Fibroblast Growth Factor Receptor 2c Signaling Is Required for Intestinal Cell Differentiation in Zebrafish

Background

There are four cell lineages derived from intestinal stem cells that are located at the crypt and villus in the mammalian intestine the non-secretory absorptive enterocytes, and the secretory cells, which include mucous-secreting goblet cells, regulatory peptide-secreting enteroendocrine cells and antimicrobial peptide-secreting Paneth cells. Although fibroblast growth factor (Fgf) signaling is important for cell proliferation and differentiation in various tissues, its role in intestinal differentiation is less well understood.

Methodology/Principal Findings

We used a loss of function approach to investigate the importance of Fgf signaling in intestinal cell differentiation in zebrafish; abnormal differentiation of goblet cells was observed when Fgf signaling was inhibited using SU5402 or in the Tg(hsp70ldnfgfr1-EGFP) transgenic line. We identified Fgfr2c as an important receptor for cell differentiation. The number of goblet cells and enteroendocrine cells was reduced in fgfr2c morphants. In addition to secretory cells, enterocyte differentiation was also disrupted in fgfr2c morphants. Furthermore, proliferating cells were increased in the morphants. Interestingly, the loss of fgfr2c expression repressed secretory cell differentiation and increased cell proliferation in the mib^ta52b mutant that had defective Notch signaling.

Conclusions/Significance

In conclusion, we found that Fgfr2c signaling derived from mesenchymal cells is important for regulating the differentiation of zebrafish intestine epithelial cells by promoting cell cycle exit. The results of Fgfr2c knockdown in mib^ta52b mutants indicated that Fgfr2c signaling is required for intestinal cell differentiation. These findings provide new evidences that Fgf signaling is required for the differentiation of intestinal cells in the zebrafish developing gut.     

Disruption of Paneth and goblet cell homeostasis and increased endoplasmic reticulum stress in Agr2−/− mice

Anterior Gradient 2 (AGR2) is a protein disulfide isomerase that plays important roles in diverse processes in multiple cell lineages as a developmental regulator, survival factor and susceptibility gene for inflammatory bowel disease. Here, we show using germline and inducible Agr2−/− mice that Agr2 plays important roles in intestinal homeostasis. Agr2−/− intestine has decreased goblet cell Mucin 2, dramatic expansion of the Paneth cell compartment, abnormal Paneth cell localization, elevated endoplasmic reticulum (ER) stress, severe terminal ileitis and colitis. Cell culture experiments show that Agr2 expression is induced by ER stress, and that siRNA knockdown of Agr2 increases ER stress response. These studies implicate Agr2 in intestinal homeostasis and ER stress and suggest a role in the etiology of inflammatory bowel disease.     

Expression of zebrafish anterior gradient 2 in the semicircular canals and supporting cells of otic vesicle sensory patches is regulated by Sox10

AGR2 is a member of the protein disulfide isomerase (PDI) family, which is implicated in cancer cell growth and metastasis, asthma, and inflammatory bowel disease. Despite the contributions of this protein to several biological processes, the regulatory mechanisms controlling expression of the AGR2 gene in different organs remain unclear. Zebrafish anterior gradient 2 (agr2) is expressed in several organs, including the otic vesicles that contain mucus-secreting cells. To elucidate the regulatory mechanisms controlling agr2 expression in otic vesicles, we generated a Tg(− 6.0 k agr2:EGFP) transgenic fish line that expressed EGFP in a pattern recapitulating that of agr2. Double immunofluorescence studies were used to demonstrate that Agr2 and GFP colocalize in the semicircular canals and supporting cells of all sensory patches in the otic vesicles of Tg(− 6.0 k agr2:EGFP) embryos. Transient/stable transgenic analyses coupled with 5′-end deletion revealed that a 100 bp sequence within the − 2.6 to − 2.5 kbp region upstream of agr2 directs EGFP expression specifically in the otic vesicles. Two HMG-binding motifs were detected in this region. Mutation of these motifs prevented EGFP expression. Furthermore, EGFP expression in the otic vesicles was prevented by knockdown of the sox10 gene. This corresponded with decreased agr2 expression in the otic vesicles of sox10 morphants during different developmental stages. Electrophoretic mobility shift assays were used to show that Sox10 binds to HMG-binding motifs located within the − 2.6 to − 2.5 kbp region upstream of agr2. These results demonstrate that agr2 expression in the otic vesicles of zebrafish embryos is regulated by Sox10.     

