David B. Melville

The ER–Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis

Autophagy is a catabolic process for bulk degradation of cytosolic materials mediated by double-membraned autophagosomes. The membrane determinant to initiate the formation of autophagosomes remains elusive. Here, we establish a cell-free assay based on LC3 lipidation to define the organelle membrane supporting early autophagosome formation. In vitro LC3 lipidation requires energy and is subject to regulation by the pathways modulating autophagy in vivo. We developed a systematic membrane isolation scheme to identify the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) as a primary membrane source both necessary and sufficient to trigger LC3 lipidation in vitro. Functional studies demonstrate that the ERGIC is required for autophagosome biogenesis in vivo. Moreover, we find that the ERGIC acts by recruiting the early autophagosome marker ATG14, a critical step for the generation of preautophagosomal membranes. DOI: http://dx.doi.org/10.7554/eLife.00947.001

An SNP-Based Linkage Map for Zebrafish Reveals Sex Determination Loci

A surprising diversity of mechanisms controls sex determination of vertebrate organisms, even among closely related species. Both genetic and temperature-dependent systems of sex determination have been described in teleost fish. In the common zebrafish model organism, heteromorphic sex chromosomes are not observed, and the potential role of a genetic component of sex determination remains largely unknown. Here we report a genome-wide linkage study of sex determination in zebrafish using a novel SNP genetic map. We identified loci on zebrafish chromosomes 5 (LOD score 7.9) and 16 (LOD score 9.3) governing sex determination as a complex trait, rather than as an XY or ZW genetic system. Each of these loci contains a prominent candidate gene with a conserved role in sex determination across additional species that suggest potential mechanisms of sex determination in zebrafish. The chromosome 5 locus harbors dmrt1, a key gene in sex determination from fruit flies to humans; mutation of the human DMRT1 ortholog is a cause of complete sex reversal of XY individuals. The chromosome 16 locus harbors cyp21a2; mutation of the human CYP21A2 ortholog is one of the more common causes of pseudohermaphroditism. Mutation detection at each of these candidate genes within the zebrafish cross identified hypomorphic variants on the female-associated allele of each locus. The two loci together accounted for 16% of variance of the trait. Interacting environmental cues are likely to be an additional important component of sex determination in zebrafish.

Sec24D-Dependent Transport of Extracellular Matrix Proteins Is Required for Zebrafish Skeletal Morphogenesis

Protein transport from endoplasmic reticulum (ER) to Golgi is primarily conducted by coated vesicular carriers such as COPII. Here, we describe zebrafish bulldog mutations that disrupt the function of the cargo adaptor Sec24D, an integral component of the COPII complex. We show that Sec24D is essential for secretion of cartilage matrix proteins, whereas the preceding development of craniofacial primordia and pre-chondrogenic condensations does not depend on this isoform. Bulldog chondrocytes fail to secrete type II collagen and matrilin to extracellular matrix (ECM), but membrane bound receptor β1-Integrin and Cadherins appear to leave ER in Sec24D-independent fashion. Consequently, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes as visualized by electron microscopy and NBD C6-ceramide staining appear functional. Consistent with the backlog of proteins in the ER, chondrocytes activate the ER stress response machinery and significantly upregulate BiP transcription. Failure of ECM secretion hinders chondroblast intercalations thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. This defect is specific to Sec24D mutants since knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial cartilage dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than in bulldog/sec24d, suggesting that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. Our study presents the first developmental perspective on Sec24D function and establishes Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects.

Tfap2a and Foxd3 regulate early steps in the development of the neural crest progenitor population

