Background: The vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-β superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects.

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BackgroundKrüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer. We previously showed that KLF4 inhibits cell cycle progression following DNA damage and that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, and the presence of DNA double strand breaks (DSBs), centrosome amplification, chromosome aberrations and aneuploidy.MethodsTo determine whether re-expression of Klf4 corrects the observed genetic instability in MEFs null for Klf4 (Klf4−/−), we transfected Klf4−/−MEFs with Klf4-expressing plasmids and compared the results to wild type (Klf4+/+) and untransfected or mock-transfected Klf4−/−MEFs.ResultsWe show that overexpression of Klf4 in Klf4−/−MEFs reduced cell proliferation rates and the proportion of cells with DSBs, abnormal centrosome numbers, aneuploidy and micronuclei. In addition, Klf4-transfected Klf4−/−MEFs exhibited a more robust DNA damage repair response as demonstrated by the greater rate in disappearance of γ-H2AX and 53BP1 foci following γ-irradiation.ConclusionTaken together these findings provide evidence that KLF4 plays a crucial role in the maintenance of genetic stability by modulating the DNA damage response and repair processes.

In many vertebrates, extra-embryonic tissues are important signaling centers that induce and pattern the germ layers. In teleosts, the mechanism by which the extra-embryonic yolk syncytial layer (YSL) patterns the embryo is not understood. Although the Nodal-related protein Squint is expressed in the YSL, its role in this tissue is not known. We generated a series of stable transgenic lines with GFP under the control of squint genomic sequences. In all species, nodal-related genes induce their own expression through a positive feedback loop. We show that two tissue specific enhancers in the zebrafish squint gene mediate the response to Nodal signals. Expression in the blastomeres depends upon a conserved Nodal response element (NRE) in the squint first intron, while expression in the extra-embryonic enveloping layer (EVL) is mediated by an element upstream of the transcription start site. Targeted depletion experiments demonstrate that the zebrafish Nodal-related proteins Squint and Cyclops are required in the YSL for endoderm and head mesoderm formation. Thus, Nodal signals mediate interactions between embryonic and extra-embryonic tissues in zebrafish that maintain nodal-related gene expression in the margin. Our results demonstrate a high degree of functional conservation between the extra-embryonic tissues of mouse and zebrafish.

Background:The pro-proliferative Krüppel-like factor 5 (KLF5) is posttranslationally regulated. Results: SMAD ubiquitination regulatory factor 2 (SMURF2) interacts with, ubiquitinates and degrades KLF5. Conclusion: SMURF2 negatively regulates KLF5. Significance: The findings increase the understanding of the mechanisms by which KLF5 is regulated posttranslationally.