Abstract
BackgroundA complex network of signaling pathways and transcription factors regulates vertebrate mesoderm development. Zebrafish mutants provide a powerful tool for examining the roles of individual genes in such a network. spadetail (spt) is a mutant with a lesion in tbx16, a T-box transcription factor involved in mesoderm development; the mutant phenotype includes disrupted primitive red blood cell formation as well as disrupted somitogenesis. Despite much recent progress, the downstream targets of tbx16 remain incompletely understood. The current study was carried out to test whether any of the five major signaling pathways are regulated by tbx16 during two specific stages of mesoderm development: primitive red blood cell formation in the intermediate mesoderm and somite formation in the tail paraxial mesoderm. This test was performed using Gene Set Enrichment Analysis, which identifies coordinated changes in expression among a priori sets of genes associated with biological features or processes.ResultsOur Gene Set Enrichment Analysis results identify Wnt and retinoic acid signaling as likely downstream targets of tbx16 in the developing zebrafish intermediate mesoderm, the site of primitive red blood cell formation. In addition, such results identify retinoic acid signaling as a downstream target of tbx16 in the developing zebrafish posterior somites. Finally, using candidate gene identification and in situ hybridization, we provide expression domain information for 25 additional genes downstream of tbx16 that are outside of both pathways; 23 were previously unknown downstream targets of tbx16, and seven had previously uncharacterized expression in zebrafish.ConclusionsOur results suggest that (1) tbx16 regulates Wnt signaling in the developing zebrafish intermediate mesoderm, the site of primitive red blood cell formation, and (2) tbx16 regulates retinoic acid signaling at two distinct embryonic locations and developmental stages, which may imply ongoing spatio-temporal regulation throughout mesoderm development.
Highlights
A complex network of signaling pathways and transcription factors regulates vertebrate mesoderm development
T-box genes are a family of transcription factors, distinguished by a DNA binding domain, that regulate numerous developmental processes; the gene family likely arose in the common ancestor of metazoans, and vertebrates possess approximately 20 members [11]. spt mutants lack trunk paraxial mesoderm because the appropriate mesodermal precursor cells mismigrate during gastrulation, localizing in the tailbud instead of converging dorsally to populate the trunk [12]. spt mutants have severely compromised primitive and definitive red blood cell formation [13,14] and irregular vasculature [15]; in some cases, they lack pectoral fins, an anus, and a pronephric kidney [14]
Gene Set Enrichment Analysis GSEA [28] was performed to determine whether specific signaling pathways specified a priori were altered in spt at different developmental stages
Summary
A complex network of signaling pathways and transcription factors regulates vertebrate mesoderm development. Spadetail (spt) is a mutant with a lesion in tbx, a T-box transcription factor involved in mesoderm development; the mutant phenotype includes disrupted primitive red blood cell formation as well as disrupted somitogenesis. The current study was carried out to test whether any of the five major signaling pathways are regulated by tbx during two specific stages of mesoderm development: primitive red blood cell formation in the intermediate mesoderm and somite formation in the tail paraxial mesoderm. This test was performed using Gene Set Enrichment Analysis, which identifies coordinated changes in expression among a priori sets of genes associated with biological features or processes. The zebrafish spt mutant is an excellent system in which to examine the molecular mechanisms underlying specific mesoderm-derived structures in an otherwise largely unaffected embryo
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