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.

Zebrafish Cdx1b modulates epithalamic asymmetry by regulating ndr2 and lft1 expression.

Nodal signaling is essential for mesoderm and endoderm formation, as well as neural plate induction and establishment of left-right asymmetry. However, the mechanisms controlling expression of Nodal pathway genes in these contexts are not fully known. Previously, we showed that Cdx1b induces expression of downstream Nodal signaling factors during early endoderm formation. In this study, we show that Cdx1b also regulates epithalamic asymmetry in zebrafish embryos by modulating expression of ndr2 and lft1. We first knocked down cdx1b with translation-blocking and splicing-blocking morpholinos (MOs). Most embryos injected with translation-blocking MOs showed absent ndr2, lft1 and pitx2c expression in the left dorsal diencephalon during segmentation and pharyngula stages accompanied by aberrant parapineal migration and habenular laterality at 72 â€‹h post fertilization (hpf). These defects were less frequent in embryos injected with splicing-blocking MO. To confirm the morphant phenotype, we next generated both zygotic (Z)cdx1b-/- and maternal zygotic (MZ)cdx1b-/- mutants by CRISPR-Cas9 mutagenesis. Expression of ndr2, lft1 and pitx2c was absent in the left dorsal diencephalon of a high proportion of MZcdx1b-/- mutants; however, aberrant dorsal diencephalic pitx2c expression patterns were observed at low frequency in Zcdx1b-/- mutant embryos. Correspondingly, dysregulated parapineal migration and habenular laterality were also observed in MZcdx1b-/- mutant embryos at 72 hpf. On the other hand, Kupffer's vesicle cilia length and number, expression pattern of spaw in the lateral plate mesoderm and pitx2c in the gut as well as left-right patterning of various visceral organs were not altered in MZcdx1b-/- mutants compared to wild-type embryos. Chromatin immunoprecipitation revealed that Cdx1b directly regulates ndr2 and lft1 expression. Furthermore, injection of cdx1b-vivo MO1 but not cdx1b-vivo 4 â€‹mm MO1 in the forebrain ventricle at 18 hpf significantly downregulated lft1 expression in the left dorsal diencephalon at 23-24 â€‹s stages. Together, our results suggest that Cdx1b regulates transcription of ndr2 and lft1 to maintain proper Nodal activity in the dorsal diencephalon and epithalamic asymmetry in zebrafish embryos.

Zebrafish Klf4 maintains the ionocyte progenitor population by regulating epidermal stem cell proliferation and lateral inhibition.

In the skin and gill epidermis of fish, ionocytes develop alongside keratinocytes and maintain body fluid ionic homeostasis that is essential for adaptation to environmental fluctuations. It is known that ionocyte progenitors in zebrafish embryos are specified from p63+ epidermal stem cells through a patterning process involving DeltaC (Dlc)-Notch-mediated lateral inhibition, which selects scattered dlc+ cells into the ionocyte progenitor fate. However, mechanisms by which the ionocyte progenitor population is modulated remain unclear. Krüppel-like factor 4 (Klf4) transcription factor was previously implicated in the terminal differentiation of mammalian skin epidermis and is known for its bifunctional regulation of cell proliferation in a tissue context-dependent manner. Here, we report novel roles for zebrafish Klf4 in the ventral ectoderm during embryonic skin development. We found that Klf4 was expressed in p63+ epidermal stem cells of the ventral ectoderm from 90% epiboly onward. Knockdown or knockout of klf4 expression reduced the proliferation rate of p63+ stem cells, resulting in decreased numbers of p63+ stem cells, dlc-p63+ keratinocyte progenitors and dlc+ p63+ ionocyte progenitor cells. These reductions subsequently led to diminished keratinocyte and ionocyte densities and resulted from upregulation of the well-known cell cycle regulators, p53 and cdkn1a/p21. Moreover, mutation analyses of the KLF motif in the dlc promoter, combined with VP16-klf4 or engrailed-klf4 mRNA overexpression analyses, showed that Klf4 can bind the dlc promoter and modulate lateral inhibition by directly repressing dlc expression. This idea was further supported by observing the lateral inhibition outcomes in klf4-overexpressing or knockdown embryos. Overall, our experiments delineate novel roles for zebrafish Klf4 in regulating the ionocyte progenitor population throughout early stem cell stage to initiation of terminal differentiation, which is dependent on Dlc-Notch-mediated lateral inhibition.

Zebrafish VCAP1X2 regulates cardiac contractility and proliferation of cardiomyocytes and epicardial cells.

