Exclusive multipotency and preferential asymmetric divisions in post-embryonic neural stem cells of the fish retina.

The potency of post-embryonic stem cells can only be addressed in the living organism, by labeling single cells after embryonic development and following their descendants. Recently, transplantation experiments involving permanently labeled cells revealed multipotent neural stem cells (NSCs) of embryonic origin in the medaka retina. To analyze whether NSC potency is affected by developmental progression, as reported for the mammalian brain, we developed an inducible toolkit for clonal labeling and non-invasive fate tracking. We used this toolkit to address post-embryonic stem cells in different tissues and to functionally differentiate transient progenitor cells from permanent, bona fide stem cells in the retina. Using temporally controlled clonal induction, we showed that post-embryonic retinal NSCs are exclusively multipotent and give rise to the complete spectrum of cell types in the neural retina. Intriguingly, and in contrast to any other vertebrate stem cell system described so far, long-term analysis of clones indicates a preferential mode of asymmetric cell division. Moreover, following the behavior of clones before and after external stimuli, such as injuries, shows that NSCs in the retina maintained the preference for asymmetric cell division during regenerative responses. We present a comprehensive analysis of individual post-embryonic NSCs in their physiological environment and establish the teleost retina as an ideal model for studying adult stem cell biology at single cell resolution.This is the final version of the article. It has been published by The Company of Biologists Ltd in Development here: http://dev.biologists.org/content/early/2014/08/19/dev.109892.long

Golden GATEway cloning--a combinatorial approach to generate fusion and recombination constructs.

The design and generation of DNA constructs is among the necessary but generally tedious tasks for molecular biologists and, typically, the cloning strategy is restricted by available restriction sites. However, increasingly sophisticated experiments require increasingly complex DNA constructs, with an intricacy that exceeds what is achievable using standard cloning procedures. Many transgenes such as inducible gene cassettes or recombination elements consist of multiple components that often require precise in-frame fusions. Here, we present an efficient protocol that facilitates the generation of these complex constructs. The golden GATEway cloning approach presented here combines two established cloning methods, namely golden Gate cloning and Multisite Gateway(TM) cloning. This allows efficient and seamless assembly as well as reuse of predefined DNA elements. The golden Gate cloning procedure follows clear and simple design rules and allows the assembly of multiple fragments with different sizes into one open reading frame. The final product can be directly integrated into the widely used Multisite Gateway(TM) cloning system, granting more flexibility when using a transgene in the context of multiple species. This adaptable and streamlined cloning procedure overcomes restrictions of "classical construct generation" and allows focusing on construct design

Complex recombination constructs and fusion proteins.

<p>A) A BBW1.0-like construct is assembled into the middle entry vector via a Golden Gate reaction 8 entry vectors are used that contain either recombination elements (LoxP, Lox2272 and FRT sites) or fluorophores. Additionally, the ORF that encodes for the tamoxifen-inducible Cre recombinase was generated with the tamoxifen-sensitive estrogen receptor (ERT2) and flanking Flag tags. The Golden Gate assemblies are made in the Gateway^TM middle entry vector. Subsequently, these are combined with the ubiquituous beta actin 2 promoter and the globin intron-SV40 polyA. B-E) Both vectors were coinjected with Tol2 mRNA. 57% (n=43) of the fish were transiently eGFP-expressing. Those were split in two groups and treated with either tamoxifen dissolved in DMSO or DMSO alone. All fish transiently injected with the BBW1.0 construct and CreERT2 retained green fluorescence after addition of DMSO. Addition of tamoxifen induced cell-specific recombination in all embryos.</p

Summary of overhangs and amino acid linkers.

<p>The entry vectors differ only in the overhangs that are created by BsaI restriction digest. These overhangs define the position of the fragment in the final assembly. Defined amino acid linker sequences are retained, since the overhangs as well as parts of the subcloning toolbox are retained in the final assembly. GS/x/GT are linkers introduced by the BamHI, KpnI restriction sites. TA-Cloning via the XcmI sites introduces the SGTA linker. Note compatible overhangs in consecutive entry vectors.</p

Golden Gate entry vector design and cloning.

<p>Cloning cassette used to fill Golden Gate entry vectors. Inserts can be introduced in two ways. XcmI restriction digest generates overhangs that can be used for TA cloning. Second, BamHI and KpnI sites can be used to clone inserts with different length either via standard ligation or an oligo annealing and cloning procedure. This cassette is flanked with BsaI sites used for the Golden Gate assembly. Each entry vector contains an SP6 promoter and a globin intron and SV40 polyA site flanking the insert for the generation of mRNA.</p

Basic nomenclature rules.

<p>The described nomenclature contains all necessary information to use the entry vectors for an assembly without the need to analyze the exact sequence. These rules are especially important for the generation of fusion proteins.</p

Golden Gate-based multisite mutagenesis.

<p>A) The FlpO ORF contains two internal BsaI sites. These are mutated by amplifying fragments of the FlpO ORF via PCR. The primers contain flanking BsaI sites that lead to compatible overhangs after restriction digest. The site-directed mutagenesis vector (pGG-sdm) contains BsaI sites for the mutagenesis assembly. Additionally, BamHI and KpnI sites allow the transfer of the mutated DNA assembly to Golden Gate entry vectors. From there, mRNA can be generated using SP6 RNA polymerase. Note that the pGG-sdm vector represents a standard Gateway^TM middle entry vector. B) A Golden Gate reaction is used to prepare a dual FRT-based recombination construct in the Gateway^TM middle entry vector. An LR reaction was prepared to generate a final expression construct. ISceI sites flank the expression cassette. The dual FRT element is driven by the zebrafish beta actin 2 promoter and a human globin intron with SV40 polyA is used in the 3’ entry vector. C) Zebrafish embryos were injected with the expression construct alone or in combination with FlpO mRNA. Without FlpO mRNA 64% (n=98) of the fish were positively injected and showed eGFP expression and the absence of mCherry. In combination with FlpO mRNA 60% (n=79) of the fish were positively injected and all of them showed the complete absence of green fluorescence and the appearance of red fluorescence, which is indicative for proper FlpO activity.</p

Generation of recombination templates using Golden Gate cloning.

<p>A) Schematic depiction of the generated recombination templates. The two recombination templates are based either on FRT or Lox elements in defined orientations. We included specific multiple cloning sites in the entry vectors -1, 3, 6 and 8’. B) Vector map of a subcloning destination vector with the restriction sites for the most common restriction endonucleases. The NcoI site (highlighted in red) is not present in the vector that lacks the ATG; otherwise restriction sites in all the vectors are identical.</p