Abstract
Body pigmentation is a major limitation for in vivo imaging and thus for the performance of longitudinal studies in biomedicine. A possibility to circumvent this obstacle is the employment of pigmentation mutants, which are used in fish species like zebrafish and medaka. To address the molecular basis of aging, the short-lived African killifish Nothobranchius furzeri has recently been established as a model organism. Despite its short lifespan, N. furzeri shows typical signs of mammalian aging including telomere shortening, accumulation of senescent cells and loss of regenerative capacity. Here, we report the generation of a transparent N. furzeri line by simultaneous inactivation of three key loci responsible for pigmentation. We demonstrate that this stable line, named klara, can serve as a tool for different in vivo applications including behavioral experiments addressing mate choice and the establishment of a senescence reporter by homology-directed repair-mediated integration of a fluorophore into the cdkn1a (p21) locus.
Introduction
In animals, pigments that can be found in specific cell types limit optical transparency and prevent the in vivo observation of processes like organogenesis, regeneration or cancer metastasis. While mammals have only one pigment cell type, the melanocyte, other vertebrates including fish develop several chromatophores that produce different colors. In one of the best-studied models for vertebrate coloration, the zebrafish (Danio rerio), the three main kinds of chromatophores are the melanophores (black), the iridophores (silvery or blue) and the xanthophores (yellow), all derived from neural crest cells 1,2. A fourth population of pigment cells, forming the retinal pigment epithelium (RPE) is derived from the optic neuroepithelium 3. Different combinations of naturally occurring mutants in pigmentation genes have been used to generate adult transparent zebrafish. The casper line lacks melanocytes and iridophores due to mutations in mitfa and mpv17, respectively 4,5. An additional mutation in the slc45a2 gene is present in crystal zebrafish, which completely lack melanin and therefore possess a transparent RPE 6. Transparent zebrafish have been used to study different aspects of cancer and stem cell biology, among others 5,7. In another model fish, the medaka (Oryzias latipes), transparent juvenile and adult animals have recently been generated through CRISPR/Cas9-mediated inactivation of oca2 and pnp4a 8.
During the last decade the turquoise killifish, Nothobranchius furzeri, has emerged as a new model for research on aging 9. With a lifespan between three and seven months, N. furzeri is the shortest-lived vertebrate that can be kept in captivity 10,11. Hatchlings grow rapidly and can reach sexual maturation already within two to three weeks 12. N. furzeri shares many hallmarks of aging with mammals, including telomere shortening, mitochondrial dysfunction, cellular senescence, loss of regenerative capacity and cognitive decline 13–19. Despite its short lifespan, N. furzeri also shows a high incidence of age-dependent neoplasias in liver and kidney 20. What makes the killifish an attractive model in addition, is the establishment of transgenesis and genome engineering 15,21–23 as well as the availability of reference sequences for the N. furzeri genome 24,25. The turquoise killifish is sexually dimorphic and dichromatic 26. Compared to females, males are larger and colorful. The latter occur in two color forms with red and yellow morphs that differ primarily in coloration of the caudal fin. Females have translucent fins and a pale greyish body with iridescent scales.
Here, we describe the generation of fully transparent juvenile and adult N. furzeri animals. We have used a single injection of three sgRNAs targeting mitfa, ltk and csf1ra, which are involved in pigment development in melanophores, iridophores and xanthophores, respectively. With the method employed we have achieved simultaneous and biallelic somatic gene disruptions of three genomic loci in a highly efficient manner. Already in the F0 generation, a fraction of animals were fully transparent. Homozygous triple mutants showed normal behavior, fertility and general health. In addition, we have used the transparent line, named klara, to inactivate additional genes, to study female and male mate choice and to generate an in vivo senescence reporter by homology-directed repair-mediated integration of an GFP allele into the locus of the senescence marker cdkn1a (p21).