Sarcomeric signaling complexes are important to sustain proper sarcomere structure and function, however, the mechanisms underlying these processes are not fully elucidated. In a gene trap experiment, we found that vascular cell adhesion protein 1 isoform X2 (VCAP1X2) mutant embryos displayed a dilated cardiomyopathy phenotype, including reduced cardiac contractility, enlarged ventricular chamber and thinned ventricular compact layer. Cardiomyocyte and epicardial cell proliferation was decreased in the mutant heart ventricle, as was the expression of pAKT and pERK. Contractile dysfunction in the mutant was caused by sarcomeric disorganization, including sparse myofilament, blurred Z-disc, and decreased gene expression for sarcomere modulators (smyd1b, mypn and fhl2a), sarcomeric proteins (myh6, myh7, vmhcl and tnnt2a) and calcium regulators (ryr2b and slc8a1a). Treatment of PI3K activator restored Z-disc alignment while injection of smyd1b mRNA restored Z-disc alignment, contractile function and cardiomyocyte proliferation in ventricles of VCAP1X2 mutant embryos. Furthermore, injection of VCAP1X2 variant mRNA rescued all phenotypes, so long as two cytosolic tyrosines were left intact. Our results reveal two tyrosine residues located in the VCAP1X2 cytoplasmic domain are essential to regulate cardiac contractility and the proliferation of ventricular cardiomyocytes and epicardial cells through modulating pAKT and pERK expression levels.

Klf8 regulates left-right asymmetric patterning through modulation of Kupffer's vesicle morphogenesis and spaw expression.

BACKGROUND: Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos. METHODS: Zebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer's vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody. RESULTS: Heart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18-22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8△zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer's vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed. Interestingly, klf8 mutant embryos, generated by a CRISPR-Cas9 system, showed bilateral spaw expression in the LPM at late somite stages. This observation may be partly attributed to compensatory upregulation of klf12b, because klf12b knockdown reduced the percentage of klf8 mutants exhibiting bilateral spaw expression. CONCLUSIONS: Our results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer's vesicle morphogenesis and spaw expression in the left LPM.

Foxa2 and Hif1ab regulate maturation of intestinal goblet cells by modulating agr2 expression in zebrafish embryos.

Mammalian anterior gradient 2 (AGR2), an endoplasmic reticulum (ER) protein disulfide-isomerase (PDI), is involved in cancer cell growth and metastasis, asthma and inflammatory bowel disease (IBD). Mice lacking Agr2 exhibit decreased Muc2 protein in intestinal goblet cells, abnormal Paneth cell development, ileitis and colitis. Despite its importance in cancer biology and inflammatory diseases, the mechanisms regulating agr2 expression in the gastrointestinal tract remain unclear. In the present study, we investigated the mechanisms that control agr2 expression in the pharynx and intestine of zebrafish by transient/stable transgenesis, coupled with motif mutation, morpholino knockdown, mRNA rescue and ChIP. A 350 bp DNA sequence with a hypoxia-inducible response element (HRE) and forkhead-response element (FHRE) within a region -4.5 to -4.2 kbp upstream of agr2 directed EGFP expression specifically in the pharynx and intestine. No EGFP expression was detected in the intestinal goblet cells of Tg(HREM:EGFP) or Tg(FHREM:EGFP) embryos with mutated HRE or FHRE, whereas EGFP was expressed in the pharynx of Tg(HREM:EGFP), but not Tg(FHREM:EGFP), embryos. Morpholino knockdown of foxa1 (forkhead box A1) reduced agr2 levels in the pharynx, whereas knockdown of foxa2 or hif1ab decreased intestinal agr2 expression and affected the differentiation and maturation of intestinal goblet cells. These results demonstrate that Foxa1 regulates agr2 expression in the pharynx, whereas both Foxa2 and Hif1ab control agr2 expression in intestinal goblet cells to regulate maturation of these cells.

Modulation of p53 and met expression by Krüppel-like factor 8 regulates zebrafish cerebellar development.

