A high-content screening assay in transgenic zebrafish identifies two novel activators of fgf signaling

Zebrafish have become an invaluable vertebrate animal model to interrogate small molecule libraries for modulators of complex biological pathways and phenotypes. We have recently described the implementation of a quantitative, high-content imaging assay in multi-well plates to analyze the effects of small molecules on Fibroblast Growth Factor (FGF) signaling in vivo. Here we have evaluated the capability of the assay to identify compounds that hyperactivate FGF signaling from a test cassette of agents with known biological activities. Using a transgenic zebrafish reporter line for FGF activity, we screened 1040 compounds from an annotated library of known bioactive agents, including FDA-approved drugs. The assay identified two molecules, 8-hydroxyquinoline sulfate and pyrithione zinc, that enhanced FGF signaling in specific areas of the brain. Subsequent studies revealed that both compounds specifically expanded FGF target gene expression. Furthermore, treatment of early stage embryos with either compound resulted in dorsalized phenotypes characteristic of hyperactivation of FGF signaling in early development. Documented activities for both agents included activation of extracellular signal-related kinase (ERK), consistent with FGF hyperactivation. To conclude, we demonstrate the power of automated quantitative high-content imaging to identify small molecule modulators of FGF.     

Essential roles of zebrafish bmp2a, fgf10, and fgf24 in the specification of the ventral pancreas

In vertebrates, pancreas and liver arise from bipotential progenitors located in the embryonic gut endoderm. Bone morphogenic protein (BMP) and fibroblast growth factor (FGF) signaling pathways have been shown to induce hepatic specification while repressing pancreatic fate. Here we show that BMP and FGF factors also play crucial function, at slightly later stages, in the specification of the ventral pancreas. By analyzing the pancreatic markers pdx1, ptf1a, and hlxb9la in different zebrafish models of BMP loss of function, we demonstrate that the BMP pathway is required between 20 and 24 h postfertilization to specify the ventral pancreatic bud. Knockdown experiments show that bmp2a, expressed in the lateral plate mesoderm at these stages, is essential for ventral pancreas specification. Bmp2a action is not restricted to the pancreatic domain and is also required for the proper expression of hepatic markers. By contrast, through the analysis of fgf10(-/-); fgf24(-/-) embryos, we reveal the specific role of these two FGF ligands in the induction of the ventral pancreas and in the repression of the hepatic fate. These mutants display ventral pancreas agenesis and ectopic masses of hepatocytes. Overall, these data highlight the dynamic role of BMP and FGF in the patterning of the hepatopancreatic region.     

Distinct roles of Shh and Fgf signaling in regulating cell proliferation during zebrafish pectoral fin development

Background  

Cell proliferation in multicellular organisms must be coordinated with pattern formation. The major signaling pathways directing pattern formation in the vertebrate limb are well characterized, and we have therefore chosen this organ to examine the interaction between proliferation and patterning. Two important signals for limb development are members of the Hedgehog (Hh) and Fibroblast Growth Factor (Fgf) families of secreted signaling proteins. Sonic hedgehog (Shh) directs pattern formation along the anterior/posterior axis of the limb, whereas several Fgfs in combination direct pattern formation along the proximal/distal axis of the limb.     

Identification of an additional protein involved in mannan biosynthesis

Galactomannans comprise a β‐1,4‐mannan backbone substituted with α‐1,6‐galactosyl residues. Genes encoding the enzymes that are primarily responsible for backbone synthesis and side‐chain addition of galactomannans were previously identified and characterized. To identify additional genes involved in galactomannan biosynthesis, we previously performed deep EST profiling of fenugreek (Trigonella foenum‐graecum L.) seed endosperm, which accumulates large quantities of galactomannans as a reserve carbohydrate during seed development. One of the candidate genes encodes a protein that is likely to be a glycosyltransferase. Because this protein is involved in mannan biosynthesis, we named it ‘mannan synthesis‐related’ (MSR). Here, we report the characterization of a fenugreek MSR gene (TfMSR) and its two Arabidopsis homologs, AtMSR1 and AtMSR2. TfMSR was highly and specifically expressed in the endosperm. TfMSR, AtMSR1 and AtMSR2 proteins were all determined to be localized to the Golgi by fluorescence confocal microscopy. The level of mannosyl residues in stem glucomannans decreased by approximately 40% for Arabidopsis msr1 single T‐DNA insertion mutants and by more than 50% for msr1 msr2 double mutants, but remained unchanged for msr2 single mutants. In addition, in vitro mannan synthase activity from the stems of msr1 single and msr1 msr2 double mutants also decreased. Expression of AtMSR1 or AtMSR2 in the msr1 msr2 double mutant completely or partially restored mannosyl levels. From these results, we conclude that the MSR protein is important for mannan biosynthesis, and offer some ideas about its role.     

The initiation and propagation of Hes7 oscillation are cooperatively regulated by Fgf and notch signaling in the somite segmentation clock

Periodic formation of somites is controlled by the segmentation clock, where the oscillator Hes7 regulates cyclic expression of the Notch modulator Lunatic fringe. Here, we show that Hes7 also regulates cyclic expression of the Fgf signaling inhibitor Dusp4 and links Notch and Fgf oscillations in phase. Strikingly, inactivation of Notch signaling abolishes the propagation but allows the initiation of Hes7 oscillation. By contrast, transient inactivation of Fgf signaling abolishes the initiation, whereas sustained inactivation abolishes both the initiation and propagation of Hes7 oscillation. We thus propose that Hes7 oscillation is initiated by Fgf signaling and propagated/maintained anteriorly by Notch signaling.     