Role of zebrafish lbx2 in embryonic lateral line development

Background

The zebrafish ladybird homeobox homologous gene 2 (lbx2) has been suggested to play a key role in the regulation of hypaxial myogenic precursor cell migration. Unlike their lbx counterparts in mammals, the function of teleost lbx genes beyond myogenesis during embryonic development remains unexplored.

Principal Findings

Abrogation of lbx2 function using a specific independent morpholino oligonucleotide (MO) or truncated lbx2 mRNA with an engrailed domain deletion (lbx2^eh-) resulted in defective formation of the zebrafish posterior lateral line (PLL). Migration of the PLL primordium was altered and accompanied by increased cell death in the primordium of lbx2-MO-injected embryos. A decreased number of muscle pioneer cells and impaired expression pattern of sdf1a in the horizontal myoseptum was observed in lbx2 morphants.

Significance

Injection of lbx2 MO or lbx2^eh- mRNA resulted in defective PPL formation and altered sdf1a expression, confirming an important function for lbx2 in sdf1a-dependent migration. In addition, the disassociation of PPL nerve extension with PLL primordial migration in some lbx2 morphants suggests that pathfinding of the PLL primordium and the lateral line nerve may be regulated independently.     

Zona pellucida domain-containing protein β-tectorin is crucial for zebrafish proper inner ear development

Background

The zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian β-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is 1 component of a gel-like structure called the tectorial membrane which is involved in transforming sound waves into neuronal signals and is important for normal auditory function. β-Tectorin is specifically expressed in the mammalian and avian inner ear.

Methodology/Principal Findings

We identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin messenger RNA was expressed in the otic placode and specialized sensory patch of the inner ear during zebrafish embryonic stages. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of β-tectorin morphants were abnormal since the development of the inner ear was compromised.

Conclusions/Significance

Our results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, and is important for regulating the development of the zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects in inner ear formation of otoliths and function.     

Anti-apoptotic effects of IGF-I on mortality and dysmorphogenesis in <Emphasis Type="Italic">tbx5</Emphasis>-deficient zebrafish embryos

Background

Tbx5 deficiency in zebrafish causes several abnormal phenotypes of the heart and pectoral fins. It has been reported that exogenous human growth hormone can enhance expression of downstream mediators in the growth hormone and insulin-like growth factor I (IGF-I) pathway and partially restore dysmorphogenesis in tbx5 morphants. This study aimed to further evaluate the effects of IGF-I on cell apoptosis and dysmorphogenesis in zebrafish embryos deficient for tbx5.

Results

Among the five studied groups of zebrafish embryos (wild-type embryos [WT], tbx5 morphants [MO], mismatched tbx5 morpholino-treated wild-type embryos [MIS], IGF-I-treated wild-type embryos [WTIGF1], and IGF-I-treated tbx5 morphants [MOIGF1]), the expression levels of the ifg1, igf1-ra, ifg-rb, erk1, and akt2 genes as well as the ERK and AKT proteins were significantly reduced in the MO group, but were partially restored in the MOIGF1 group. These expression levels remained normal in the WT, MIS, and WTIGF1 groups. Exogenous human IGF-I also reduced the incidence of phenotypic anomalies, decreased the expression levels of apoptotic genes and proteins, suppressed cell apoptosis, and improved survival of the MOIGF1 group.

Conclusions

These results suggest that IGF-I has an anti-apoptotic protective effect in zebrafish embryos with tbx5 deficiency.

     

Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis

Background

MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis.

Methods and Findings

By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1β, TNF-α, and IFN-γ was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-γ, TNF-α, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis.

Conclusions

Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.     

AGR2, an Endoplasmic Reticulum Protein,Is Secreted into the Gastrointestinal Mucus

The MUC2 mucin is the major constituent of the two mucus layers in colon. Mice lacking the disulfide isomerase-like protein Agr2 have been shown to be more susceptible to colon inflammation. The Agr2^−/− mice have less filled goblet cells and were now shown to have a poorly developed inner colon mucus layer. We could not show AGR2 covalently bound to recombinant MUC2 N- and C-termini as have previously been suggested. We found relatively high concentrations of Agr2 in secreted mucus throughout the murine gastrointestinal tract, suggesting that Agr2 may play extracellular roles. In tissue culture (CHO-K1) cells, AGR2 is normally not secreted. Replacement of the single Cys in AGR2 with Ser (C81S) allowed secretion, suggesting that modification of this Cys might provide a mechanism for circumventing the KTEL endoplasmic reticulum retention signal. In conclusion, these results suggest that AGR2 has both intracellular and extracellular effects in the intestine.     

A key role for E-cadherin in intestinal homeostasis and Paneth cell maturation

Background

E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn''s disease.

Methods and Findings

To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen.

Conclusion

These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells.     