Krüppel-like factor 8 (Klf8) is a zinc-finger transcription factor implicated in cell proliferation, and cancer cell survival and invasion; however, little is known about its role in normal embryonic development. Here, we show that Klf8 is required for normal cerebellar development in zebrafish embryos. Morpholino knockdown of klf8 resulted in abnormal cerebellar primordium morphology and the induction of p53 in the brain region at 24 hours post-fertilization (hpf). Both p53-dependent reduction of cell proliferation and augmentation of apoptosis were observed in the cerebellar anlage of 24 hpf-klf8 morphants. In klf8 morphants, expression of ptf1a in the ventricular zone was decreased from 48 to 72 hpf; on the other hand, expression of atohla in the upper rhombic lip was unaffected. Consistent with this finding, Purkinje cell development was perturbed and granule cell number was reduced in 72 hpf-klf8 morphants; co-injection of p53 MO(sp) or klf8 mRNA substantially rescued development of cerebellar Purkinje cells in klf8 morphants. Hepatocyte growth factor/Met signaling is known to regulate cerebellar development in zebrafish and mouse. We observed decreased met expression in the tectum and rhombomere 1 of 24 hpf-klf8 morphants, which was largely rescued by co-injection with klf8 mRNA. Moreover, co-injection of met mRNA substantially rescued formation of Purkinje cells in klf8 morphants at 72 hpf. Together, these results demonstrate that Klf8 modulates expression of p53 and met to maintain ptf1a-expressing neuronal progenitors, which are required for the appropriate development of cerebellar Purkinje and granule cells in zebrafish embryos.

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.

Low temperature mitigates cardia bifida in zebrafish embryos.

The coordinated migration of bilateral cardiomyocytes and the formation of the cardiac cone are essential for heart tube formation. We investigated gene regulatory mechanisms involved in myocardial migration, and regulation of the timing of cardiac cone formation in zebrafish embryos. Through screening of zebrafish treated with ethylnitrosourea, we isolated a mutant with a hypomorphic allele of mil (s1pr2)/edg5, called s1pr2(as10) (as10). Mutant embryos with this allele expressed less mil/edg5 mRNA and exhibited cardia bifida prior to 28 hours post-fertilization. Although the bilateral hearts of the mutants gradually fused together, the resulting formation of two atria and one tightly-packed ventricle failed to support normal blood circulation. Interestingly, cardia bifida of s1pr2(as10) embryos could be rescued and normal circulation could be restored by incubating the embryos at low temperature (22.5°C). Rescue was also observed in gata5 and bon cardia bifida morphants raised at 22.5 °C. The use of DNA microarrays, digital gene expression analyses, loss-of-function, as well as mRNA and protein rescue experiments, revealed that low temperature mitigates cardia bifida by regulating the expression of genes encoding components of the extracellular matrix (fibronectin 1, tenascin-c, tenascin-w). Furthermore, the addition of N-acetyl cysteine (NAC), a reactive oxygen species (ROS) scavenger, significantly decreased the effect of low temperature on mitigating cardia bifida in s1pr2(as10) embryos. Our study reveals that temperature coordinates the development of the heart tube and somitogenesis, and that extracellular matrix genes (fibronectin 1, tenascin-c and tenascin-w) are involved.

Nitrogen-doped graphene sheets grown by chemical vapor deposition: synthesis and influence of nitrogen impurities on carrier transport.

A significant advance toward achieving practical applications of graphene as a two-dimensional material in nanoelectronics would be provided by successful synthesis of both n-type and p-type doped graphene. However, reliable doping and a thorough understanding of carrier transport in the presence of charged impurities governed by ionized donors or acceptors in the graphene lattice are still lacking. Here we report experimental realization of few-layer nitrogen-doped (N-doped) graphene sheets by chemical vapor deposition of organic molecule 1,3,5-triazine on Cu metal catalyst. When reducing the growth temperature, the atomic percentage of nitrogen doping is raised from 2.1% to 5.6%. With increasing doping concentration, N-doped graphene sheet exhibits a crossover from p-type to n-type behavior accompanied by a strong enhancement of electron-hole transport asymmetry, manifesting the influence of incorporated nitrogen impurities. In addition, by analyzing the data of X-ray photoelectron spectroscopy, Raman spectroscopy, and electrical measurements, we show that pyridinic and pyrrolic N impurities play an important role in determining the transport behavior of carriers in our N-doped graphene sheets.

Gene expression profiling of breast cancer survivability by pooled cDNA microarray analysis using logistic regression, artificial neural networks and decision trees.

