Transcription activator-like effector nucleases (TALENs) are an approach for directed gene disruption and have been proved to be effective in various animal models. Here, we report that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germ-line transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for RNA transcription and microinjection into Xenopus embryos. Eight pairs of TALENs were constructed to target eight Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci. Furthermore, mutations induced by TALENs were highly efficiently passed through the germ line to F 1 frogs. Together with simple and reliable PCR-based approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus.genome editing | heritable mutagenesis | mutagenesis detection | reverse genetics | genome engineering A mong current animal models, Xenopus laevis and Xenopus tropicalis are classical animal models widely used in the study of embryonic development. However, because of the lack of methodologies for homologous recombination and embryonic stem cell derivation, it is difficult to perform specific gene targeting in these two models, which has impeded their use in genetic studies. Recently, site-specific gene targeting with transcription activator-like effector nucleases (TALENs) has been successfully applied in several animal models including rat, zebrafish, and Caenorhabditis elegans (1-4). Similar to zinc finger nucleases (ZFNs) (5), TALENs are engineered DNA nucleases that consist of a custom-designed DNA-binding domain and a nonspecific nuclease domain derived from Fok I endonuclease. Binding of adjacent TALENs allows dimerization of the endonuclease domains, leading to double-strand breaks at the predetermined site (6). These double-strand DNA breaks are frequently repaired through nonhomologous end joining (NHEJ) (7, 8), resulting in deletion or insertion (indel) mutations. The DNA binding specificity of TALENs, as distinct from ZFNs, is based on the transcription activator-like effectors (TALEs) from Xanthomonas plant pathogens (9, 10). The TALE proteins consist of an N-terminal translocation domain, a nuclear localization signal, and various numbers of tandem 34-aa repeats that determine the DNA binding specificity. Each repeat in the tandem array is identical except for two variable amino acid residues at positions 12 and 13 called repeat variable di-residues (RVDs), through which each repeat independently determines the targeted base (11,12). It is known that the RVDs NI, NG, HD, and NN preferentially recognize adenine (A), thymine (T), cytosine (C), and guanine (G)/adenine (A), respectively (13). With a given repeat combination, the TALE recognizes a specific target sequence predicted by this code. A pair of TALENs can then cleave doublestrand DNA between the two targ...

The competence of a cell to respond to the signalling molecule retinoic acid (RA) is thought to depend largely on its repertoire of cognate zinc finger nuclear receptors. XCYP26 is an RA hydroxylase that is expressed differentially during early Xenopus development. In Xenopus embryos, XCYP26 can rescue developmental defects induced by application of exogenous RA, suggesting that the enzymatic modifications introduced inhibit RA signalling activities in vivo. Alterations in the expression pattern of a number of different molecular markers for neural development induced upon ectopic expression of XCYP26 reflect a primary function of RA signalling in hindbrain development. Progressive inactivation of RA signalling results in a stepwise anteriorization of the molecular identity of individual rhombomeres. The expression pattern of XCYP26 during gastrulation appears to define areas within the prospective neural plate that develop in response to different concentrations of RA. Taken together, these observations appear to reflect an important regulatory function of XCYP26 for RA signalling; XCYP26‐mediated modification of RA modulates its signalling activity and helps to establish boundaries of differentially responsive and non‐responsive territories.

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Retinoic acid (RA) metabolizing enzymes play important roles in RA signaling during vertebrate embryogenesis. We have previously reported on a RA degrading enzyme, XCYP26, which appears to be critical for the anteroposterior patterning of the central nervous system (EMBO J. 17 (1998) 7361). Here, we report on the sequence, expression and function of its counterpart, XRALDH2, a RA generating enzyme in Xenopus. During gastrulation and neurulation, XRALDH2 and XCYP26 show non-overlapping, complementary expression domains. Upon misexpression, XRALDH2 is found to reduce the forebrain territory and to posteriorize the molecular identity of midbrain and individual hindbrain rhombomeres in Xenopus embryos. Furthermore, ectopic XRALDH2, in combination with its substrate, all-trans-retinal (ATR), can mimic the RA phenotype to result in microcephalic embryos. Taken together, our data support the notion that XRALDH2 plays an important role in RA homeostasis by the creation of a critical RA concentration gradient along the anteroposterior axis of early embryos, which is essential for proper patterning of the central nervous system in Xenopus.

Retinoic acid (RA) is a morphogen derived from retinol (vitamin A) that plays important roles in cell growth, differentiation, and organogenesis. The production of RA from retinol requires two consecutive enzymatic reactions catalyzed by different sets of dehydrogenases. The retinol is first oxidized into retinal, which is then oxidized into RA. The RA interacts with retinoic acid receptor (RAR) and retinoic acid X receptor (RXR) which then regulate the target gene expression. In this review, we have discussed the metabolism of RA and the important components of RA signaling pathway, and highlighted current understanding of the functions of RA during early embryonic development.

