Density of the waterborne parasite Ceratomyxa shasta and its biological effects on salmon.

The myxozoan parasite Ceratomyxa shasta is a significant pathogen of juvenile salmonids in the Pacific Northwest of North America and is limiting recovery of Chinook (Oncorhynchus tshawytscha) and coho (O. kisutch) salmon populations in the Klamath River. We conducted a 5-year monitoring program that comprised concurrent sentinel fish exposures and water sampling across 212 river kilometers of the Klamath River. We used percent mortality and degree-days to death to measure disease severity in fish. We analyzed water samples using quantitative PCR and Sanger sequencing, to determine total parasite density and relative abundance of C. shasta genotypes, which differ in their pathogenicity to salmonids. We detected the parasite throughout the study zone, but parasite density and genetic composition fluctuated spatially and temporally. Chinook and coho mortality increased with density of their specific parasite genotype, but mortality-density thresholds and time to death differed. A lethality threshold of 40% mortality was reached with 10 spores liter(-1) for Chinook but only 5 spores liter(-1) for coho. Parasite density did not affect degree-days to death for Chinook but was negatively correlated for coho, and there was wider variation among coho individuals. These differences likely reflect the different life histories and genetic heterogeneity of the salmon populations. Direct quantification of the density of host-specific parasite genotypes in water samples offers a management tool for predicting host population-level impacts.

WNT8 and BMP2B co-regulate non-axial mesoderm patterning during zebrafish gastrulation.

During vertebrate mesoderm formation, fates are established according to position in the dorsoventral (D/V) axis of the embryo. Initially, maternal signaling divides nascent mesoderm into axial (dorsal) and non-axial (ventral) domains. Although the subsequent subdivision of non-axial mesoderm into multiple D/V fate domains is known to involve zygotic Wnt8 and BMP signaling as well as the Vent/Vox/Ved family of transcriptional repressors, how levels of signaling activity are translated into differential regulation of fates is not well understood. To address this question, we have analyzed zebrafish embryos lacking Wnt8 and BMP2b. Zebrafish wnt8; swr (bmp2b) double mutants display a progressive loss of non-axial mesoderm and a concomitant expansion of axial mesoderm during gastrulation. Mesoderm induction and specification of the axial domain occur normally in wnt8; swr mutants, but dorsal mesoderm genes eventually come to be expressed throughout the mesoderm, suggesting that the establishment of non-axial mesoderm identity requires continual repression of dorsal mesoderm factors, including repressors of ventral genes. Loss-of-function for Vent, Vox, and Ved phenocopies the wnt8; swr mutant phenotype, consistent with Wnt8 and BMP2b maintaining non-axial mesoderm identity during gastrulation through the regulation of these three transcriptional repressors. We postulate that timely differentiation of the mesoderm requires the maintenance of non-axial mesoderm identity by Wnt8 and BMP2b at the onset of gastrulation followed by subdivision of the non-axial mesoderm into different functional domains during gastrulation.

Rhombomere boundaries are Wnt signaling centers that regulate metameric patterning in the zebrafish hindbrain.

The vertebrate hindbrain develops from a series of segments (rhombomeres) distributed along the anteroposterior axis. We are studying the roles of Wnt and Delta-Notch signaling in maintaining rhombomere boundaries as organizing centers in the zebrafish hindbrain. Several wnt genes (wnt1, wnt3a, wnt8b, and wnt10b) show elevated expression at rhombomere boundaries, whereas several delta genes (dlA, dlB, and dlD) are expressed in transverse stripes flanking rhombomere boundaries. Partial disruption of Wnt signaling by knockdown of multiple wnt genes, or the Wnt mediator tcf3b, ablates boundaries and associated cell types. Expression of dlA is chaotic, and cell types associated with rhombomere centers are disorganized. Similar patterning defects are observed in segmentation mutants spiel-ohne-grenzen (spg) and valentino (val), which fail to form rhombomere boundaries due to faulty interactions between adjacent rhombomeres. Stripes of wnt expression are variably disrupted, with corresponding disturbances in metameric patterning. Mutations in dlA or mind bomb (mib) disrupt Delta-Notch signaling and cause a wide range of patterning defects in the hindbrain. Stripes of wnt1 are initially normal but subsequently dissipate, and metameric patterning becomes increasingly disorganized. Driving wnt1 expression using a heat-shock construct partially rescues metameric patterning in mib mutants. Thus, rhombomere boundaries act as Wnt signaling centers required for precise metameric patterning, and Delta signals from flanking cells provide feedback to maintain wnt expression at boundaries. Similar feedback mechanisms operate in the Drosophila wing disc and vertebrate limb bud, suggesting coaptation of a conserved signaling module that spatially organizes cells in complex organ systems.