BACKGROUND: Microarray technology can acquire information about thousands of genes simultaneously. We analyzed published breast cancer microarray databases to predict five-year recurrence and compared the performance of three data mining algorithms of artificial neural networks (ANN), decision trees (DT) and logistic regression (LR) and two composite models of DT-ANN and DT-LR. The collection of microarray datasets from the Gene Expression Omnibus, four breast cancer datasets were pooled for predicting five-year breast cancer relapse. After data compilation, 757 subjects, 5 clinical variables and 13,452 genetic variables were aggregated. The bootstrap method, Mann-Whitney U test and 20-fold cross-validation were performed to investigate candidate genes with 100 most-significant p-values. The predictive powers of DT, LR and ANN models were assessed using accuracy and the area under ROC curve. The associated genes were evaluated using Cox regression. RESULTS: The DT models exhibited the lowest predictive power and the poorest extrapolation when applied to the test samples. The ANN models displayed the best predictive power and showed the best extrapolation. The 21 most-associated genes, as determined by integration of each model, were analyzed using Cox regression with a 3.53-fold (95% CI: 2.24-5.58) increased risk of breast cancer five-year recurrence. CONCLUSIONS: The 21 selected genes can predict breast cancer recurrence. Among these genes, CCNB1, PLK1 and TOP2A are in the cell cycle G2/M DNA damage checkpoint pathway. Oncologists can offer the genetic information for patients when understanding the gene expression profiles on breast cancer recurrence.

Zebrafish Agr2 is required for terminal differentiation of intestinal goblet cells.

BACKGROUND: Mammalian Anterior Gradient 2 (AGR2) is a protein disulfide isomerase that is required for the production of intestinal mucus and Paneth and goblet cell homeostasis. However, whether increased endoplasmic reticulum (ER) stress occurs in Agr2(-/-) mice remains a controversial issue. METHODOLOGY/PRINCIPAL FINDINGS: We characterized the function of zebrafish agr2 by both morpholino antisense oligomer-mediated knockdown and agr2 mRNA overexpression. Fluorescent whole-mount double in situ hybridization indicated that in the intestine, agr2 was only expressed in goblet cells. Significantly increased numbers of immature Alcian blue-stained goblet cells were observed in the intestines of 104- and 120-hours post fertilization (hpf) agr2 morphants. Transmission electron microscopy analyses further confirmed the existence of immature pre-goblet cells containing few mucous granules in the mid-intestines of 104- and 120-hpf agr2 morphants. agr2 expression was not significantly induced by an ER stress inducer, tunicamycin. Expression of the ER chaperone gene hspa5, the spliced form of xbp1s, c/enhancer binding protein homologous protein chop, and the activating transcription factor 4b1 atf4b1 were not significantly induced in either 104-hpf agr2 morphants or agr2-overexpressed embryos. Similar percentages of P-Histone H3-stained M phase cells were identified in intestines of 104-hpf agr2 morphants and control embryos. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that in contrast to mouse AGR2, zebrafish Agr2 is expressed in only one intestinal secretory cell type - the goblet cells. Agr2 is essential for terminal differentiation of intestinal goblet cells in zebrafish embryos. Either knockdown of agr2 function or agr2 overexpression could not extensively induce expression of members of the unfolded protein response pathway.

ß-Lapachone induces heart morphogenetic and functional defects by promoting the death of erythrocytes and the endocardium in zebrafish embryos.

BACKGROUND: ß-Lapachone has antitumor and wound healing-promoting activities. To address the potential influences of various chemicals on heart development of zebrafish embryos, we previously treated zebrafish embryos with chemicals from a Sigma LOPAC1280™ library and found several chemicals including ß-lapachone that affected heart morphogenesis. In this study, we further evaluated the effects of ß-lapachone on zebrafish embryonic heart development. METHODS: Embryos were treated with ß-lapachone or dimethyl sulfoxide (DMSO) at 24 or 48 hours post fertilization (hpf) for 4 h at 28°C. Heart looping and valve development was analyzed by whole-mount in situ hybridization and histological analysis. For fractional shortening and wall shear stress analyses, AB and Tg (gata1:DsRed) embryos were recorded for their heart pumping and blood cell circulations via time-lapse fluorescence microscopy. Dextran rhodamine dye injection into the tail reticular cells was used to analyze circulation. Reactive oxygen species (ROS) was analyzed by incubating embryos in 5-(and 6-)-chloromethyl-2',7'-dichloro-dihydrofluorescein diacetate (CM-H2DCFDA) and recorded using fluorescence microscopy. o-Dianisidine (ODA) staining and whole mount in situ hybridization were used to analyze erythrocytes. TUNEL assay was used to examine DNA fragmentation. RESULTS: We observed a linear arrangement of the ventricle and atrium, bradycardia arrhythmia, reduced fractional shortening, circulation with a few or no erythrocytes, and pericardial edema in ß-lapachone-treated 52-hpf embryos. Abnormal expression patterns of cmlc2, nppa, BMP4, versican, and nfatc1, and histological analyses showed defects in heart-looping and valve development of ß-lapachone-treated embryos. ROS production was observed in erythrocytes and DNA fragmentation was detected in both erythrocytes and endocardium of ß-lapachone-treated embryos. Reduction in wall shear stress was uncovered in ß-lapachone-treated embryos. Co-treatment with the NQO1 inhibitor, dicoumarol, or the calcium chelator, BAPTA-AM, rescued the erythrocyte-deficiency in circulation and heart-looping defect phenotypes in ß-lapachone-treated embryos. These results suggest that the induction of apoptosis of endocardium and erythrocytes by ß-lapachone is mediated through an NQO1- and calcium-dependent pathway. CONCLUSIONS: The novel finding of this study is that ß-lapachone affects heart morphogenesis and function through the induction of apoptosis of endocardium and erythrocytes. In addition, this study further demonstrates the importance of endocardium and hemodynamic forces on heart morphogenesis and contractile performance.