Results
Multiple genes can be simultaneously inactivated in N. furzeri
For the generation of a transparent Nothobranchius furzeri line, we selected the genes mitfa, ltk, and csf1ra as targets to interfere with the formation of melanophores, iridophores and xanthophores, respectively. The expression of those three genes was analyzed in skin tissue from fish of both sexes at the age of 1, 2, 3 and 6 weeks post hatching (wph). In male fish, we observed a significant up-regulation of mitfa at the age of 6 wph, whereas mitfa expression did not change in female fish (Fig. 1a). The expression of ltk increased steadily with age in both, male and female fish (Fig. 1b). In contrast, csf1ra expression did not differ significantly over time in males and females (Fig. 1c). Direct comparison of mitfa, ltk and csf1ra expression between females and males did not reveal sex-specific differences except for mitfa at 6 wph (Extended Data Fig. 1a-c). To induce mutations in the selected genes, single guide RNAs were designed based on the genome sequence provided by the Nothobranchius furzeri Genome Browser 24. Since three genes should be inactivated at the same time, we used one sgRNA per gene. To facilitate mutation detection PAM sequences were chosen that had a restriction site directly upstream, assuming that the restriction site would be lost upon the introduction of a mutation. After characterization of different sgRNAs for each gene, sgRNAs were synthesized targeting mitfa in exon 6, ltk in exon 22 and csf1ra in exon 9 (Extended Data Fig. 1d-f). To simultaneously inactivate mitfa, ltk and csf1ra, we injected Cas9 mRNA, the three different sgRNAs and GFP mRNA into one-cell-stage embryos of the long-lived N. furzeri strain MZCS-08/122 27. GFP mRNA was used to indicate properly injected embryos one day after the injection. An injection mold, stabilizing the eggs during the injection procedure, has already been reported 28. To further improve the injection procedure, we developed a new type of injection mold having single slots for each embryo. Moreover, the wall of these slots pointing towards the direction of the injection needle is sloped facilitating the access of the needle to the egg (Extended Data Fig. 1g). Using this injection mold, we were able to inject close to 600 embryos within two days, which were sorted for a GFP signal one day post injection. One third each of the embryos were GFP-positive, GFP-negative or dead (Extended Data Fig. 1h). Seven days after injection, GFP-positive eggs were transferred onto coconut coir plates, mimicking the dry phase N. furzeri eggs undergo in their natural habitat. Since it is possible to detect melanophores already in embryos, GFP-positive and GFP-negative embryos were phenotypically analyzed. While melanophores were clearly detected in GFP-negative embryos, a reduction or an almost complete loss of melanophores was observed on the head and along the body axis in a fraction of GFP-positive embryos (Fig. 1d). Iridophores and xanthophores are not detectable at this stage of development.
In order to analyze if the sgRNAs targeting mitfa, ltk and csf1ra had induced mutations, regions around the expected mutation sites were amplified via PCR using DNA extracts from 8 randomly selected GFP-positive and 6 GFP-negative embryos. Those amplicons were used in restriction enzyme digests to determine the presence of mutations. For all three genes. we observed a non-cleaved PCR fragment in all samples of GFP-positive embryos, suggesting that mutations had been introduced that made the amplicons resistant to restriction enzyme digest. The analysis of mutations in the mitfa sequence revealed, that 75% (6/8) of the GFP-positive samples only showed one undigested fragment, whereas 25% (2/8) were mosaic, since they also showed two additional fragments that only occur in the presence of the wild type sequence. For ltk, we observed mosaicism in 37.5% of embryos (3/8) and for csf1ra in 87.5% (7/8) (Extended Data Fig. 1i-k). For the GFP-negative embryos, we identified digested fragments in all samples, as in the respective wild type controls. However, the first two GFP-negative embryos also showed non-cleaved fragments, indicating the presence of mutations. This suggests that the sorting into GFP-positive and GFP-negative embryos is not fully stringent.
Next, we hatched and raised GFP-positive embryos from the F0 generation. Compared to wild type larvae and adult animals, the majority of fish from the F0 generation of successfully injected eggs showed a mosaic loss of pigment cells. However, in some of the fish we could already observe an almost complete loss of pigment cells, resulting in transparency of the animals (Fig. 1e-h). This already allowed us to have a clear view on inner organs. At an age of 25 days post hatching (dph), the stomach in those transparent fish shows an orange color due to artemia, small crustaceans, which are used as food at this age. Moreover, the swim bladder and in females the ovaries were clearly visible. One could also observe the blood flow in the cardinal vein and in the small vessels of the caudal fin. Additionally, we could identify single eggs including lipid droplets in the ovaries. Besides this phenotypical analysis of fish from the F0 generation, we also analyzed the mutation rates. Based on results from the previously mentioned restriction analysis, we observed that all 85 fish had a mutation in mitfa, ltk and csf1ra, at least in a mosaic fashion. Moreover, this analysis revealed that already in the F0 generation biallelic mutations were observed (mitfa: 48.2%, ltk: 67.1%, csf1ra: 23.5%) (Extended Data Fig. 1l). These data indicate a high efficiency of the CRISPR/Cas9 tool in N. furzeri and show that it is possible to simultaneously induce mutations in three genes of interest.