The interaction of epithelial Ihha and mesenchymal Fgf10 in zebrafish esophageal and swimbladder development

Developmental patterning and growth of the vertebrate digestive and respiratory tracts requires interactions between the epithelial endoderm and adjacent mesoderm. The esophagus is a specialized structure that connects the digestive and respiratory systems and its normal development is critical for both. Shh signaling from the epithelium regulates related aspects of mammalian and zebrafish digestive organ development and has a prominent effect on esophageal morphogenesis. The mechanisms underlying esophageal malformations, however, are poorly understood. Here, we show that zebrafish Ihha signaling from the epithelium acting in parallel, but independently of Shh, controls epithelial and mesenchymal cell proliferation and differentiation of smooth muscles and neurons in the gut and swimbladder. In zebrafish ihha mutants, the esophageal and swimbladder epithelium is dysmorphic, and expression of fgf10 in adjacent mesenchymal cells is affected. Analysis of the development of the esophagus and swimbladder in fgf10 mutant daedalus (dae) and compound dae/ihha mutants shows that the Ihha–Fgf10 regulatory interaction is realized through a signaling feedback loop between the Ihha-expressing epithelium and Fgf10-expressing mesenchyme. Disruption of this loop further affects the esophageal and swimbladder epithelium in ihha mutants, and Ihha acts in parallel to but independently of Shha in this process. These findings contribute to the understanding of epithelial–mesenchymal interactions and highlight an interaction between Hh and Fgf signaling pathways during esophagus and swimbladder development.     

Genomic pedigree reconstruction identifies predictors of mating and reproductive success in an invasive vertebrate

The persistence of an invasive species is influenced by its reproductive ecology, and a successful control program must operate on this premise. However, the reproductive ecology of invasive species may be enigmatic due to factors that also limit their management, such as cryptic coloration and behavior. We explored the mating and reproductive ecology of the invasive Brown Treesnake (BTS: Boiga irregularis) by reconstructing a multigenerational genomic pedigree based on 654 single nucleotide polymorphisms for a geographically closed population established in 2004 on Guam (N = 426). The pedigree allowed annual estimates of individual mating and reproductive success to be inferred for snakes in the study population over a 14‐year period. We then employed generalized linear mixed models to gauge how well phenotypic and genomic data could predict sex‐specific annual mating and reproductive success. Average snout–vent length (SVL), average body condition index (BCI), and trappability were significantly related to annual mating success for males, with average SVL also related to annual mating success for females. Male and female annual reproductive success was positively affected by SVL, BCI, and trappability. Surprisingly, the degree to which individuals were inbred had no effect on annual mating or reproductive success. When juxtaposed with current control methods, these results indicate that baited traps, a common interdiction tool, may target fecund BTS in some regards but not others. Our study emphasizes the importance of reproductive ecology as a focus for improving BTS control and promotes genomic pedigree reconstruction for such an endeavor in this invasive species and others.     

The you gene encodes an EGF-CUB protein essential for Hedgehog signaling in zebrafish

Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed you-class mutations, that share common defects in somite shape and in most cases disrupt Hedgehog signaling. These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.     

Conserved features and evolutionary shifts of the EDA signaling pathway involved in vertebrate skin appendage development

It is widely accepted that evolutionary changes in conserved developmental signaling pathways play an important role in morphological evolution. However, few in silico studies were interested in tracking such changes in a signaling pathway. The Ectodysplasin (EDA) pathway provides an opportunity to fill this gap because it is involved in vertebrate skin appendage development such as scales, teeth, hair, and feathers that take an obvious part in the adaptation of species to their environment. We benefited from the large amount of genomic data now available to explore the evolution of the upstream genes of the EDA pathway. In mammals, these genes are eda (encoding 2 ligands, EDA-A1 and EDA-A2), edar (EDA-A1 receptor), edaradd (EDA receptor [EDAR] adapter), xedar (EDA-A2 receptor), and troy (a XEDAR-related receptor). We show that the evolution of EDA pathway genes combines both strongly conserved features and evolutionary shifts. These shifts are found at different signaling levels (from the ligand to intracellular signaling) and at different taxonomic levels (class, suborder, and genera). Although conserved features likely participate to the similarities found in the early development of vertebrate skin appendages, these shifts might account for innovations and specializations. Moreover, our study demonstrates that we can now benefit from the large number of sequenced vertebrate genomes to explore the evolution of specific signaling pathways and thereby to open new perspectives for developmental biology and evolutionary developmental biology.     

The zebrafish floating head mutant demonstrates podocytes play an important role in directing glomerular differentiation

In zebrafish, the pronephric glomerulus occupies a midline position underneath the notochord and is vascularized through angiogenic capillary ingrowth from the dorsal aorta. The midline mutants floating head (flh), sonic you (syu), and you-too (yot) provide the opportunity to study glomerular differentiation in the absence of the notochord and vascularization from the dorsal aorta. In flh, syu, and yot mutants, glomeruli differentiate at ectopic lateral positions within the embryo and contain morphologically identifiable podocyte and endothelial cell types. In the absence of the dorsal aorta, endothelia from an alternate source are recruited by podocytes during glomerular vascularization to make functional glomeruli. Our results suggest that midline signals are required for proper glomerular morphogenesis but not for the differentiation of podocytes. Podocytes appear to play an important role in directing cellular recruitment events leading to glomerular differentiation. Furthermore, we find defects in sclerotomal development that correlate with defects in glomerular morphogenesis suggesting a possible link between the formation of these embryonic structures.