Zebrafish krüppel-like factor 4a represses intestinal cell proliferation and promotes differentiation of intestinal cell lineages.

BACKGROUND: Mouse krüppel-like factor 4 (Klf4) is a zinc finger-containing transcription factor required for terminal differentiation of goblet cells in the colon. However, studies using either Klf4(-/-) mice or mice with conditionally deleted Klf4 in their gastric epithelia showed different results in the role of Klf4 in epithelial cell proliferation. We used zebrafish as a model organism to gain further understanding of the role of Klf4 in the intestinal cell proliferation and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: We characterized the function of klf4a, a mammalian klf4 homologue by antisense morpholino oligomer knockdown. Zebrafish Klf4a shared high amino acid similarities with human and mouse Klf4. Phylogenetic analysis grouped zebrafish Klf4a together with both human and mouse Klf4 in a branch with high bootstrap value. In zebrafish, we demonstrate that Klf4a represses intestinal cell proliferation based on results of BrdU incorporation, p-Histone 3 immunostaining, and transmission electron microscopy analyses. Decreased PepT1 expression was detected in intestinal bulbs of 80- and 102-hours post fertilization (hpf) klf4a morphants. Significant reduction of alcian blue-stained goblet cell number was identified in intestines of 102- and 120-hpf klf4a morphants. Embryos treated with γ-secretase inhibitor showed increased klf4a expression in the intestine, while decreased klf4a expression and reduction in goblet cell number were observed in embryos injected with Notch intracellular domain (NICD) mRNA. We were able to detect recovery of goblet cell number in 102-hpf embryos that had been co-injected with both klf4a and Notch 1a NICD mRNA. CONCLUSIONS/SIGNIFICANCE: This study provides in vivo evidence showing that zebrafih Klf4a is essential for the repression of intestinal cell proliferation. Zebrafish Klf4a is required for the differentiation of goblet cells and the terminal differentiation of enterocytes. Moreover, the regulation of differentiation of goblet cells in zebrafish intestine by Notch signaling at least partially mediated through Klf4a.

Zebrafish cdx1b regulates differentiation of various intestinal cell lineages.

Both antisense morpholino oligonucleotide (MO)-mediated knockdown and overexpression experiments were performed to analyze zebrafish cdx1b's function in intestinal cell differentiation. Substantial reductions in goblet cell numbers were detected in intestines of 102- and 120-hours post-fertilization (hpf) cdx1b MO-injected embryos (morphants) compared to cdx1b-4-base mismatched (4mm)-MO-injected and wild type embryos. A significant decrease in enteroendocrine cell numbers was also observed in intestines of 96-hpf cdx1b morphants. Furthermore, ectopic cdx1b expression caused notable increases in respective cell numbers of enteroendocrine and goblet cells in intestines of 96- and 98-hpf injected embryos. Decreased PepT1 expression was detected in enterocytes of intestines in cdx1b morphants from 80 to 102 hr of development. In addition, increased cell proliferation was detected in intestines of cdx1b morphants. Overall, our results suggest that zebrafish cdx1b plays important roles in regulating intestinal cell proliferation and the differentiation of various intestinal cell lineages.

Zebrafish cdx1b regulates expression of downstream factors of Nodal signaling during early endoderm formation.

