Genetic Analysis of Fin Development in Zebrafish Identifies Furin and Hemicentin1 as Potential Novel Fraser Syndrome Disease Genes

Using forward genetics, we have identified the genes mutated in two classes of zebrafish fin mutants. The mutants of the first class are characterized by defects in embryonic fin morphogenesis, which are due to mutations in a Laminin subunit or an Integrin alpha receptor, respectively. The mutants of the second class display characteristic blistering underneath the basement membrane of the fin epidermis. Three of them are due to mutations in zebrafish orthologues of FRAS1, FREM1, or FREM2, large basement membrane protein encoding genes that are mutated in mouse bleb mutants and in human patients suffering from Fraser Syndrome, a rare congenital condition characterized by syndactyly and cryptophthalmos. Fin blistering in a fourth group of zebrafish mutants is caused by mutations in Hemicentin1 (Hmcn1), another large extracellular matrix protein the function of which in vertebrates was hitherto unknown. Our mutant and dose-dependent interaction data suggest a potential involvement of Hmcn1 in Fraser complex-dependent basement membrane anchorage. Furthermore, we present biochemical and genetic data suggesting a role for the proprotein convertase FurinA in zebrafish fin development and cell surface shedding of Fras1 and Frem2, thereby allowing proper localization of the proteins within the basement membrane of forming fins. Finally, we identify the extracellular matrix protein Fibrillin2 as an indispensable interaction partner of Hmcn1. Thus we have defined a series of zebrafish mutants modelling Fraser Syndrome and have identified several implicated novel genes that might help to further elucidate the mechanisms of basement membrane anchorage and of the disease's aetiology. In addition, the novel genes might prove helpful to unravel the molecular nature of thus far unresolved cases of the human disease

Patterning and Stabilizing the Zebrafish Pharyngeal Arch Intermediate Domain

xv, 76 p. : ill. (some col.) Includes 4 video files.Improved understanding of pharyngeal arch (PA) patterning and morphogenesis can reveal critical insights into the origins of craniofacial diseases, such as Fraser syndrome. PAs contain mesenchymal condensations, which give rise to most of the facial skeleton in vertebrates. Studies of Endothelin1 signaling reveal that the skeleton derived from the first two PAs are patterned into dorsal, intermediate, and ventral domains. Previous work has indicated that endothelin targets, including the Dlx genes, homeotically pattern dorsal versus ventral PA identity. I show that the Dlx gene family plays a vital role in PA intermediate-domain identity establishment. In WT fish, the PA intermediate domain is delineated by combined expression of all Dlx genes. Reduction of Dlx gene function results in loss of intermediate-domain identity. Conversely, ventral expansion of Dlx expression, seen in hand2 mutants, results in ventral expansion of intermediate-domain identity. Hence, PA intermediate-domain identity is defined by co-expression of Dlx genes. Epithelial-mesenchymal interactions play an important part in PA intermediate-domain morphogenesis. Zebrafish fras1 (epithelially expressed) and itga8 (mesenchymally expressed) mutants also show specific defects within intermediate-domain skeleton and epithelia. Facial phenotypes in fras1;itga8 double mutants look extremely similar to either single mutant, suggesting that fras1 and itga8 might participate in the same epithelial-mesenchymal interaction during PA intermediate-domain formation. Our developmental studies reveal that fras1 - and itga8 -dependent epithelial segmentation of the PA intermediate domain stabilizes developing skeletal elements. Lesions in human FRAS1 underlie many cases of Fraser syndrome, and this work provides an excellent developmental model for the craniofacial defects found in Fraser syndrome. Loss of either Dlx or fras1 function produces defects in the PA intermediate domain, yet seemingly during different developmental periods. Nonetheless, combined reduction of both Dlx and fras1 function synergistically increases skeletal defects, implying a molecular connection between early (Dlx -mediated) pattern formation and later (fras1 -mediated) pattern stabilization. Elucidation of the Dlx-fras1 interaction is an interesting topic which may unveil new molecules pertinent to Fraser syndrome. Supplemental movies highlighting skeletal and epithelial morphogenesis accompany this dissertation.Committee in charge: Judith S. Eisen, Chairperson; Charles B. Kimmel, Advisor; John H. Postlethwait, Member; Chris Q. Doe, Member; Kennith E. Prehoda, Outside Membe

A Streamlined CRISPR Pipeline to Reliably Generate Zebrafish Frameshifting Alleles

(NCBI Gene ID 494168), with the target sequence downstream of potential alter-native start sites and upstream of the homeodomain. (C) Polymerase chain reaction products from a subset of injected embryos (green lines) are subjected to a high-resolution melt curve and normalized to uninjected curves (red lines), revealing the melting difference caused by injection. (D) High-resolution melt analysis (HRMA) difference curves identifying three potentially different lesions in six1a were (E) sequenced from heterozygous F1 generation embryos. HRMA curves identified in this initial analysis are confirmed by (F) genotyping adult F1 generation fish fin DNA. (G) Homozygous mutant F2 generation sequence reads confirm the lesions inferred from heterozygous F1 generation sequences. Once confirmed, lesions are given allele designations. The mutation in six1a lesion A is six1aoz8, a 2 bp deletion that frameshifts Six1a after amino acid 93 of 284 and introduces 37 aberrant aa before terminating. Sequence analysis for lesion B (six1aoz9), which frameshifts Six1a after amino acid 94 and introduces only 12 aberrant aa, is described in the Supplementary Data. Lesion C is another 2 bp deletion (not shown). Color images available online at www.liebertpub.com/ze

hand2 and Dlx genes specify dorsal, intermediate and ventral domains within zebrafish pharyngeal arches

