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TALENs mediate efficient and heritable mutation of endogenous genes in the marine annelid Platynereis dumerilii.

ABSTRACT: Platynereis dumerilii is a marine polychaete and an established model system for studies of evolution and development. Platynereis is also a re-emerging model for studying the molecular basis of circalunar reproductive timing: a biological phenomenon observed in many marine species. While gene expression studies have provided new insight into patterns of gene regulation, a lack of reverse genetic tools has so far limited the depth of functional analyses in this species. To address this need, we established customized transcriptional activator-like effector nucleases (TALENs) as a tool to engineer targeted modifications in Platynereis genes. By adapting a workflow of TALEN construction protocols and mutation screening approaches for use in Platynereis, we engineered frameshift mutations in three endogenous Platynereis genes. We confirmed that such mutations are heritable, demonstrating that TALENs can be used to generate homozygous knockout lines in P. dumerilii. This is the first use of TALENs for generating genetic knockout mutations in an annelid model. These tools not only open the door for detailed in vivo functional analyses, but also can facilitate further technical development, such as targeted genome editing.

SUBMITTER: Bannister S

PROVIDER: S-EPMC4012502 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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TALENs mediate efficient and heritable mutation of endogenous genes in the marine annelid Platynereis dumerilii.

Bannister Stephanie S Antonova Olga O Polo Alessandra A Lohs Claudia C Hallay Natalia N Valinciute Agne A Raible Florian F Tessmar-Raible Kristin K

Genetics 20140320 1

Platynereis dumerilii is a marine polychaete and an established model system for studies of evolution and development. Platynereis is also a re-emerging model for studying the molecular basis of circalunar reproductive timing: a biological phenomenon observed in many marine species. While gene expression studies have provided new insight into patterns of gene regulation, a lack of reverse genetic tools has so far limited the depth of functional analyses in this species. To address this need, we ...[more]

PMID: 24653002

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Project description:Background:Wnt/β-catenin (or canonical) signalling pathway activity is necessary and used independently several times for specification of vegetal fate and endoderm, gut differentiation, maintenance of epithelium in adult intestine and the development of gut-derived organs in various vertebrate and non-vertebrate organisms. However, its conservation in later stages of digestive tract development still remains questionable due to the lack of detailed data, mainly from Spiralia. Results:Here we characterize the Pdu-Tcf gene, a Tcf/LEF orthologue and a component of Wnt/β-catenin pathway from Platynereis dumerilii, a spiralian, marine annelid worm. Pdu-Tcf undergoes extensive alternative splicing in the C-terminal region of the gene generating as many as eight mRNA isoforms some of which differ in the presence or absence of a C-clamp domain which suggests a distinct DNA binding activity of individual protein variants. Pdu-Tcf is broadly expressed throughout development which is indicative of many functions. One of the most prominent domains that exhibits rather strong Pdu-Tcf expression is in the putative precursors of endodermal gut cells which are detected after 72 h post-fertilization (hpf). At day 5 post-fertilization (dpf), Pdu-Tcf is expressed in the hindgut and pharynx (foregut), whereas at 7 dpf stage, it is strongly transcribed in the now-cellularized midgut for the first time. In order to gain insight into the role of Wnt/β-catenin signalling, we disrupted its activity using pharmacological inhibitors between day 5 and 7 of development. The inhibition of Wnt/β-catenin signalling led to the loss of midgut marker genes Subtilisin-1, Subtilisin-2, α-Amylase and Otx along with a drop in β-catenin protein levels, Axin expression in the gut and nearly the complete loss of proliferative activity throughout the body of larva. At the same time, a hindgut marker gene Legumain was expanded to the midgut compartment under the same conditions. Conclusions:Our findings suggest that high Wnt/β-catenin signalling in the midgut might be necessary for proper differentiation of the endoderm to an epithelium capable of secreting digestive enzymes. Together, our data provide evidence for the role of Wnt/β-catenin signalling in gut differentiation in Platynereis.

Dataset Information

TALENs mediate efficient and heritable mutation of endogenous genes in the marine annelid Platynereis dumerilii.

Publications

TALENs mediate efficient and heritable mutation of endogenous genes in the marine annelid Platynereis dumerilii.

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