Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.

The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

Non-cell autonomous induction of apoptosis and loss of posterior structures by activation domain-specific interactions of Oct-1 in the Xenopus embryo.

Oct-1, a member of the POU family of transcription factors, is expressed at relatively high levels in ectodermal and mesodermal cell lineages during early Xenopus embryogenesis (Veenstra et al, 1995). Here we show that overexpression of Oct-1 induces programmed cell death concomitant with the loss of the posterior part of the body axis. Truncated Oct-1 variants, missing either the C-terminal or N-terminal trans-activation domain, exhibit a different capacity to cause such developmental defects. Oct-1-induced cell death is rescued in unilaterally injected embryos by non-injected cells, indicative of the non-cell autonomous character of the developmental effects of Oct-1. This was confirmed by marker gene analysis, which showed a significant decrease in brachyury expression, suggesting that Oct-1 interferes with an FGF-type signalling pathway.

XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos.

XTcf-3 is a maternally expressed Xenopus homolog of the mammalian HMG box factors Tcf-1 and Lef-1. The N-terminus of XTcf-3 binds to beta-catenin. Microinjection of XTcf-3 mRNA in embryos results in nuclear translocation of beta-catenin. The beta-catenin-XTcf-3 complex activates transcription in a transient reporter gene assay, while XTcf-3 by itself is silent. N-terminal deletion of XTcf-3 (delta N) abrogates the interaction with beta-catenin, as well as the consequent transcription activation. This dominant-negative delta N mutant suppresses the induction of axis duplication by microinjected beta-catenin. It also suppresses endogenous axis specification upon injection into the dorsal blastomeres of a 4-cell-stage embryo. We propose that signaling by beta-catenin involves complex formation with XTcf-3, followed by nuclear translocation and activation of specific XTcf-3 target genes.

Dynamic and differential Oct-1 expression during early Xenopus embryogenesis: persistence of Oct-1 protein following down-regulation of the RNA.

As a first step towards the elucidation of the role of the transcription factor Oct-1 in development, we prepared a monoclonal antibody to study the spatio-temporal distribution of Oct-1 protein in vivo. Here we report differential expression of the Oct-1 gene in the Xenopus embryo both at the RNA and the protein level. Transcripts and protein are detected in ectodermal and mesodermal cell lineages, in which the expression exhibits a pattern of progressive spatial restriction in the course of development. The Oct-1 expression as reported here is not correlated with cell density or cell proliferation in the embryo. Our results suggest a role of Oct-1 in the specification and differentiation of neuronal and neural crest cells. In many other cells, the developmental decision to down regulate Oct-1 is delayed, probably due to a high stability of the protein.

Expression of the glucocorticoid receptor gene is regulated during early embryogenesis of Xenopus laevis.

To study the possible role of the glucocorticoid receptor (GR) in early embryogenesis, we isolated a Xenopus glucocorticoid receptor cDNA from an embryonic stage 17 cDNA library. Overexpression of this Xenopus GR in COS cells confers the ability to transactivate a GRE-tk CAT promoter construct in a ligand dependent manner. Expression of the Xenopus GR gene at the RNA level was analyzed by Northern blot hybridization. Transcripts of 4 and 6 kb are present in oocytes. The 4 kb mRNA is abundant and is degraded together with the 6 kb mRNA during cleavage stages of early development. Between stages 17 and 24, GR messengers are extremely rare. From stage 32 onwards, both GR transcripts start to be expressed again at intermediate levels. These results provide the first evidence that expression of the GR gene is regulated during early embryonic development.

The promoter of the Xwnt-5C gene contains octamer and AP-2 motifs functional in Xenopus embryos.

The Xwnt-5C gene is expressed in Xenopus embryos from the early gastrula stage onwards. The transcription of Xwnt-5C is regulated differentially with respect to transcript size, timing and localization. To gain insight into the generation of the Xwnt-5C expression pattern, we started to analyze the transcriptional regulation of this gene. We isolated Xwnt-5C genomic DNA sequences. By microinjection of chimaeric reporter constructs into Xenopus embryos we demonstrate that the upstream region contains a promoter functional in vivo. Of the several putative binding sites for trans-acting factors, present in a minimal promoter fragment, some have been studied in more detail. Mutations in an octamer motif and in an AP-2 consensus sequence interfere with the activity of the Xwnt-5C minimal promoter. In vitro binding assays with extracts from gastrula stage Xenopus embryos show that the octamer motif of the Xwnt-5C promoter can bind several Octamer binding factors, one of which is Oct1.