For the emerging amphibian genetic model Xenopus tropicalis targeted gene disruption is dependent on zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), which require either complex design and selection or laborious construction. Thus, easy and efficient genome editing tools are still highly desirable for this species. Here, we report that RNA-guided Cas9 nuclease resulted in precise targeted gene disruption in all ten X. tropicalis genes that we analyzed, with efficiencies above 45% and readily up to 100%. Systematic point mutation analyses in two loci revealed that perfect matches between the spacer and the protospacer sequences proximal to the protospacer adjacent motif (PAM) were essential for Cas9 to cleave the target sites in the X. tropicalis genome. Further study showed that the Cas9 system could serve as an efficient tool for multiplexed genome engineering in Xenopus embryos. Analysis of the disruption of two genes, ptf1a/p48 and tyrosinase, indicated that Cas9-mediated gene targeting can facilitate direct phenotypic assessment in X. tropicalis embryos. Finally, five founder frogs from targeting of either elastase-T1, elastase-T2 or tyrosinase showed highly efficient transmission of targeted mutations into F1 embryos. Together, our data demonstrate that the Cas9 system is an easy, efficient and reliable tool for multiplex genome editing in X. tropicalis.

Patterning of the embryonic endoderm into distinct sets of precursor cells involves the precisely regulated activities of key transcription regulators. Ectopic, pan-endodermal activation of XPtf1a/p48 during pancreas precursor cell stages of Xenopus embryogenesis results in an expansion of the pancreatic territory, precisely within the borders of XlHbox8 expression. A combination of both activities is sufficient to expand the pancreatic precursor cell population also into more posterior portions of the endoderm. Both treatments result in the formation of a giant pancreas that persists up to late tadpole stages of development and carries both supernumerary endocrine and exocrine cells. A combination of XPtf1a/ p48 and XlHbox8 is thus sufficient to convert nonpancreatic endodermal cells into pancreatic precursor cells. The vertebrate pancreas develops from one dorsal and two ventral evaginations in the endodermal epithelium (Slack 1995). Early pancreatic precursor cells express Pdx1, a homeodomain protein that is also expressed in the adjacent presumptive stomach and duodenum (Ohlsson et al. 1993;Jonsson et al. 1994), as well as Ptf1a/p48, a basic helix-loop-helix (bHLH) transcription factor (Kawaguchi et al. 2002). Lineage tracing studies have indicated that the Pdx1-expressing cells represent precursor cells that contribute to the formation of both endocrine and exocrine lineages of the mature pancreas (Gu et al. 2002). In Pdx1-homozygous mutant mice, pancreatic buds seem to be arrested in their development; however, early glucagon-and insulin-expressing cells can still be detected (Ahlgren et al. 1996;Offield et al. 1996). Such mice exhibit additional malformations in stomach and duodenum, where Pdx1 is normally expressed. Interestingly, and of primary relevance for the results reported in this communication, ectopic expression of Pdx1 in nonpancreatic chicken endoderm resulted in the initiation of pancreatic budding, but it was not sufficient to promote differentiation of either exocrine or endocrine cells (Grapin-Botton et al. 2001). Ptf1a/p48 is a bHLH transcription factor, which was originally identified as a part of a heterotrimeric pancreas transcription factor complex, referred to as PTF1 that activates transcription of exocrine specific pancreatic genes in the mature pancreas (Cockell et al. 1989;Beres et al. 2006). Mice bearing a null mutation of Ptf1a/ p48 are completely devoid of exocrine pancreas, while endocrine pancreatic cells still form, but are found to be translocated to the spleen (Krapp et al. 1998). More recent studies have revealed that Ptf1a/p48 is already expressed in pancreatic precursor cells which contribute to all pancreatic cell types, and that, in the absence of Ptf1a/p48, pancreatic precursor cells adopt a duodenal fate (Kawaguchi et al. 2002). These findings suggest a role for Ptf1a/p48 that is not solely in exocrine differentiation, but that is also relevant for cells of the endocrine lineages.Here, we report on observations that are in strong support of the concept that a combi...

Cancer cells are immature cells resulting from cellular reprogramming by gene misregulation, and redifferentiation is expected to reduce malignancy. It is unclear, however, whether cancer cells can undergo terminal differentiation. Here, we show that inhibition of the epigenetic modification enzyme enhancer of zeste homolog 2 (EZH2), histone deacetylases 1 and 3 (HDAC1 and -3), lysine demethylase 1A (LSD1), or DNA methyltransferase 1 (DNMT1), which all promote cancer development and progression, leads to postmitotic neuron-like differentiation with loss of malignant features in distinct solid cancer cell lines. The regulatory effect of these enzymes in neuronal differentiation resided in their intrinsic activity in embryonic neural precursor/progenitor cells. We further found that a major part of pan-cancer-promoting genes and the signal transducers of the pan-cancer-promoting signaling pathways, including the epithelial-to-mesenchymal transition (EMT) mesenchymal marker genes, display neural specific expression during embryonic neurulation. In contrast, many tumor suppressor genes, including the EMT epithelial marker gene that encodes cadherin 1 (), exhibited non-neural or no expression. This correlation indicated that cancer cells and embryonic neural cells share a regulatory network, mediating both tumorigenesis and neural development. This observed similarity in regulatory mechanisms suggests that cancer cells might share characteristics of embryonic neural cells.