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

Abstract

Endothelin1 (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 Proprotein Convertase Subtilisin/Kexin FurinA Regulates Zebrafish Host Response against Mycobacterium marinum

“…This leads to a loss-of-function FurinA mutant protein and enables the design of qRT-PCR primers, which can be used to specifically quantify native furinA mRNA molecules. In accordance with the developmental lethality of other homozygous furinA zebrafish mutants (Ͼ98% lethality of furinA tg419/tg419 mutant) (30), no homozygous furinA td204e/td204e mutant fish could be obtained in our fish crosses (up to ϳ450 genotyped fish), suggesting that in homozygous form this allele is also lethal. In contrast, the heterozygous furinA td204e/ϩ mutants were born in normal Mendelian ratios and did not show signs of developmental defects or spontaneous autoimmunity.…”

“…In the furinA td204e mutant fish, a specific thymidine (T)-to-adenosine (A) splice site mutation results in a skipped exon 9 during the transcription of the furinA gene (see Fig. S1 in the supplemental material) (30). This leads to a loss-of-function FurinA mutant protein and enables the design of qRT-PCR primers, which can be used to specifically quantify native furinA mRNA molecules.…”

“…We have previously characterized the expression of seven pcsk genes in developing embryos and multiple adult zebrafish tissues (32). Two orthologous genes of mammalian FURIN, furinA and furinB (30), were found to be ubiquitously expressed, and biochemical analyses The M. marinum burden of furinA td204e/ϩ mutants (n ϭ 17 to 20) and WT controls (n ϭ 13 to 18) was quantified with DNA qRT-PCR at 4 and 9 wpi. Bacterial load is represented as the median of total bacterial copies (log 10 ).…”

“…furinA td204e mutation-bearing zebrafish in an AB genetic background (Zebrafish Information Network [ZFIN] ID: ZDB-GENO-080606-310) were purchased from the Zebrafish International Resource Center (Oregon). The genotypes of the furinA td204e/ϩ mutant zebrafish and their wild-type (WT) siblings were confirmed by sequencing (30). The fish were kept in a standardized flowthrough system (Aquatic Habitats, Florida, USA) with a light/dark cycle of 14 h and 10 h and fed with SDS 400 food twice a day.…”

“…Zebrafish has two FURIN orthologs: furinA and furinB. furinA, like the mammalian FURIN gene, has a critical, nonredundant role in organism development (30). Here, we have silenced the expression of furin genes in developing fish and used a furinA td204e/ϩ mutant zebrafish strain to study how FurinA regulates the development of adult zebrafish immune cells and the host response against mycobacteria.…”