Mucociliary epithelia are essential for homeostasis of many organs and consist of mucus-secreting goblet cells and ciliated cells. Here, we present the ciliated epidermis of Xenopus embryos as a facile model system for in vivo molecular studies of mucociliary epithelial development. Using an in situ hybridization-based approach, we identified numerous genes expressed differentially in mucus-secreting cells or in ciliated cells. Focusing on genes expressed in ciliated cells, we have identified new candidate ciliogenesis factors, including several not present in the current ciliome. We find that TTC25-GFP is localized to the base of cilia and to ciliary axonemes, and disruption of TTC25 function disrupts ciliogenesis. Mig12-GFP localizes very strongly to the base of cilia and confocal imaging of this construct allows for simple visualization of the planar polarity of basal bodies that underlies polarized ciliary beating. Knockdown of Mig12 disrupts ciliogenesis. Finally, we show that ciliogenesis factors identified in the Xenopus epidermis are required in the midline to facilitate neural tube closure. These results provide further evidence of a requirement for cilia in neural tube morphogenesis and suggest that genes identified in the Xenopus epidermis play broad roles in ciliogenesis. The suites of genes identified here will provide a foundation for future studies, and may also contribute to our understanding of pathological changes in mucociliary epithelia that accompany diseases such as asthma.
Xenopus laevis tadpoles are capable of limb regeneration after amputation, in a process that initially involves the formation of a blastema. However, Xenopus has full regenerative capacity only through premetamorphic stages. We have used the Affymetrix Xenopus laevis Genome Genechip microarray to perform a large-scale screen of gene expression in the regeneration-complete, stage 53 (st53), and regeneration-incomplete, stage 57 (st57), hindlimbs at 1 and 5 days postamputation. Through an exhaustive reannotation of the Genechip and a variety of comparative bioinformatic analyses, we have identified genes that are differentially expressed between the regeneration-complete and -incomplete stages, detected the transcriptional changes associated with the regenerating blastema, and compared these results with those of other regeneration researchers. We focus particular attention on striking transcriptional activity observed in genes associated with patterning, stress response, and inflammation. Overall, this work provides the most comprehensive views yet of a regenerating limb and different transcriptional compositions of regeneration-competent and deficient tissues.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant that causes altered heart morphology, circulatory impairment, edema, hemorrhage, and early life stage mortality in fish. TCDD toxicity is dependent, in large part, on the aryl hydrocarbon receptor (AHR), but understanding of the molecular mechanism of cardiovascular embryotoxicity remains incomplete. To identify genes potentially involved in cardiovascular effects, we constructed custom cDNA microarrays consisting of 4896 zebrafish adult heart cDNA clones and over 200 genes with known developmental, toxicological and housekeeping roles. Gene expression profiles were obtained for 3-day-old zebrafish after early embryonic exposure to either 0.5 or 5.0 nM TCDD. In all, 516 clones were significantly differentially expressed (p < 0.005) under at least one treatment condition; 123 high-priority clones were selected for further investigation. Cytochromes P450 1A and 1B1, and other members of the AHR gene battery, were strongly and dose-dependently induced by TCDD. Importantly, altered expression of cardiac sarcomere components, including cardiac troponin T2 and multiple myosin isoforms, was consistent with the hypothesis that TCDD causes dilated cardiomyopathy. Observed increases in expression levels of mitochondrial energy transfer genes also may be related to cardiomyopathy. Other TCDD-responsive genes included fatty acid and steroid metabolism enzymes, ribosomal and signal-transduction proteins, and 18 expressed sequence tags (ESTs) with no known protein homologs. As the first broad-scale study of TCDD-modulated gene expression in a non-mammalian system, this work provides an important perspective on mechanisms of TCDD toxicity.
In Drosophila, the tinman gene is absolutely required for development of the dorsal vessel, the insect equivalent of the heart. In vertebrates, the tinman gene is represented by a small family of tinman-related sequences, some of which are expressed during embryonic heart development. At present however, the precise importance of this gene family for vertebrate heart development is unclear. Using the Xenopus embryo, we have employed a dominant inhibitory strategy to interfere with the function of the endogenous tinman-related genes. In these experiments, suppression of tinman gene function can result in the complete elimination of myocardial gene expression and the absence of cell movements associated with embryonic heart development. This inhibition can be rescued by expression of wild-type tinman sequences. These experiments indicate that function of tinman family genes is essential for development of the vertebrate heart.
The Drosophila bagpipe (bap) gene is involved in the specification of the musculature of the embryonic midgut. We report the isolation and characterization of a Xenopus sequence, Xbap, which is closely related to bap. Xbap is also expressed in the developing musculature of the midgut, suggesting that this developmental role of bagpipe is evolutionarily conserved. However, a second, novel role in development is suggested by the observation that Xbap is also expressed in a region of the developing facial cartilage. Using a combination of cartilage staining and comparison to the goosecoid head expression pattern, we show that Xbap expression marks the precursors to the basihyobranchial, palatoquadrate, and possibly Meckel's cartilages. This vertebrate bagpipe sequence therefore is expressed in both mesodermally and neural crest-derived tissues.
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