and trunk vessel circulation. The phenotype is partially rescued by microinjection of human perlecan or endorepellin. These findings indicate that perlecan is essential for the integrity of somitic muscle and developmental angiogenesis and that endorepellin mediates most of these biological activities.

Zebrafish acvr2a and acvr2b exhibit distinct roles in craniofacial development

To examine the roles of activin type II receptor signaling in craniofacial development, full-length zebrafish acvr2a and acvr2b clones were isolated. Although ubiquitously expressed as maternal mRNAs and in early embryogenesis, by 24 hr postfertilization (hpf), acvr2a and acvr2b exhibit restricted expression in neural, hindbrain, and neural crest cells (NCCs). A morpholino-based targeted protein depletion approach was used to reveal discrete functions for each acvr2 gene product. The acvr2a morphants exhibited defects in the development of most cranial NCC-derived cartilage, bone, and pharyngeal tooth structures, whereas acvr2b morphant defects were largely restricted to posterior arch structures and included the absence and/or aberrant migration of posterior NCC streams, defects in NCC-derived posterior arch cartilages, and dysmorphic pharyngeal tooth development. These studies revealed previously uncharacterized roles for acvr2a and acvr2b in hindbrain and NCC patterning, in NCC derived pharyngeal arch cartilage and joint formation, and in tooth development.