The neural crest is a stem cell-like population exclusive to vertebrates that gives rise to many different cell types including chondrocytes, neurons and melanocytes. Arising from the neural plate border at the intersection of Wnt and Bmp signaling pathways, the complexity of neural crest gene regulatory networks has made the earliest steps of induction difficult to elucidate. Here, we report that tfap2a and foxd3 participate in neural crest induction and are necessary and sufficient for this process to proceed. Double mutant tfap2a (mont blanc, mob) and foxd3 (mother superior, mos) mob;mos zebrafish embryos completely lack all neural crest-derived tissues. Moreover, tfap2a and foxd3 are expressed during gastrulation prior to neural crest induction in distinct, complementary, domains; tfap2a is expressed in the ventral non-neural ectoderm and foxd3 in the dorsal mesendoderm and ectoderm. We further show that Bmp signaling is expanded in mob;mos embryos while expression of dkk1, a Wnt signaling inhibitor, is increased and canonical Wnt targets are suppressed. These changes in Bmp and Wnt signaling result in specific perturbations of neural crest induction rather than general defects in neural plate border or dorso-ventral patterning. foxd3 overexpression, on the other hand, enhances the ability of tfap2a to ectopically induce neural crest around the neural plate, overriding the normal neural plate border limit of the early neural crest territory. Although loss of either Tfap2a or Foxd3 alters Bmp and Wnt signaling patterns, only their combined inactivation sufficiently alters these signaling gradients to abort neural crest induction. Collectively, our results indicate that tfap2a and foxd3, in addition to their respective roles in the differentiation of neural crest derivatives, also jointly maintain the balance of Bmp and Wnt signaling in order to delineate the neural crest induction domain.

The feelgood mutation in zebrafish dysregulates COPII-dependent secretion of select extracellular matrix proteins in skeletal morphogenesis

SUMMARY Craniofacial and skeletal dysmorphologies account for the majority of birth defects. A number of the disease phenotypes have been attributed to abnormal synthesis, maintenance and composition of extracellular matrix (ECM), yet the molecular and cellular mechanisms causing these ECM defects remain poorly understood. The zebrafish feelgood mutant manifests a severely malformed head skeleton and shortened body length due to defects in the maturation stage of chondrocyte development. In vivo analyses reveal a backlog of type II and type IV collagens in rough endoplasmic reticulum (ER) similar to those found in coat protein II complex (COPII)-deficient cells. The feelgood mutation hinders collagen deposition in the ECM, but trafficking of small cargos and other large ECM proteins such as laminin to the extracellular space is unaffected. We demonstrate that the zebrafish feelgood mutation causes a single amino acid substitution within the DNA-binding domain of transcription factor Creb3l2. We show that Creb3l2 selectively regulates the expression of genes encoding distinct COPII proteins (sec23a, sec23b and sec24d) but find no evidence for its regulation of sec24c expression. Moreover, we did not detect activation of ER stress response genes despite intracellular accumulation of collagen and prominent skeletal defects. Promoter trans-activation assays show that the Creb3l2 feelgood variant is a hypomorphic allele that retains approximately 50% of its transcriptional activity. Transgenic rescue experiments of the feelgood phenotype restore craniofacial development, illustrating that a precise level of Creb3l2 transcriptional activity is essential for skeletogenesis. Our results indicate that Creb3l2 modulates the availability of COPII machinery in a tissue- and cargo-specific manner. These findings could lead to a better understanding of the etiology of human craniofacial and skeletal birth defects as well as adult-onset diseases that are linked to dysregulated ECM deposition, such as arthritis, fibrosis or osteoporosis.

Functional modeling in zebrafish demonstrates that the atrial-fibrillation-associated gene GREM2 regulates cardiac laterality, cardiomyocyte differentiation and atrial rhythm

SUMMARY Atrial fibrillation (AF) is the most common cardiac arrhythmia and carries a significant risk of stroke and heart failure. The molecular etiologies of AF are poorly understood, leaving patients with limited therapeutic options. AF has been recognized as an inherited disease in almost 30% of patient cases. However, few genetic loci have been identified and the mechanisms linking genetic variants to AF susceptibility remain unclear. By sequencing 193 probands with lone AF, we identified a Q76E variant within the coding sequence of the bone morphogenetic protein (BMP) antagonist gremlin-2 (GREM2) that increases its inhibitory activity. Functional modeling in zebrafish revealed that, through regulation of BMP signaling, GREM2 is required for cardiac laterality and atrial differentiation during embryonic development. GREM2 overactivity results in slower cardiac contraction rates in zebrafish, and induction of previously identified AF candidate genes encoding connexin-40, sarcolipin and atrial natriuretic peptide in differentiated mouse embryonic stem cells. By live heart imaging in zebrafish overexpressing wild-type or variant GREM2, we found abnormal contraction velocity specifically in atrial cardiomyocytes. These results implicate, for the first time, regulators of BMP signaling in human AF, providing mechanistic insights into the pathogenesis of the disease and identifying potential new therapeutic targets.

