Ela W. Knapik

Neural crest survival and differentiation in zebrafish depends on mont blanc/tfap2a gene function.

Neural crest progenitor cells are the main contributors to craniofacial cartilage and connective tissue of the vertebrate head. These progenitor cells also give rise to the pigment, neuronal and glial cell lineages. To study the molecular basis of neural crest differentiation, we have cloned the gene disrupted in the mont blanc (mobm610) mutation, which affects all neural crest derivatives. Using a positional candidate cloning approach we identified an A to G transition within the 3′ splice site of the sixth intron of the tfap2a gene that abolishes the last exon encoding the crucial protein dimerization and DNA-binding domains. Neural crest induction and specification are not hindered in mobm610 mutant embryos, as revealed by normal expression of early neural crest specific genes such as snail2, foxd3 and sox10. In addition, the initial stages of cranial neural crest migration appear undisturbed, while at a later phase the craniofacial primordia in pharyngeal arches two to seven fail to express their typical set of genes (sox9a, wnt5a, dlx2, hoxa2/b2). In mobm610 mutant embryos, the cell number of neuronal and glial derivatives of neural crest is greatly reduced, suggesting that tfap2a is required for their normal development. By tracing the fate of neural crest progenitors in live mont blanc (mobm610) embryos, we found that at 24 hpf neural crest cells migrate normally in the first pharyngeal arch while the preotic and postotic neural crest cells begin migration but fail to descend to the pharyngeal region of the head. TUNEL assay and Acridine Orange staining revealed that in the absence of tfap2a a subset of neural crest cells are unable to undergo terminal differentiation and die by apoptosis. Furthermore, surviving neural crest cells in tfap2a/mobm610 mutant embryos proliferate normally and later differentiate to individual derivatives. Our results indicate that tfap2a is essential to turn on the normal developmental program in arches 2-7 and in trunk neural crest. Thus, tfap2a does not appear to be involved in early specification and cell proliferation of neural crest, but it is a key regulator of an early differentiation phase and is required for cell survival in neural crest derived cell lineages.

Secretory COPII coat component Sec23a is essential for craniofacial chondrocyte maturation

An increasing number of human disorders have been linked to mutations in genes of the secretory pathway. The chemically induced zebrafish crusher variant results in malformed craniofacial skeleton, kinked pectoral fins and a short body length. By positional cloning, we identified a nonsense mutation converting leucine to a stop codon (L402X) in the sec23a gene, an integral component of the COPII complex, which is critical for anterograde protein trafficking between endoplasmic reticulum and Golgi apparatus. Zebrafish crusher mutants develop normally until the onset of craniofacial chondrogenesis. crusher chondrocytes accumulate proteins in a distended endoplasmic reticulum, resulting in severe reduction of cartilage extracellular matrix (ECM) deposits, including type II collagen. We demonstrate that the paralogous gene sec23b is also an essential component of the ECM secretory pathway in chondrocytes. In contrast, knockdown of the COPI complex does not hinder craniofacial morphogenesis. As SEC23A lesions cause the cranio-lenticulo-sutural dysplasia syndrome, crusher provides the first vertebrate model system that links the biology of endoplasmic reticulum to Golgi trafficking with a clinically relevant dysmorphology.

Noradrenergic neurons in the zebrafish hindbrain are induced by retinoic acid and require tfap2a for expression of the neurotransmitter phenotype

Tfap2a is a transcriptional activator expressed in many different cell types, including neurons, neural crest derivatives and epidermis. We show that mutations at the zebrafish locus previously called mont blanc (mob) or lockjaw (low) encode tfap2a. The mutant phenotype reveals that tfap2a is essential for the development of hindbrain noradrenergic (NA) neurons of the locus coeruleus, medulla and area postrema, as well as for sympathetic NA neurons, epibranchial placode derived visceral sensory ganglia, and craniofacial and trunk crest derivatives. We focus our analysis on the role of tfap2a NA differentiation in the CNS. In the locus coeruleus, Phox2a and Tfap2a are co-expressed and are both required for NA development. By contrast, in the medulla Phox2a and Tfap2a are expressed in adjacent overlapping domains, but only tfap2a activity is required for NA differentiation, as NA neurons develop normally in soulless/phox2a mutant medulla. phox2a and tfap2a do not appear to affect each others expression. Our studies show that two distinct inductive mechanisms control NA development in the zebrafish hindbrain. For the posterior hindbrain, we identify retinoic acid as an important signal to induce NA differentiation in the medulla oblongata and area postrema, where it expands the tfap2a expression domain and thus acts upstream of tfap2a. By contrast, previous work revealed Fgf8 to be involved in specification of NA neurons in the locus coeruleus. Thus, although the inductive signals may be distinct, hindbrain NA neurons of the locus coeruleus and the posterior groups both require Tfap2a to establish their noradrenergic identity.

