Sheng-Ping L. Hwang

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.

Recapitulation of zebrafish sncga expression pattern and labeling the habenular complex in transgenic zebrafish using green fluorescent protein reporter gene

Human synuclein family consists of α‐, β‐, and γ‐synucleins. Here, we cloned three genes, sncb, sncga and sncgb from zebrafish. They encode β‐, γ1‐, and γ2‐synucleins, respectively. The zSyn‐β, zSyn‐γ1, and zSyn‐γ2 proteins display 69%, 47%, and 50% identity to human β‐synuclein and γ‐synuclein, respectively. By reverse transcriptase‐polymerase chain reaction, we demonstrated that sncb and sncga mRNA were abundant in brain and eye, while sncgb expression was moderate in brain, kidney, ovary and testis. The 1.8‐kb 5′‐upstream/promoter region of the sncga gene was sufficient to direct green fluorescent protein (GFP) expression in the central nervous system and cranial ganglions. A transgenic line, Tg(sncga:GFP), was generated and its GFP expression is similar to that of endogenous sncga mRNA. Moreover, this line also labels the habenular complex and the domain of GFP expression is larger in the left than in the right habenula. Thus, this line can be used to study sncga gene regulation and for left–right asymmetry study in zebrafish brain. Developmental Dynamics 238:746–754, 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 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.

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.

Control of Wnt5b secretion by Wntless modulates chondrogenic cell proliferation through fine-tuning fgf3 expression.

ABSTRACT Wnts and Fgfs regulate various tissues development in vertebrates. However, how regional Wnt or Fgf activities are established and how they interact in any given developmental event is elusive. Here, we investigated the Wnt-mediated craniofacial cartilage development in zebrafish and found that fgf3 expression in the pharyngeal pouches is differentially reduced along the anteroposterior axis in wnt5b mutants and wntless (wls) morphants, but its expression is normal in wnt9a and wnt11 morphants. Introducing fgf3 mRNAs rescued the cartilage defects in Wnt5b- and Wls-deficient larvae. In wls morphants, endogenous Wls expression is not detectable but maternally deposited Wls is present in eggs, which might account for the lack of axis defects in wls morphants. Secretion of endogenous Wnt5b but not Wnt11 was affected in the pharyngeal tissue of Wls morphants, indicating that Wls is not involved in every Wnt secretion event. Furthermore, cell proliferation but not apoptosis in the developing jaw was affected in Wnt5b- and Wls-deficient embryos. Therefore, Wnt5b requires Wls for its secretion and regulates the proliferation of chondrogenic cells through fine-tuning the expression of fgf3 during jaw cartilage development. Summary: Wntless-mediated Wnt5b secretion modulates the proliferation of chondrogenic cells through fine-tuning regional Fgf3 expression during the development of craniofacial cartilages.

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.

Protein tyrosine phosphatase receptor type O (Ptpro) regulates cerebellar formation during zebrafish development through modulating Fgf signaling

Protein activities controlled by receptor protein tyrosine phosphatases (RPTPs) play comparably important roles in transducing cell surface signals into the cytoplasm by protein tyrosine kinases. Previous studies showed that several RPTPs are involved in neuronal generation, migration, and axon guidance in Drosophila, and the vertebrate hippocampus, retina, and developing limbs. However, whether the protein tyrosine phosphatase type O (ptpro), one kind of RPTP, participates in regulating vertebrate brain development is largely unknown. We isolated the zebrafish ptpro gene and found that its transcripts are primarily expressed in the embryonic and adult central nervous system. Depletion of zebrafish embryonic Ptpro by antisense morpholino oligonucleotide knockdown resulted in prominent defects in the forebrain and cerebellum, and the injected larvae died on the 4th day post-fertilization (dpf). We further investigated the function of ptpro in cerebellar development and found that the expression of ephrin-A5b (efnA5b), a Fgf signaling induced cerebellum patterning factor, was decreased while the expression of dusp6, a negative-feedback gene of Fgf signaling in the midbrain-hindbrain boundary region, was notably induced in ptpro morphants. Further analyses demonstrated that cerebellar defects of ptpro morphants were partially rescued by inhibiting Fgf signaling. Moreover, Ptpro physically interacted with the Fgf receptor 1a (Fgfr1a) and dephosphorylated Fgfr1a in a dose-dependant manner. Therefore, our findings demonstrate that Ptpro activity is required for patterning the zebrafish embryonic brain. Specifically, Ptpro regulates cerebellar formation during zebrafish development through modulating Fgf signaling.

Identification and characterization of bone morphogenetic protein 2/4 gene from the starfish Archaster typicus

A bone morphogenetic protein 2/4 (BMP2/4) gene has been cloned from the starfish, Archaster typicus, for the purpose of investigating the expression pattern of the BMP4 gene in echinoderm embryos which do not produce micromeres. The isolated gene (named AtBMP2/4) contained two exons that encoded the entire coding region. The deduced AtBMP2/4 protein sequence contained 509 amino acids. Sequence comparison showed that it shared high amino acid similarity with sea urchin BMP2/4 and Xenopus BMP2 and BMP4. Northern blot analyses indicated that AtBMP2/4 mRNA initially appears at the blastula stage and has a maximal expression level at the gastrula stage. Whole-mount in situ hybridization revealed that AtBMP2/4 mRNA is expressed in the archenteron, coelomic vesicles, and ectodermal cells of gastrula stage embryos. The observed spatial distribution pattern vastly differs from that of sea urchin SpBMP2/4, which is expressed mainly in the oral ectoderm region of the mesenchyme blastula and early gastrula embryos.

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.