Delta/Jagged-mediated Notch signaling induces the differentiation of agr2-positive epidermal mucous cells in zebrafish embryos

Teleosts live in aquatic habitats, where they encounter ionic and acid-base fluctuations as well as infectious pathogens. To protect from these external challenges, the teleost epidermis is composed of living cells, including keratinocytes and ionocytes that maintain body fluid ionic homeostasis, and mucous cells that secret mucus. While ionocyte progenitors are known to be specified by Delta-Notch-mediated lateral inhibition during late gastrulation and early segmentation, it remains unclear how epidermal mucous cells (EMCs) are differentiated and maintained. Here, we show that Delta/Jagged-mediated activation of Notch signaling induces the differentiation of agr2-positive (agr2⁺) EMCs in zebrafish embryos during segmentation. We demonstrated that agr2⁺ EMCs contain cytoplasmic secretory granules and express muc5.1 and muc5.2. Reductions in agr2⁺ EMC number were observed in mib mutants and notch3 MOs-injected notch1a mutants, while increases in agr2⁺ cell number were detected in notch1a- and X-Su(H)/ANK-overexpressing embryos. Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of agr2⁺ EMCs. Increased agr2⁺ EMC numbers were also observed in jag1a-, jag1b-, jag2a- and dlc-overexpressing, but not jag2b-overexpressing embryos. Meanwhile, reductions in agr2⁺ EMC numbers were detected in jag1a morphants, jag1b mutants, jag2a mutants and dlc morphants, but not jag2b mutants. Reduced numbers of pvalb8-positive epidermal cells were also observed in mib or jag2a mutants and jag1a or jag1b morphants, while increased pvalb8-positive epidermal cell numbers were detected in notch1a-overexpressing, but not dlc-overexpressing embryos. BrdU labeling further revealed that the agr2⁺ EMC population is maintained by proliferation. Cell lineage experiments showed that agr2⁺ EMCs are derived from the same ectodermal precursors as keratinocytes or ionocytes. Together, our results indicate that specification of agr2⁺ EMCs in zebrafish embryos is induced by DeltaC/Jagged-dependent activation of Notch1a/3 signaling, and the cell population is maintained by proliferation.     

Platelet-Derived Growth Factor Receptor β Is Critical for Zebrafish Intersegmental Vessel Formation

Background

Platelet-derived growth factor receptor β (PDGFRβ) is a tyrosine kinase receptor known to affect vascular development. The zebrafish is an excellent model to study specific regulators of vascular development, yet the role of PDGF signaling has not been determined in early zebrafish embryos. Furthermore, vascular mural cells, in which PDGFRβ functions cell autonomously in other systems, have not been identified in zebrafish embryos younger than 72 hours post fertilization.

Methodology/Principal Findings

In order to investigate the role of PDGFRβ in zebrafish vascular development, we cloned the highly conserved zebrafish homolog of PDGFRβ. We found that pdgfrβ is expressed in the hypochord, a developmental structure that is immediately dorsal to the dorsal aorta and potentially regulates blood vessel development in the zebrafish. Using a PDGFR tyrosine kinase inhibitor, a morpholino oligonucleotide specific to PDGFRβ, and a dominant negative PDGFRβ transgenic line, we found that PDGFRβ is necessary for angiogenesis of the intersegmental vessels.

Significance/Conclusion

Our data provide the first evidence that PDGFRβ signaling is required for zebrafish angiogenesis. We propose a novel mechanism for zebrafish PDGFRβ signaling that regulates vascular angiogenesis in the absence of mural cells.     

Calycosin Promotes Angiogenesis Involving Estrogen Receptor and Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Zebrafish and HUVEC

Background

Angiogenesis plays an important role in a wide range of physiological processes, and many diseases are associated with the dysregulation of angiogenesis. Radix Astragali is a Chinese medicinal herb commonly used for treating cardiovascular disorders and has been shown to possess angiogenic effect in previous studies but its active constituent and underlying mechanism remain unclear. The present study investigates the angiogenic effects of calycosin, a major isoflavonoid isolated from Radix Astragali, in vitro and in vivo.

Methodology

Tg(fli1:EGFP) and Tg(fli1:nEGFP) transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 µM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment. Zebrafish embryos were exposed to calycosin (10, 100 µM) from 72 hpf to 78 hpf before gene-expression analysis. The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis were studied using 72 hpf Tg(fli1:EGFP) and Tg(fli1:nEGFP) zebrafish embryos. The pro-angiogenic effects of calycosin were compared with raloxifene and tamoxifen in 72 hpf Tg(fli1:EGFP) zebrafish embryos. The binding affinities of calycosin to estrogen receptors (ERs) were evaluated by cell-free and cell-based estrogen receptor binding assays. Human umbilical vein endothelial cell cultures (HUVEC) were pretreated with different concentrations of calycosin (3, 10, 30, 100 µM) for 48 h then tested for cell viability and tube formation. The role of MAPK signaling in calycosin-induced angiogenesis was evaluated using western blotting.