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.

The mother superior mutation ablates foxd3 activity in neural crest progenitor cells and depletes neural crest derivatives in zebrafish.

The zebrafish mutation mother superior (mosm188) leads to a depletion of neural crest (NC) derivatives including the craniofacial cartilage skeleton, the peripheral nervous system (sympathetic neurons, dorsal root ganglia, enteric neurons), and pigment cells. The loss of derivatives is preceded by a reduction in NC‐expressed transcription factors, snail1b, sox9b, sox10, and a specific loss of foxd3 expression in NC progenitor cells. We employed genetic linkage analysis and physical mapping to place the mosm188 mutation on zebrafish chromosome 6 in the vicinity of the foxd3 gene. Furthermore, we found that mosm188 does not complement the sym1/foxd3 mutation, indicating that mosm188 resides within the foxd3 locus. Injection of PAC clones containing the foxd3 gene into mosm188 embryos restored foxd3 expression in NC progenitors and suppressed the mosm188 phenotype. However, sequencing the foxd3 transcribed area in mosm188 embryos did not reveal nucleotide changes segregating with the mosm188 phenotype, implying that the mutation most likely resides outside the foxd3‐coding region. Based on these findings, we propose that the mosm188 mutation perturbs a NC‐specific foxd3 regulatory element. Further analysis of mosm188 mutants and foxd3 morphants revealed that NC cells are initially formed, suggesting that foxd3 function is required to maintain the pool of NC progenitors. Developmental Dynamics 235:3199–3212, 2006.

ENU mutagenesis in zebrafish-from genes to complex diseases.

The Human Genome Project is in full swing, and every day we know more of the sequences that define us (Burris et al. 1998). Some smaller bacterial genomes are already sequenced and so are the yeast ( S. cerevisiae ) and worm ( C. elegans ) genomes. Progress in sequencing technologies promises that within the next few years we will know the entire sequence of the 3000 Mb human genome, and shortly afterwards the genome sequence of “the queen” of the genetic animal model systems, the mouse. At that point the work on understanding the biological existence of organisms will only begin. The function of genes in patterning, organogenesis, and physiology will need to be uncovered and the best current tools to accomplish these goals lie in mutational analysis of mouse, zebrafish, worm, fly, rat, and, for plants– Arabidopsis thaliana . As the complete sequence of each of the model systems becomes known, comparative analyses of gene function will provide clues towards understanding normal development and physiology as well as monogenic traits and complex diseases. Zebrafish was first discovered as a potentially excellent model system to study embryology owing to its transparent embryos available year round in plenitude. The pivotal point in the zebrafish field was the landmark paper of the late George Streisinger published in the journal Nature in 1981. It reported that this small teleost fish is suitable for random mutagenesis and mutant screens (Sterisinger et al. 1981). Coincidentally, the Streisinger paper appeared around the same time as the Nobel prize-winning work of Nüsslein-Volhard and Wieschaus on induction and recovery of mutants in the fruit fly (Nu ̈sslein-Volhard and Wieschaus 1980). The significance of the zebrafish work by scientists in Oregon was originally not widely recognized, but the paper “Production of clones of homozygous diploid zebra fish ( Brachydanio rerio)” marked the birth of a new animal model system. Streisinger’s pioneering developmental and genetics work has been continued by Chuck Kimmel and his colleagues at the University of Oregon. Over the past decade the zebrafish has become the vertebrate of choice for random, genome-wide, large-scale mutagenesis of genes crucial for development. After some groundwork to determine the most effective method of inducing mutations in zebrafish, it became clear that ENU would lead the pack (Solnica-Krezel et al. 1994; Mullins et al. 1994; Riley and Grunwald 1995). The establishment of methods for mutagenesis and screening protocols was fast and benefited greatly from experience in the mouse, fly, and worm fields (Russell et al. 1979; Ashburner 1989; Wood 1988). ENU mutations are not tagged in any easy to recognize way; therefore, one needs to resort to a long and tedious positional cloning process. Here the ever perfect fish comes to aid; its tremendous fecundity allows collection of over 5,000 meioses in less than half a year (Kimmel 1989). At this mapping resolution, one can narrow the genetic interval containing the mutation to a readily sequenced BAC clone. At the time when hundreds of ENU-mutagenized males were swimming in Boston and Tu ̈bingen, a genetic linkage map of the zebrafish had not yet been established, although Goff and others at Harvard and MIT had shown that microsatellite markers were p sent in the zebrafish genome (Goff et al. 1992). In 1994 the first RAPD-based map for zebrafish was welcome, although it provided only a temporary solution (Postlethwait et al 1994). The 1996 special Zebrafish Issue of Development revealed it all, thousands of mutations in genes essential for early patterning and organogenesis, along with the first zebrafish reference cross and framework microsatellites genetic linkage map (Development 1996). Today the field of zebrafish genetics is quite comparable to other, longer-established animal model systems. The infrastructure development is centered on fast gene discovery, especially from ENU-induced mutations (see Infobox 1). These resources are growing very rapidly and ensure that positional cloning of zebrafish mutants should become a routine technique in many laboratories. These efforts will supply a multitude of genes annotated by function in their specific domains that are essential for normal development of different tissues. Subsequently, we will be able to use this information, together with ease of constructing double and triple mutants, to study gene interactions, gene networks, and gene hierarchy during embryonic development in vertebrates.