We identified a zebrafish caudal-related homeobox (cdx1b) gene, which shares syntenic conservation with both human and mouse Cdx1. Zebrafish cdx1b transcripts are maternally deposited. cdx1b is uniformly expressed in both epiblast and hypoblast cells from late gastrulation to the 1-2s stages and can be identified in the retinas, brain and somites during 18-22 hpf stages. After 28 hours of development, cdx1b is exclusively expressed in the developing intestine. Both antisense morpholino oligonucleotide-mediated knockdown and overexpression experiments were conducted to analyze cdx1b function. Hypoplastic development of the liver and pancreas and intestinal abnormalities were observed in 96 hpf cdx1b morphants. In 85% epiboly cdx1b morphants, twofold decreases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors were identified. Furthermore, ectopic cdx1b expression caused substantial increases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors and altered their distribution patterns in 85% epiboly injected embryos. Conserved Cdx1-binding motifs were identified in both gata5 and foxa2 genes by interspecific sequence comparisons. Cdx1b can bind to the Cdx1-binding motif located in intron 1 of the foxa2 gene based on an electrophoretic mobility shift assay. Co-injection of either zebrafish or mouse foxa2 mRNA with the cdx1b MO rescued the expression domains of ceruloplasmin in the liver of 53 hpf injected embryos. These results indicate that zebrafish cdx1b regulates foxa2 expression and may also modulate gata5 expression, thus affecting early endoderm formation. This study underscores a novel role of zebrafish cdx1b in the development of different digestive organs compared with its mammalian homologs.

Characterization of the agr2 gene, a homologue of X. laevis anterior gradient 2, from the zebrafish, Danio rerio.

We characterized a zebrafish (Danio rerio) anterior gradient 2 homologue (agr2) gene. agr2 contains an open reading frame of 513bp encoding 171 amino acids. Deduced amino acid sequence comparison showed that the zebrafish agr2 protein shares high (80-89%) amino acid sequence similarity with those homologues of anterior gradient 2 (HAGR2, MAgr2, Tagr2, and Sagr2) from the human, mouse, pufferfish, and Atlantic salmon, while sharing less (67-71%) sequence similarity with those anterior gradient 2 genes (XAG-2, XAG-1, XAgr2, MAgr3, and HAGR3) from Xenopus laevis, mouse, and human. Both phylogenetic and syntenic analyses indicate that zebrafish agr2 is the orthologue of human AGR2 and mouse Agr2 genes. Whole-mount in situ hybridization indicated that zebrafish agr2 is expressed in most organs, such as epidermis, olfactory bulbs, otic vesicles, pharynx, esophagus, pneumatic duct, swim bladder, and intestine, which contain mucus-secreting cells. Moreover, semi-quantitative RT-PCR demonstrated agr2 is expressed in the gill, pharynx/esophagus, swim bladder/pneumatic duct, and intestine in the adult fish. In contrast, Xenopus anterior gradient 2 homologues are mainly expressed in ectoderm-derived organs including the cement gland and otic vesicles, while human and mouse anterior gradient 2 orthologues are mainly distributed in endoderm-derived organs including the trachea, lungs, stomach, intestines, and colon.

Heart-targeted overexpression of Nip3a in zebrafish embryos causes abnormal heart development and cardiac dysfunction.

We transiently expressed a proapoptotic protein, Nip3a, by a heart-specific BMP4 promoter in zebrafish embryos and generated two variants of embryos with abnormal heart phenotypes (A and B). Embryos with phenotype A heart defects showed hypoplastic or elongated ventricles, elongated or enlarged atriums with no normal cardiac looping resulting a significant longer SV-BA distance, and bradycardia at 48 h post-fertilization (hpf). Embryos with phenotype B heart defects showed an enlarged fluid-filled pericardium, severe hypoplasia, non-contracting ventricles, and elongated or enlarged slowly beating atriums with no normal looping. Histological sections further revealed the absence of a proper atrioventricular boundary and no endocardial cells lining this region in both 48- and 72-hpf Nip3a-overexpressing embryos, implicating defective endocardial cushion formation. These phenotypes are reminiscent of atrioventricular canal defects in humans. In addition, induced apoptotic myocardium cells were clustered in the presumptive atrioventricular boundary as well as in the adjacent ventricle and atrium of 48- and 72-hpf Nip3a-overexpressing embryos. Nip3a expression was readily detected in 80% epiboly BMP4-Nip3a-injected embryos, and defects in heart development were observed in both the linear heart tube and subsequent chamber formation stages. These results showed that myocyte apoptosis is a universal pathogenic factor for congenital heart failure using zebrafish as a model organism.