Generation of a stable, transparent killifish line
For the generation of a stable, transparent N. furzeri line and to reduce potential off-target effects, we performed an outcross of selected F0 fish with wild type animals. As expected, all of the obtained F1 progeny showed a normal pigmentation pattern and hence were phenotypically not distinguishable from wild type animals. To assess the presence of mutations, we extracted DNA from fin biopsies for genotyping. Among 60 analyzed fish, 14 animals were triple-heterozygous (mitfa+/−, ltk+/−, csf1ra+/−). The targeted loci in those fish were analyzed via sequencing. Various deletion mutations were detected, but two fish carried the same mutation at the mitfa (Δ11 bp), ltk (Δ4 bp) and csf1ra (Δ5 bp) locus (Extended Data Fig. 2a-c). Those animals were used for a subsequent incross, from which according to Mendelian ratio 1/64 (1.56%) of the F2 offspring was expected to be triple homozygous. We first checked whether triple homozygous embryos were viable and could be detected among the F2 eggs. Hence, we randomly selected embryos to assess their genotype. We observed a lack of melanophores in a proportion of embryos. Those embryos carried a homozygous mutation in mitfa, whereas embryos with only a heterozygous mitfa mutation had melanophores (Extended Data Fig. 2d-k). The genotypes for ltk and csf1ra could only be assessed via molecular analysis at this developmental stage. Notably, one triple homozygous embryo was detected, indicating that at least until the hatching stage those embryos were viable. We then proceeded with hatching of F2 eggs. In this generation, different genotypes have been observed to result in different phenotypical appearances. Female fish with a heterozygous mutation in ltk and homozygous mutations in mitfa and csf1ra resembled at first glance wild type females, whereas in male fish especially the lack of xanthophores resulted in a silver-blue appearance (Fig. 2a,a’). In contrast to this, the lack of melanophores and iridophores in fish with the genotype mitfa-/-, ltk-/-, csf1ra+/− allowed a view on inner organs, particularly in females (Fig. 2b,b’). In order to increase the likelihood to obtain transparent, triple homozygous fish, we crossed two fish with the genotype mitfa-/-, ltk-/-, csf1ra+/−. From this cross we genotyped 50 individuals via high-resolution melting analysis (HRMA) and could identify 13 triple homozygous fish. We named the transparent N. furzeri line klara (Fig. 2c,c’).
Characterization of klara animals
In zebrafish, the role of csf1ra in xanthophore development has been described 29. In addition, csf1ra is also known to play a role in the immune system, in particular in the survival, proliferation and differentiation of monocytes and macrophage 30,31. For this reason, we wondered whether the inactivation of csf1ra has an effect on the immune cell population of klara. Since the kidney is the primary hematopoietic organ in teleost fish, we analyzed the whole kidney marrow (WKM) of fish with homozygous mutations in mitfa and ltk, which had in addition either no mutation, a heterozygous or a homozygous mutation in csf1ra. Based on a published gating strategy 32, we could identify four subpopulations in the WKM of N. furzeri using flow cytometry (Extended Data Fig. 2l). The strongest csf1ra expression was detected in the myeloid cell population, which contains macrophages (Fig. 2d). Comparing the number of immune cells in the three sub-populations, we did not observe any differences among the csf1ra genotypes (Fig. 2e and Extended Data Fig. 2m).
During raising of klara fish, we observed that around the age of approximately four weeks, i.e., at the time of sexual maturation, melanophores appeared in male fish, in particular on fin appendages. However, this was not detected in female klara fish (Fig. 2f). With age the melanophores spread over the whole fins and resulted e.g., in a fully black caudal fin. This, however, did not interfere with the overall transparency of klara animals.
Males and females prefer pigmented mating partners
Besides its role as camouflage, protection from UV damage or for recognition, body pigmentation also plays an important role for the choice of mating partners. We wanted to investigate whether the lack of body pigmentation affects the breeding behavior of klara fish. We set up three breeding groups consisting of one klara male and two klara females, at the age of 14 weeks, which had never been used for breeding before. Klara fish showed a normal mating behavior, whereby the male uses its caudal fin to push the female into the sand and thus induces egg laying (Extended Data Movies 1,2). We also assessed the quantity and quality of collected eggs, which did not differ from wild type fish, so that we could maintain the klara line in a triple homozygous state (Fig. 3a). To assess the role of body pigmentation for mate choice in killifish, we set up different combinations of breeding trios consisting of wild type and klara fish, thus that a wild type or a klara animal of each sex had the choice between a wild type or a klara animal of the other sex (Extended data Fig. 3). For each of the four combinations, we analyzed three tanks. Fish were put together and a sand box, which is required for egg deposition, was put into the tank. After 10 days the sand box was removed for the following two days. Subsequently, the box was added again and for the following four weeks we collected eggs once per week for further analysis. In order to decide who produced the egg or who had fertilized it, we determined the genotype of the fertilized eggs by HRMA. We observed that in the presence of a klara and a wild type female fish, irrespective of whether the male was a wild type or a klara animal, approximately 75% of fertilized eggs originated from the wild type female (Fig. 3b). This indicated that both klara and wild type males showed a preference for the pigmented wild type female. This mate choice was not influenced by size or weight, since both parameters were indistinguishable between wild type and klara females (Fig. 3c). Similarly, in the breeding groups, in which a klara and a wild type male was present, more than 90% of eggs were fertilized by the wild type male (Fig. 3b). Again, wild type and klara males did not show a difference in size, although klara males had less weight (Fig. 3c). Taken together, this competitive breeding experiments indicated that pigmented fish were the preferred mating partner for both sexes.
