Search

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.

Studies in various animals have shown that asymmetrically localized maternal transcripts play important roles in axial patterning and cell fate specification in early embryos. However, comprehensive analyses of the maternal transcriptomes with spatial information are scarce and limited to a handful of model organisms. In cephalochordates (amphioxus), an early branching chordate group, maternal transcripts of germline determinants form a compact granule that is inherited by a single blastomere during cleavage stages. Further blastomere separation experiments suggest that other transcripts associated with the granule are likely responsible for organizing the posterior structure in amphioxus; however, the identities of these determinants remain unknown. In this study, we used high-throughput RNA sequencing of separated blastomeres to examine asymmetrically localized transcripts in two-cell and eight-cell stage embryos of the amphioxus Branchiostoma floridae. We identified 111 and 391 differentially enriched transcripts at the 2-cell stage and the 8-cell stage, respectively, and used in situ hybridization to validate the spatial distribution patterns for a subset of these transcripts. The identified transcripts could be categorized into two major groups: (1) vegetal tier/germ granule-enriched and (2) animal tier/anterior-enriched transcripts. Using zebrafish as a surrogate model system, we showed that overexpression of one animal tier/anterior-localized amphioxus transcript, zfp665, causes a dorsalization/anteriorization phenotype in zebrafish embryos by downregulating the expression of the ventral gene, eve1, suggesting a potential function of zfp665 in early axial patterning. Our results provide a global transcriptomic blueprint for early-stage amphioxus embryos. This dataset represents a rich platform to guide future characterization of molecular players in early amphioxus development and to elucidate conservation and divergence of developmental programs during chordate evolution.

Areca nut (AN) is a popular carcinogen used by about 0.6-1.2 billion people worldwide. Although AN contains apoptosis-inducing ingredients, we previously demonstrated that both AN extract (ANE) and its 30-100 kDa fraction (ANE 30-100K) predominantly induce autophagic cell death in both normal and malignant cells. In this study, we further explored the action mechanism of ANE 30-100K-induced autophagy (AIA) in Jurkat T lymphocytes and carcinoma cell lines including OECM-1 (mouth), CE81T/VGH (esophagus), SCC25 (tongue), and SCC-15 (tongue). The results showed that chemical- and small hairpin RNA (shRNA)-mediated inhibition of AMP-activated protein kinase (AMPK) resulted in the attenuation of AIA in Jurkat T but not in OECM-1 cells. Knockdown of Atg5 and Beclin 1 expressions ameliorated AIA in OECM-1/CE81T/VGH/Jurkat T and OECM-1/SCC25/SCC-15, respectively. Furthermore, ANE 30-100K could activate caspase-3 after inhibition of Beclin 1 expression in OECM-1/SCC25/SCC15 cells. Meanwhile, AMPK was demonstrated to be the upstream activator of the extracellular-regulated kinase (ERK) in Jurkat T cells, and inhibition of MEK attenuated AIA in Jurkat T/OECM-1/CE81T/VGH cells. Finally, we also found that multiple myeloma RPMI8226, lymphoma U937, and SCC15 cells survived from long-term non-cytotoxic ANE 30-100K treatment exhibited stronger resistance against serum deprivation through upregulated autophagy. Collectively, our studies indicate that Beclin-1 and Atg5 but not AMPK are commonly required for AIA, and MEK/ERK pathway is involved in AIA. Meanwhile, it is also suggested that long-term AN usage might increase the resistance of survived tumor cells against serum-limited conditions.

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.

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.