Yu-Fen Lu

Zebrafish cdx1b regulates differentiation of various intestinal cell lineages.

Both antisense morpholino oligonucleotide (MO)‐mediated knockdown and overexpression experiments were performed to analyze zebrafish cdx1bs 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. Developmental Dynamics 238:1021–1032, 2009.

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.

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.

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 MOsp 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.

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 s1pr2as10 (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 s1pr2as10 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 s1pr2as10 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.

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.