Klara fish can serve as a platform for further genetic manipulation
The simultaneous inactivation of mitfa, ltk and csf1ra resulted in a loss of body pigmentation, however, the eyes were still normally pigmented (Fig. 4a). Transparent zebrafish from the casper line 5 also still have pigmented eyes, while fish of the fully transparent crystal line lack those pigments. This was achieved via an inactivation of the slc45a2 gene 6. In order to get rid of retinal pigmentation in klara animals we designed a sgRNA targeting the slc45a2 locus (Extended Data Fig. 4a). We first tested this sgRNA in one-cell stage eggs from the wild type strain. Compared to GFP-negative embryos, we observed a loss of pigmentation in the eye of GFP-positive embryos (Fig. 4b). Notably, also the appearance of melanophores on the head and body of those embryos was reduced. This phenotypical analysis indicated inactivation of slc45a2, which was subsequently confirmed via restriction enzyme digest (Extended Data Fig. 4b). We then performed microinjections using the sgRNA targeting slc45a2 into klara one-cell stage embryos and hatched F0 fish (n=7). Besides the transparent body, the black pigmentation of the eye was absent, while we still observed a silver-pigmented ring around the eye of the animals (Fig. 4c). The presence of a mutation in the slc45a2 locus of all seven F0 fish was confirmed via restriction enzyme digest (Extended Data Fig. 4c). Subsequently, we performed an outcross of F0 fish with klara and identified the presence of various indel mutations in F1 offspring (Extended Data Fig. 4d,e). This experiment showed that the klara line can serve as a platform for further genetic manipulations.
Generation of an in vivo senescence reporter
The accumulation of senescent cells is one of the hallmarks of the aging process 33. We wanted to take advantage of the transparent klara line and generate a senescence reporter line. To this end, we planned to insert a reporter construct, consisting of an eGFP and nitroreductase (NTR) cassette into the cdkn1a (p21) locus of klara via homology-directed repair (Fig. 5a). The cdkna1 gene is a senescence marker and upregulated in old killifish 14. With this reporter/NTR cassette, cdkn1a expressing cells can be labelled via eGFP and can also be ablated via the NTR/Mtz system 34. To facilitate HDR, we added flanking arms of 903 bp and 901 bp on the 5’ and 3’ ends of the construct, respectively. Since it has been reported that 5’ modifications of double-stranded donor templates increase HDR efficiency 35,36, we amplified the template using biotinylated oligonucleotides (Fig. 5b). Subsequently, we performed microinjections into one-cell stage klara eggs using an injection solution with a sgRNA that induces a DNA double-strand break in close proximity to the intended insertion site and the biotinylated HDR donor template. In 1 out of 16 randomly selected GFP-positive (successfully injected) embryos, we detected the presence of the reporter cassette. Its proper insertion was verified via sequencing (Extended Data Fig. 5a). We subsequently set up the remaining GFP-positive embryos for hatching and detected three fish with a proper insertion among 19 F0 animals. Next, we performed an outcross of the F0 animals with klara fish. To assess whether the reporter was functional, the obtained F1 embryos were exposed to γ–irradiation of 10 Gy. This should induce DNA damage and lead to activation of TP53 and up-regulation of cdkn1a. To analyze, whether the up-regulated cdkn1a expression would also be linked to an up-regulation of GFP, we analyzed the embryos 1h before as well as 24h post irradiation (hpi) via fluorescence microscopy (Extended Data Fig. 5b). Before irradiation, autofluorescence originating from the yolk was observed in cdkn1a+/+ and cdkn1aki/+ embryos (Fig. 5c). In contrast, at 24 hpi we observed GFP-positive cells in the optic tectum and other parts of the developing brain and fin buds of cdkn1aki/+ embryos (Fig. 5d). Gene expression analysis revealed an upregulation of endogenous cdkn1a as well as both GFP and ntr expression upon γ–irradiation in cdkn1aki/+ embryos, which further significantly increased at 48 hpi (Fig. 5e-g). Thus, we were able to insert a functional 1.5 kb reporter cassette into the cdkn1a locus of klara fish.
After the initial characterization of the cdkn1aki/+ embryos, we raised them to adulthood and generated F2 offspring. Subsequently, we used immunofluorescence and flow cytometry to detect and quantify the occurrence of GFP-positive cells and their potential accumulation upon aging. As organs for analysis we selected liver and kidney from young and old cdkn1aki/+ and cdkn1aki/ki animals. Staining for GFP revealed many more GFP+ cells in the liver of a 30 weeks-old cdkn1aki/ki fish than in a 4 weeks-old animal (Fig. 5h). Of note, GFP+ cells in older tissue were larger and often appeared clustered as opposed to smaller and scattered GFP+ cells in young tissue. Using FACS, we detected 0.4 and 0.2% GFP+ cells in the liver and kidney of 5 weeks-old cdkn1aki/+ fish, respectively. This numbers increased to 2.4 and 0.5% in the liver and kidney of 32 weeks-old fish (Fig. 5i,j). These data suggest that the reporter that we have generated can be used to monitor cdkn1a (p21)-positive cells in vivo and that these cells accumulate upon aging.
Discussion
Here, we report the generation of a transparent killifish line that is lacking melanophores, iridophores and xanthophores. Based on published literature, we have selected the three genes mitfa, ltk and csf1ra as targets for CRISPR/Cas9-mediated inactivation in Nothobranchius furzeri 29,37,38. We employed a single injection with three single guide RNAs that had been pre-selected and characterized. The observation that some of the injected F0 embryos showed a complete loss of melanophores demonstrated that the CRISPR/Cas9 system acts very efficiently in N. furzeri. This has been observed before 22 and is most likely explained by the long duration of the one-cell stage in this species of 2-3 hours during which Cas9 can act 39. Cas9 efficiency was further confirmed by the fact that for all three genes both alleles were inactivated in the F0 animals, many of which showed an almost complete transparency. This efficiency is comparable to the one reported for zebrafish, whereby a codon-optimized Cas9 protein was employed to target a reporter transgene and four endogenous loci. In this case, mutagenesis rates reached 75–99% 40.