Mutations in fam20b and xylt1 Reveal That Cartilage Matrix Controls Timing of Endochondral Ossification by Inhibiting Chondrocyte Maturation

Differentiating cells interact with their extracellular environment over time. Chondrocytes embed themselves in a proteoglycan (PG)-rich matrix, then undergo a developmental transition, termed “maturation,” when they express ihh to induce bone in the overlying tissue, the perichondrium. Here, we ask whether PGs regulate interactions between chondrocytes and perichondrium, using zebrafish mutants to reveal that cartilage PGs inhibit chondrocyte maturation, which ultimately dictates the timing of perichondral bone development. In a mutagenesis screen, we isolated a class of mutants with decreased cartilage matrix and increased perichondral bone. Positional cloning identified lesions in two genes, fam20b and xylosyltransferase1 (xylt1), both of which encode PG synthesis enzymes. Mutants failed to produce wild-type levels of chondroitin sulfate PGs, which are normally abundant in cartilage matrix, and initiated perichondral bone formation earlier than their wild-type siblings. Primary chondrocyte defects might induce the bone phenotype secondarily, because mutant chondrocytes precociously initiated maturation, showing increased and early expression of such markers as runx2b, collagen type 10a1, and ihh co-orthologs, and ihha mutation suppressed early perichondral bone in PG mutants. Ultrastructural analyses demonstrated aberrant matrix organization and also early cellular features of chondrocyte hypertrophy in mutants. Refining previous in vitro reports, which demonstrated that fam20b and xylt1 were involved in PG synthesis, our in vivo analyses reveal that these genes function in cartilage matrix production and ultimately regulate the timing of skeletal development.

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.

An exclusively mesodermal origin of fin mesenchyme demonstrates that zebrafish trunk neural crest does not generate ectomesenchyme.

The neural crest is a multipotent stem cell population that arises from the dorsal aspect of the neural tube and generates both non-ectomesenchymal (melanocytes, peripheral neurons and glia) and ectomesenchymal (skeletogenic, odontogenic, cartilaginous and connective tissue) derivatives. In amniotes, only cranial neural crest generates both classes, with trunk neural crest restricted to non-ectomesenchyme. By contrast, it has been suggested that anamniotes might generate derivatives of both classes at all axial levels, with trunk neural crest generating fin osteoblasts, scale mineral-forming cells and connective tissue cells; however, this has not been fully tested. The cause and evolutionary significance of this cranial/trunk dichotomy, and its absence in anamniotes, are debated. Recent experiments have disputed the contribution of fish trunk neural crest to fin osteoblasts and scale mineral-forming cells. This prompted us to test the contribution of anamniote trunk neural crest to fin connective tissue cells. Using genetics-based lineage tracing in zebrafish, we find that these fin mesenchyme cells derive entirely from the mesoderm and that neural crest makes no contribution. Furthermore, contrary to previous suggestions, larval fin mesenchyme cells do not generate the skeletogenic cells of the adult fin, but persist to form fibroblasts associated with adult fin rays. Our data demonstrate that zebrafish trunk neural crest does not generate ectomesenchymal derivatives and challenge long-held ideas about trunk neural crest fate. These findings have important implications for the ontogeny and evolution of the neural crest.