Conservation of structure and functional divergence of duplicated Wnt8s in pufferfish.

The zebrafish wnt8 locus differs from its tetrapod counterparts in that it produces two functionally overlapping but distinct Wnt8 proteins. Studies of zebrafish wnt8 have suggested that the two major Wnt8 proteins produced are functionally similar yet may behave differently depending on the assay context. To determine whether the bicistronic wnt8 and its accompanying unique protein activities found in zebrafish are more widespread (and perhaps universal) among teleosts, we have extended our studies to the pufferfish Takifugu rubripes. We have found that Takifugu wnt8 is also bicistronic, indicating that the wnt8 duplication occurred before the divergence of these teleosts approximately 150 million years ago. Furthermore, overexpression assays in zebrafish embryos show that functional differences between the zebrafish Wnt8.1 and Wnt8.2 proteins are conserved in their Takifugu orthologs. Thus, despite the fact that Wnt8.1 and Wnt8.2 proteins are as similar to each other as each is to Xenopus Xwnt-8, Wnt8 family members can behave quite differently in the context of zebrafish embryos. This finding suggests that zebrafish (and possibly teleost in general) Wnt8 receptors are able to discriminate between highly related ligands.

Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation.

Wnt signaling is known to be required for the normal development of the vertebrate midbrain and hindbrain, but genetic loss of function analyses in the mouse and zebrafish yield differing results regarding the relative importance of specific Wnt loci. In the zebrafish, Wnt1 and Wnt10b functionally overlap in their control of gene expression in the ventral midbrain-hindbrain boundary (MHB), but they are not required for the formation of the MHB constriction. Whether other wnt loci are involved in zebrafish MHB development is unclear, although the expression of at least two wnts, wnt3a and wnt8b, is maintained in wnt1/wnt10b mutants. In order to address the role of wnt3a in zebrafish, we have isolated a full length cDNA and examined its expression and function via knockdown by morpholino antisense oligonucleotide (MO)-mediated knockdown. The expression pattern of wnt3a appears to be evolutionarily conserved between zebrafish and mouse, and MO knockdown shows that Wnt3a, while not uniquely required for MHB development, is required in the absence of Wnt1 and Wnt10b for the formation of the MHB constriction. In zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b, the expression of engrailed orthologs, pax2a and fgf8 is not maintained after mid-somitogenesis. In contrast to acerebellar and no isthmus mutants, in which midbrain and hindbrain cells acquire new fates but cell number is not significantly affected until late in embryogenesis, zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b undergo extensive apoptosis in the midbrain and cerebellum anlagen beginning in mid-somitogenesis, which results in the absence of a significant portion of the midbrain and cerebellum. Thus, the requirement for Wnt signaling in forming the MHB constriction is evolutionarily conserved in vertebrates and it is possible in zebrafish to dissect the relative impact of multiple Wnt loci in midbrain and hindbrain development.

Wnt1 and wnt10b function redundantly at the zebrafish midbrain-hindbrain boundary.

Wnt signals have been shown to be involved in multiple steps of vertebrate neural patterning, yet the relative contributions of individual Wnts to the process of brain regionalization is poorly understood. Wnt1 has been shown in the mouse to be required for the formation of the midbrain and the anterior hindbrain, but this function of wnt1 has not been explored in other model systems. Further, wnt1 is part of a Wnt cluster conserved in all vertebrates comprising wnt1 and wnt10b, yet the function of wnt10b during embryogenesis has not been explored. Here, we report that in zebrafish wnt10b is expressed in a pattern overlapping extensively with that of wnt1. We have generated a deficiency allele for these closely linked loci and performed morpholino antisense oligo knockdown to show that wnt1 and wnt10b provide partially redundant functions in the formation of the midbrain-hindbrain boundary (MHB). When both loci are deleted, the expression of pax2.1, en2, and her5 is lost in the ventral portion of the MHB beginning at the 8-somite stage. However, wnt1 and wnt10b are not required for the maintenance of fgf8, en3, wnt8b, or wnt3a expression. Embryos homozygous for the wnt1-wnt10b deficiency display a mild MHB phenotype, but are sensitized to reductions in either Pax2.1 or Fgf8; that is, in combination with mutant alleles of either of these loci, the morphological MHB is lost. Thus, wnt1 and wnt10b are required to maintain threshold levels of Pax2.1 and Fgf8 at the MHB.