The animals did not show any anomalies regarding phenotype and behavior and could be bred to homozygosity and kept as a stable line. While klara animals were initially fully transparent, male animals developed black pigments, particularly on fin appendages, which increased during their lifespan. This suggests that there is a second, mitfa-independent population of melanophores in killifish that appears at later life. In zebrafish it has been shown that the paralogous gene, mitfb might fulfill this role by activating tyrosinase expression 41. Whether or not this is also the case in killifish and whether mitfb might also be responsible for the remaining pigmentation in the retina remains to be determined. By inactivating slc45a2 in klara fish, we obtained animals that were fully transparent regarding their body and the eye. This quadruple mutant might be particularly interesting for research on eye and retina regeneration and shows that klara animals can be used as a background for further gene inactivation.
We used the klara line for addressing mate choice in killifish. While breeding behavior was normal among klara animals, both wild type and klara animals preferred pigmented mating partners in competitive breeding situations. Here, mate choice for pigmented partners was more pronounced in females (>90%) than in males (approx. 75%). This might be explained by the fact that the difference in appearance of wild type and klara males is much more distinct than between the respective females. The choice for pigmented males might also be influenced by the larger weight of wild type males. Our observation is in line with an earlier report on the two-spotted gobies. In this case, males preferred to mate with more colourful females, which have bright yellow-orange bellies during the breeding season 42. It is very surprising that for mate choice N. furzeri seems to rely on visible cues, as their natural habitat is turbid with limited visibility 43. It is tempting to speculate that mate choice in N. furzeri could also be influenced by other traits like chemical signals including pheromones.
The main motivation to generate a transparent killifish line was the possibility to perform longitudinal studies regarding aging and regeneration and to be able to observe respective processes in real-time without having to sacrifice cohorts of animals at distinct time points. One of the hallmarks of aging is the accumulation of senescent cells that are characterized by the expression of specific markers including cdkn1a (p21) and cdkn2a (p16) 33. It is still a matter of debate whether senescent cells limit or extend lifespan 44,45. To visualize and address the role of senescent cells, we have integrated a cassette encompassing a fluorescence reporter as well as a nitroreductase allele into the cdkn1a locus of klara animals. The respective HDR template was flanked by 0.9 kb homology arms and carried biotinylated 5’-ends, as those modifications of double-stranded donor templates have been reported to increase HDR efficiency 35,36. Out of 35 injected embryos, four showed proper integration of the construct, corresponding to 11%. At least three F0 animals passed on the engineered allele to the next generation. Our analysis of klara embryos harboring the GFP allele in the cdkn1a locus had shown that the integrated reporter is functional and can be activated upon γ-irradiation. The characterization of respective adult animals demonstrated an accumulation of GFP+ cells with age, as determined by flow cytometry and immunofluorescence. Whether those cells are truly senescent cells remains to be determined. Since the reporter also encompasses a ntr allele, it shall be possible to delete the GFP+ and thus presumably senescent cells by administration of Metronidazole, a prodrug that is converted to a cytotoxic agent by NTR. With the transparent senescence reporter line, it will be possible to further characterize the function of senescent cells in development, aging and regeneration. The generation of additional reporters into different loci, e.g. cdkn2a/b or other senescent markers will also allow to address the possible heterogeneity of senescent cells 46.
We consider the klara fish that we describe here a valuable and versatile tool for research on aging, regeneration and behavior. This fish line will also be beneficial for colleagues interested in cancer biology and ecology. Beyond its potential to be used for the investigation of questions in biology klara animals can also contribute to the reduction of animal numbers, an aspect that gains increasing importance in biomedical research.
Materials and Methods
Fish husbandry
All the work reported here was performed in the wild type N. furzeri strain MZCS-08/122, which is originally derived from southern Mozambique 27, or the klara line. Fish are kept in single-housing at 26°C on a light:dark cycle of 12 hours each. Adult fish are fed once a day ad libitum with red mosquito larvae, whereas juvenile fish (up to 5 weeks post hatching) are fed with artemia twice a day. To obtain a high number of fertilized wild type oocytes for injections, multiple breeding groups consisting of ten fish (2 males, 8 females) were set up in 40 liter tanks. In order to obtain oocytes from klara fish, breeding groups of 1 male and 3 female were set up in tanks containing approximately 8.5 l. The sand box, which is necessary for the deposition of eggs, was always removed two days before and put back into the tank two hours before the injection. The eggs were collected with a sieve and were then used for microinjections. The routinely collection of eggs for line maintenance was done on a weekly basis. Eggs were put on coconut coir plates and stored at 29°C.