The ventrally expressed secreted polypeptide endothelin1 (Edn1) patterns the skeleton derived from the first two pharyngeal arches into dorsal, intermediate and ventral domains. Edn1 activates expression of many genes, including hand2 and Dlx genes. We wanted to know how hand2/Dlx genes might generate distinct domain identities. Here, we show that differential expression of hand2 and Dlx genes delineates domain boundaries before and during cartilage morphogenesis. Knockdown of the broadly expressed genes dlx1a and dlx2a results in both dorsal and intermediate defects, whereas knockdown of three intermediate-domain restricted genes dlx3b, dlx4b and dlx5a results in intermediate-domain-specific defects. The ventrally expressed gene hand2 patterns ventral identity, in part by repressing dlx3b/4b/5a. The jaw joint is an intermediate-domain structure that expresses nkx3.2 and a more general joint marker, trps1. The jaw joint expression of trps1 and nkx3.2 requires dlx3b/4b/5a function, and expands in hand2 mutants. Both hand2 and dlx3b/4b/5a repress dorsal patterning markers. Collectively, our work indicates that the expression and function of hand2 and Dlx genes specify major patterning domains along the dorsoventral axis of zebrafish pharyngeal arches

Zebrafish furin mutants reveal intricacies in regulating Endothelin1 signaling in craniofacial patterning

AbstractEndothelin1 (Edn1) signaling promotes ventral character to the facial skeleton. In zebrafish edn1 mutants, the ventral jaw structures are severely reduced and fused to their dorsal counterparts, with a loss of joints that normally form at an intermediate dorsal–ventral position. Loss of function at another locus, sturgeon, also yields joint losses, but only mild reductions in the ventral jaw structures. We show that sturgeon encodes one of two orthologs of Furin present in zebrafish, and that both furin genes may function partially redundantly to activate Edn1 signaling. Supporting this hypothesis, early expression of edn1-dependent genes is downregulated in sturgeon (furinA) mutants. Later in development, expression of most of these genes recovers to near wild-type levels in furinA mutants but not in edn1 mutants. The recovery explains the less severe furinA mutant skeletal phenotype and suggests that late gene expression is dependent on a critical level of Edn1 signaling not present in the more severe edn1 mutants. However, expression defects in the intermediate joint-forming domains in both mutants persist, explaining the joint losses observed later in both mutants. We further show that in both mutants the arches fail to correctly undergo ventral elongation before skeletogenesis begins and propose a model in which this failure is largely responsible for the loss of an Edn1-dependent compartmentation of the arch into the intermediate and ventral domains

The ascl1a and dlx genes have a regulatory role in the development of GABAergic interneurons in the zebrafish diencephalon

AbstractDuring development of the mouse forebrain interneurons, the Dlx genes play a key role in a gene regulatory network (GRN) that leads to the GABAergic phenotype. Here, we have examined the regulatory relationships between the ascl1a, dlx, and gad1b genes in the zebrafish forebrain. Expression of ascl1a overlaps with dlx1a in the telencephalon and diencephalon during early forebrain development. The loss of Ascl1a function results in a loss of dlx expression, and subsequent losses of dlx5a and gad1b expression in the diencephalic prethalamus and hypothalamus. Loss of Dlx1a and Dlx2a function, and, to a lesser extent, of Dlx5a and Dlx6a, impairs gad1b expression in the prethalamus and hypothalamus. We conclude that dlx1a/2a act downstream of ascl1a but upstream of dlx5a/dlx6a and gad1b to activate GABAergic specification. This pathway is conserved in the diencephalon, but has diverged between mammals and teleosts in the telencephalon

Pharyngeal morphogenesis requires fras1 - itga8 -dependent epithelial-mesenchymal interaction

Both Fras1 and Itga8 connect mesenchymal cells to epithelia by way of an extracellular ‘Fraser protein complex’ that functions in signaling and adhesion; these proteins are vital to the development of several vertebrate organs. We previously found that zebrafish fras1 mutants have craniofacial defects, specifically, shortened symplectic cartilages and cartilage fusions that spare joint elements. During a forward mutagenesis screen, we identified a new zebrafish mutation, b1161, that we show here disrupts itga8, as confirmed using CRISPR-generated itga8 alleles. fras1 and itga8 single mutants and double mutants have similar craniofacial phenotypes, a result expected if loss of either gene disrupts function of the Fraser protein complex. Unlike fras1 mutants or other Fraser-related mutants, itga8 mutants do not show blistered tail fins. Thus, the function of the Fraser complex differs in the craniofacial skeleton and the tail fin. Focusing on the face, we find that itga8 mutants consistently show defective outpocketing of a late-forming portion of the first pharyngeal pouch, and variably express skeletal defects, matching previously characterized fras1 mutant phenotypes. In itga8 and fras1 mutants, skeletal severity varies markedly between sides, indicating that both mutants have increased developmental instability. Whereas fras1 is expressed in epithelia, we show that itga8 is expressed complementarily in facial mesenchyme. Paired with the observed phenotypic similarity, this expression indicates that the genes function in epithelial-mesenchymal interactions. Similar interactions between Fras1 and Itga8 have previously been found in mouse kidney, where these genes both regulate Nephronectin (Npnt) protein abundance. We find that zebrafish facial tissues express both npnt and the Fraser gene fibrillin2b (fbn2b), but their transcript levels do not depend on fras1 or itga8 function. Using a revertible fras1 allele, we find that the critical window for fras1 function in the craniofacial skeleton is between 1.5 and 3 days post fertilization, which coincides with the onset of fras1-dependent and itga8-dependent morphogenesis. We propose a model wherein Fras1 and Itga8 interact during late pharyngeal pouch morphogenesis to sculpt pharyngeal arches through epithelial-mesenchymal interactions, thereby stabilizing the developing craniofacial skeleton