Conclusion

Calycosin was shown to induce angiogenesis in human umbilical vein endothelial cell cultures (HUVEC) in vitro and zebrafish embryos in vivo via the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 mRNA expression. It was demonstrated that calycosin acted similar to other selective estrogen receptor modulators (SERMs), such as raloxifene and tamoxifen, by displaying selective potency and affinity to estrogen receptors ERα and ERβ. Our results further indicated that calycosin promotes angiogenesis via activation of MAPK with the involvement of ERK1/2 and ER. Together, this study revealed, for the first time, that calycosin acts as a selective estrogen receptor modulator (SERM) to promote angiogenesis, at least in part through VEGF-VEGFR2 and MAPK signaling pathways.     

Expression and Functional Characterization of Smyd1a in Myofibril Organization of Skeletal Muscles

Background

Smyd1, the founding member of the Smyd family including Smyd-1, 2, 3, 4 and 5, is a SET and MYND domain containing protein that plays a key role in myofibril assembly in skeletal and cardiac muscles. Bioinformatic analysis revealed that zebrafish genome contains two highly related smyd1 genes, smyd1a and smyd1b. Although Smyd1b function is well characterized in skeletal and cardiac muscles, the function of Smyd1a is, however, unknown.

Methodology/Principal Findings

To investigate the function of Smyd1a in muscle development, we isolated smyd1a from zebrafish, and characterized its expression and function during muscle development via gene knockdown and transgenic expression approaches. The results showed that smyd1a was strongly expressed in skeletal muscles of zebrafish embryos. Functional analysis revealed that knockdown of smyd1a alone had no significant effect on myofibril assembly in zebrafish skeletal muscles. However, knockdown of smyd1a and smyd1b together resulted in a complete disruption of myofibril organization in skeletal muscles, a phenotype stronger than knockdown of smyd1a or smyd1b alone. Moreover, ectopic expression of zebrafish smyd1a or mouse Smyd1 transgene could rescue the myofibril defects from the smyd1b knockdown in zebrafish embryos.

Conclusion/Significance

Collectively, these data indicate that Smyd1a and Smyd1b share similar biological activity in myofibril assembly in zebrafish embryos. However, Smyd1b appears to play a major role in this process.     

Role of intestinal bacteria in gliadin-induced changes in intestinal mucosa: study in germ-free rats

Background and Aims

Celiac disease (CD) is a chronic inflammatory disorder of the small intestine that is induced by dietary wheat gluten proteins (gliadins) in genetically predisposed individuals. The overgrowth of potentially pathogenic bacteria and infections has been suggested to contribute to CD pathogenesis. We aimed to study the effects of gliadin and various intestinal bacterial strains on mucosal barrier integrity, gliadin translocation, and cytokine production.

Methodology/Principal Findings

Changes in gut mucosa were assessed in the intestinal loops of inbred Wistar-AVN rats that were reared under germ-free conditions in the presence of various intestinal bacteria (enterobacteria and bifidobacteria isolated from CD patients and healthy children, respectively) and CD-triggering agents (gliadin and IFN-γ) by histology, scanning electron microscopy, immunofluorescence, and a rat cytokine antibody array. Adhesion of the bacterial strains to the IEC-6 rat cell line was evaluated in vitro.Gliadin fragments alone or together with the proinflammatory cytokine interferon (IFN)-γ significantly decreased the number of goblet cells in the small intestine; this effect was more pronounced in the presence of Escherichia coli CBL2 and Shigella CBD8. Shigella CBD8 and IFN-γ induced the highest mucin secretion and greatest impairment in tight junctions and, consequently, translocation of gliadin fragments into the lamina propria. Shigella CBD8 and E. coli CBL2 strongly adhered to IEC-6 epithelial cells. The number of goblet cells in small intestine increased by the simultaneous incubation of Bifidobacterium bifidum IATA-ES2 with gliadin, IFN-γ and enterobacteria. B. bifidum IATA-ES2 also enhanced the production of chemotactic factors and inhibitors of metalloproteinases, which can contribute to gut mucosal protection.

Conclusions

Our results suggest that the composition of the intestinal microbiota affects the permeability of the intestinal mucosa and, consequently, could be involved in the early stages of CD pathogenesis.