All fish were maintained in the Nothobranchius facility of the Leibniz Institute on Aging – Fritz Lipmann Institute Jena according to the German Animal Welfare Law. The performed experiments reported here were covered by the animal license FLI-17-016, FLI-20-001 and FLI-20-102, which were approved by the local authorities (Thüringer Landesamt für Verbraucherschutz).
Design and synthesis of single-guide RNAs (sgRNAs)
Single-guide RNAs were designed based on the genome sequence provided by the Nothobranchius furzeri Genome Browser 24. Target sequences for sgRNAs had a length of 20 nucleotides followed by the PAM sequence-NGG. Only sequences containing a restriction site directly upstream of the PAM sequences were selected. A TAGG-overhang was added to the 5’-end of the forward sgRNA oligonucleotide and an AAAC-overhang to the 5’-end of the reverse complementary oligonucleotide (sg_mitfa_1: 5’-TAGG-TGAAATGGATTTCCTGATGG-3’ sg_mitfa_2: 5’-AAAC-CCATCAGGAAATCCATTTCA-3’, sg_ltk_1: 5’-TAGG-AACATCAAAAGGGAATTCAC-3’ sg_ltk_2: 5’-AAAC-GTGAATTCCCTTTTGATGTT-3’, sg_csf1ra_1: 5’-TAGG-CAGAGACACTTTTTCCATGG-3’ sg_csf1ra_2: 5’-AAAC-CCATGGAAAAAGTGTCTCTG-3’, sg_slc45a2_1: 5’-TAGG-TGACTACTGCCGCTCACAGT-3’ sg_slc45a2_2: 5’-AAAC-ACTGTGAGCGGCAGTAGTCA-3’). Complementary sgRNA oligonucleotides were annealed by heating them up to 95°C followed by gradually cooling by 1°C per 30 seconds. The annealed oligonucleotides were ligated into the BsaI-linearized pDR274 vector (Addgene, plasmid #42250). After the ligation, this vector, containing the sgRNA sequence, was transformed into E. coli TOP10 cells. Isolated plasmids were checked via sequencing for the correct presence of the sgRNA sequence. Using the DraI restriction enzyme a fragment of approximately 300 bp was excised from the plasmid containing the sgRNA and the T7 promoter sequence. This fragment was used as a template for the in vitro transcription, which was performed according to the manufacturer’s protocol of the mMESSAGE mMACHINE™ T7 Transcription Kit (Thermo Fisher Scientific Inc.). Quality of in vitro transcribed sgRNAs was controlled via RNA agarose gel electrophoresis.
Design and synthesis of DNA donor templates for HDR
The assembly of the donor template for the insertion of a P2A-eGFP-P2A-NTR cassette into the cdkn1a locus of klara was done using the NEBuilder® HiFi DNA Assembly Cloning Kit. P2A sites were added to the eGFP (derived from the tol2 kit plasmid #395) and the NTR sequence (obtained from plasmid: Myl7-LoxP-myctagBFP-LoxPNTRmCherry 47) via PCR. The NEBuilder Assembly Tool was used for the design of oligonucleotides containing overlap sequences, which are required for the assembly of individual PCR fragments. 0.05 pmol of each fragment (flanking arms, P2A-eGFP, P2A-NTR, pGGC (pUC57-BsaI) backbone vector 48) were used together with 10 μl of the NEBuilder® HiFi DNA Assembly Master Mix. The NEBuilder assembly reaction was performed for one hour at 50°C. 5 µl of this reaction were used for a subsequent transformation into chemically competent TOP10 E. coli. Isolated plasmids were checked via sequencing for correct template assembly. 5’-biotinylated oligonucleotides (bio_cdkn1a_fw: 5’-TCTTACACCAAACACCACAA-3’ bio_cdkn1a_rv: 5’-TAAAACATGCAGGATACCGG-3’) were used to amplify the template from the isolated and then linearized plasmid. The amplicon with the expected size was excised from the agarose gel and purified using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel). In order to induce a DNA double-strand break in close proximity to the site of insertion, the following oligonucleotides for sgRNA synthesis were used: sg_cdkn1a_1: 5’-TAGG-AATATCACTCCCCGGATTTC-3’ sg_cdkn1a_2: 5’-AAAC-GAAATCCGGGGAGTGATATT-3’. Synthesis of this sgRNA was done as described above.
Microinjections into N. furzeri oocytes
For microinjections, an injection mold (manufactured by GT-Labortechnik, Arnstein, Germany) forming single slots for the size of an N. furzeri embryo was used. Injection plates were freshly prepared by dissolving 1.5 g of agarose in 50 ml of 0.3 x Danieau’s medium. This solution was boiled in a microwave oven and then poured into a petri dish (94 mm x 16 mm). The injection mold was put in while the solution is still liquid and as soon as the agarose is hardened (after approximately 30 min), the stamp can be removed and the plate is ready to use. Fertilized embryos were lined up individually in each slot in a way that the cell was facing towards the direction of the injection needle. 0.3 x Danieau’s medium was added onto the plate until the eggs were completely covered. The injection was performed under a stereomicroscope using glass capillary needles, a pressure injector (World Precision Instruments) and a micromanipulator (Saur). The injection solution for the inactivation of target genes contained the sgRNAs (30 ng/µl each), Cas9 mRNA (300 ng/µl), GFP mRNA (200 ng/µl) and phenol red. For knock-in approaches, the concentration of sgRNA and Cas9 mRNA was kept the same, whereas GFP mRNA was reduced to 100 ng/µl and 20 ng/µl of the HDR template were added. After injections, the embryos remained on the plate and were stored at 29°C until the next day. Using a fluorescence microscope, the injected embryos were sorted into GFP-positive and GFP-negative embryos. To reduce the risk of cross contaminations, GFP-positive embryos were transferred into single wells of a 96-well plate containing 0.3 x Danieau’s medium.
