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.

Study of Highly Efficient Au/Pt Nanoparticles for Rapid Screening of Clostridium difficile.

This study synthesized core/shell gold-platinum nanoparticles and characterized their colorimetric properties; ultraviolet-visible spectroscopy revealed that the synthesized nanoparticles exhibited distinct colors from conventional gold nanoparticles. Furthermore, the nanoparticles were subjected to lateral flow assays using Protein A, and the results revealed that they outperformed conventional spherical gold nanoparticles in terms of color development. This improvement can be attributed to the distinct core/shell structures of our nanoparticles. Further evaluation revealed that these nanoparticles could facilitate the detection of Clostridium difficile Toxin B visually at an extremely low concentration (1 ng/mL) without the requirement for advanced instrumentation. This substantial improvement in sensitivity can be attributed to the meticulous design and nanoscale engineering of the structure of the nanoparticles.

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.

FASTR3D: a fast and accurate search tool for similar RNA 3D structures.

FASTR3D is a web-based search tool that allows the user to fast and accurately search the PDB database for structurally similar RNAs. Currently, it allows the user to input three types of queries: (i) a PDB code of an RNA tertiary structure (default), optionally with specified residue range, (ii) an RNA secondary structure, optionally with primary sequence, in the dot-bracket notation and (iii) an RNA primary sequence in the FASTA format. In addition, the user can run FASTR3D with specifying additional filtering options: (i) the released date of RNA structures in the PDB database, and (ii) the experimental methods used to determine RNA structures and their least resolutions. In the output page, FASTR3D will show the user-queried RNA molecule, as well as user-specified options, followed by a detailed list of identified structurally similar RNAs. Particularly, when queried with RNA tertiary structures, FASTR3D provides a graphical display to show the structural superposition of the query structure and each of identified structures. FASTR3D is now available online at http://bioalgorithm.life.nctu.edu.tw/FASTR3D/.

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.

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.