Animal model of Sar1b deficiency presents lipid absorption deficits similar to Anderson disease

Anderson disease (ANDD) or chylomicron retention disease (CMRD) is a rare, hereditary lipid malabsorption syndrome associated with mutations in the SAR1B gene that is characterized by failure to thrive and hypocholesterolemia. Although the SAR1B structure has been resolved and its role in formation of coat protein II (COPII)-coated carriers is well established, little is known about the requirement for SAR1B during embryogenesis. To address this question, we have developed a zebrafish model of Sar1b deficiency based on antisense oligonucleotide knockdown. We show that zebrafish sar1b is highly conserved among vertebrates; broadly expressed during development; and enriched in the digestive tract organs, brain, and craniofacial skeleton. Consistent with ANDD symptoms of chylomicron retention, we found that dietary lipids in Sar1b-deficient embryos accumulate in enterocytes. Transgenic expression analysis revealed that Sar1b is required for growth of exocrine pancreas and liver. Furthermore, we found abnormal differentiation and maturation of craniofacial cartilage associated with defects in procollagen II secretion and absence of select, neuroD-positive neurons of the midbrain and hindbrain. The model presented here will help to systematically dissect developmental roles of Sar1b and to discover molecular and cellular mechanisms leading to organ-specific ANDD pathology.Key messagesSar1b depletion phenotype in zebrafish resembles Anderson disease deficits.Sar1b deficiency results in multi-organ developmental deficits.Sar1b is required for dietary cholesterol uptake into enterocytes.

TANGO1 assembles into rings around COPII coats at ER exit sites

TANGO1 (transport and Golgi organization 1) interacts with CTAGE5 and COPII components Sec23/Sec24 and recruits ERGIC-53 (endoplasmic reticulum [ER]–Golgi intermediate compartment 53)–containing membranes to generate a mega-transport carrier for export of collagens and apolipoproteins from the ER. We now show that TANGO1, at the ER, assembles in a ring that encircles COPII components. The C-terminal, proline-rich domains of TANGO1 molecules in the ring are initially tilted onto COPII coats but appear to be pushed apart as the carrier grows. These findings lend support to our suggestion that growth of transport carriers for exporting bulky cargoes requires addition of membranes and not simply COPII-mediated accretion of a larger surface of ER. TANGO1 remains at the neck of the newly forming transport carrier, which grows in size by addition of ERGIC-53–containing membranes to generate a transport intermediate for the export of bulky collagens.

Trafficking mechanisms of extracellular matrix macromolecules: Insights from vertebrate development and human diseases

Cellular life depends on protein transport and membrane traffic. In multicellular organisms, membrane traffic is required for extracellular matrix deposition, cell adhesion, growth factor release, and receptor signaling, which are collectively required to integrate the development and physiology of tissues and organs. Understanding the regulatory mechanisms that govern cargo and membrane flow presents a prime challenge in cell biology. Extracellular matrix (ECM) secretion remains poorly understood, although given its essential roles in the regulation of cell migration, differentiation, and survival, ECM secretion mechanisms are likely to be tightly controlled. Recent studies in vertebrate model systems, from fishes to mammals and in human patients, have revealed complex and diverse loss-of-function phenotypes associated with mutations in components of the secretory machinery. A broad spectrum of diseases from skeletal and cardiovascular to neurological deficits have been linked to ECM trafficking. These discoveries have directly challenged the prevailing view of secretion as an essential but monolithic process. Here, we will discuss the latest findings on mechanisms of ECM trafficking in vertebrates.

Traffic jams in fish bones: ER-to-Golgi protein transport during zebrafish development.

Extracellular matrix (ECM) proteins, cell adhesion molecules, cytokines, morphogens and membrane receptors are synthesized in the ER and transported through the Golgi complex to the cell surface and the extracellular space. The first leg in this journey from the ER to Golgi is facilitated by the Coat Protein II (COPII) vesicular carriers. Genetic defects in genes encoding various COPII components cause a broad spectrum of human diseases, from anemia to skeletal deformities. Here, we summarize our findings in zebrafish and discuss how mutations in COPII elements may cause specific cellular and developmental defects.