DNA sampling
DNA samples were obtained either from caudal fin biopsies or whole embryos. For fin biopsies, a small part of the caudal fin was cut off from the anesthetized fish. For the extraction of DNA from whole embryos, the embryos were mechanically disrupted using a pipet tip. For fin biopsy samples 100 µl and for embryos 50 µl of NaOH (50 mM) was added to the sample and subsequently incubated at 95°C for 45 min. Afterwards, 10 µl or 5 µl respectively of Tris-HCl (1 M, pH 8.0) were added.
Restriction enzyme digest
The sgRNA design enabled the use of restriction enzymes to check for the presence of mutations in the targeted genes. The region flanking the potential mutation site was amplified via PCR using the following oligonucleotides: mitfa_fw: 5’-TGCTTCACATACGTTTGCAG-3’ mitfa_rv: 5’-CAAAGGTCTGAGGGCTTTCC-3’, ltk_fw: 5’-TGTTCTGTCACCACCCTTGT-3’ ltk_rv: 5’-ACACTGCTATTACCAGGTTTGAC-3’, csf1ra_fw: 5’-CATAGATACCGTGCAAGCCTG-3’ csf1ra_rv: 5’-AGCCCAGGTATGAAATCCGT-3’, slc45a2_fw: 5’-GGATTTGGTGTTTTGGCCCT-3’ slc45a2_rv: 5’-GTAACTCGGCTCTAATCGTGC-3’. For the restriction enzyme digest, 20 µl of the PCR reaction were incubated over night at 37°C after adding 6.75 µl of ddH2O, 0.25 µl of the corresponding enzyme and 3 µl of the respective enzyme buffer (mitfa: EcoRI, ltk: EcoNI, csf1ra: NcoI-HF, slc45a2: HypCH4III). Samples from the control digest were analyzed on a 1% agarose gel. In the presence of a mutation, the restriction enzyme was not able to cleave the PCR fragment, whereas non-mutated sequences were still cleaved. As positive control, a PCR amplicon from a wild type fish was always included in order to verify that the restriction enzyme digest worked properly.
High-resolution melting analysis (HRMA)
As soon as the exact mutations in the targeted gene loci were identified (from F2 generation on), genotyping was performed via HRMA. For this analysis, the CFX384™ Real-time PCR Detection System (Bio-Rad) was used. DNA samples obtained from either fin biopsies or whole embryo lysates were diluted 1:50 with H2O before use. The HRM analysis was done according to the protocol provided by the Precision Melt Supermix Kit (Bio-Rad; Catalog #172-5112). The following oligonucleotides were used for the HRM analysis: HRMA_mitfa_fw: 5’-CCTCACGAGTCTCTCTATCA-3’ HRMA_mitfa_rv: 5’-GCCCCATGAACCCAATATAA-3’, HRMA_ltk_fw: 5’-CCACAGACTCTTCCAGAAAT-3’ HRMA_ltk_rv: 5’-CTGATTATGAGGTGCGACTA-3’, HRMA_csf1ra_fw: 5’-AGTGTGTGGCTTTCAATTTG-3’ HRMA_csf1ra_rv: 5’-TTTCTGGTGAGTGTTTGTTA-3’. For the assessment of the genotypes, melt curves were analyzed using the Precision Melt AnalysisTM software (Bio-Rad).
Isolation of nucleic acids
RNA isolation from fish tissues was done according to the manufacturer’s protocol of the RNeasy Mini Kit (Qiagen). Tissue homogenization using ceramic beats was performed with the TissueLyser II (2 min at 30 Hz). The optional on-column DNase digestion step was included. 20 µl of DEPC H2O were used for final elution. RNA isolation from FACS sorted cells was done according to the protocol of the MagMaxTM-96 Total RNA Isolation Kit. Isolation of RNA and DNA from whole embryos was done via phenol-chloroform extraction. The chorion of the embryos was mechanically disrupted using a pipet tip before 500 µl of TRIzol were added. Homogenization of the embryos was performed with ceramic beats using the TissueLyser II (2 min at 30 Hz). After an incubation at RT for 5 min, 200 µl of chloroform were added. Samples were then mixed for 15 s, incubated at RT for 3 min and then centrifuged at 12,000 x g for 20 min (4°C). The upper, aqueous phase, which contains the RNA, was transferred into a fresh tube and 1.1 volumes of isopropanol, 0.16 volumes of NaAc (2M, pH 4.0) and 1 µl of GlycoBlue were added. Samples were incubated at RT for 10 min and then centrifuged at 12,000 x g for 20 min (4°C). The supernatant was removed and the pellet was washed with 1 ml of 80% EtOH and centrifuged at 7,500 x g for 10 min (4°C). The supernatant was discarded and the pellet was air-dried. RNA pellet was dissolved in 20 µl of DEPC-H2O and stored at −80°C. The inter- and organic phase were used for extraction of DNA (required for genotyping PCR). 300 µl of EtOH (100%) were added, samples were incubated at RT for 2-3 min and then centrifuged at 2,000 x g for 5 min (4°C). Supernatant was discarded and pellet was incubated in 1 ml of 0.1 M sodium citrate in 10% EtOH for 30 min before centrifugation at 2,000 x g for 5 min (4°C). Supernatant was discarded and 1 ml of EtOH (75%) was added for 15 min before centrifugation at 2,000 x g for 5 min (4°C). Pellet was air-dried and afterwards dissolved in 15 µl of NaOH (8 mM).
cDNA synthesis and gene expression analysis
For cDNA synthesis, 500 ng of RNA from fish tissues and whole embryos or 25 ng of RNA from FACS sorted cells were used. cDNA synthesis was performed according to the instructions of the iScript cDNA Synthesis Kit (Bio-Rad). qRT-PCRs were performed in 384-well plates using 2x SYBR Green Mix and the CFX384 Real-Time System (Bio-Rad). Reaction mix included 3 µl of cDNA (diluted 1:5 in DEPC H2O), 0.4 µl of each oligonucleotide, 1.2 µl DEPC H2O and 5 µl of 2x SYBR Green Mix. Gene expression levels were determined using the following oligonucleotides: q_mitfa_fw: 5’-TGAAGCAAGTACTGGACAAG-3’ q_mitfa_rv: 5’-TCCAGTAGAGTCAGAAGTCC-3’, q_ltk_fw: 5’-CTGGGAGGAATCCGCTTA-3’ q_ltk_rv: 5’-AGTGAGACCAGTGCAGAG-3’, q_csf1ra_fw: 5’-AGTTCAAATGTATCAGAGACCT-3’ q_csf1ra_rv: 5’-TATCCTGCTCCGAGAATCAT-3’, q_gfp_fw: 5’-AAGGGCATCGACTTCAAGGA-3’ q_gfp_rv: 5’-GGCGGATCTTGAAGTTCACC-3’, q_ntr_fw: 5’-CTTTTGATGCCAGCAAGAAA-3’ q_ntr_rv: 5’-GAAGCCACAATAAAATGCCA-3’, q_cdkn1a_fw: 5’-ATGTGCAGAGGGATGGCTAC-3’ q_cdkn1a_rv: 5’-CCTCCAGATCTTTACGCAG-3’. For normalization the housekeeping gene rpl13a (q_rpl13a_fw: 5’-ACTGTCAGAGGCATGCTTCC-3’ q_rpl13a_rv: 5’-TGCTCTGAAAATTGTGCGCC-3’) was used.
Whole kidney marrow analysis via flow cytometry
Kidneys were dissected from fish and immediately pressed through a 40 μm cell strainer (placed on top of a 50 ml falcon) using a syringe plunger. Strainer and plunger were rinsed with 1 ml of PBS each. Cells were pelleted by centrifugation at 330 x g for 5 min (4°C). Supernatant was discarded and the cell pellet was dissolved in 300 μl of PBS. Analysis of the WKM was done using a BD FACS AriaTM IIIu. The gating strategy was chosen as described for the analysis of the WKM of zebrafish 32.
Irradiation of N. furzeri eggs
For γ–irradiation of F1 eggs from the cdkn1a-reporter line, eggs were placed into 12-well plates (one egg per well) containing 1 ml of 0.3x Danieau’s medium. Eggs were irradiated in the 12-well plate with a dose of 10 Gy using a Gammacell® 40 Exactor (Best Theratronics Ltd.). The presence of an eGFP signal was checked using an Axio Zoom.V16 with ApoTome (Zeiss).
Genotyping of eggs and fish from the cdkn1a-reporter line
DNA was extracted from eggs or fin biopsies as described above. To check for the presence of the reporter construct in the cdkn1a locus, the following primer pair was used: cdkn1a_insertion_fw: 5’-TATTTCTCTGGTGTTTGCCT-3’ egfp_insertion_rv: 5’-TGATATAGACGTTGTGGCTG-3’. To discriminate between the three different genotypes, the following primer pair was used: cdkn1a_genotyping_fw: 5’-CTACAGATCCAGCGTCATC-3’ cdkn1a_genotyping_rv: 5’-CCAAGAGAACCAGACAAAGA-3’. For cdkn1a+/+ animals an amplicon with a size of 393 bp was expected, whereas a 1,893 bp fragment occurred in cdkn1aki/ki fish. In cdkn1aki/+ animals both amplicons were present.
Analysis of GFP-positive cells from the cdkn1a-reporter line
Organs (liver and kidney) were removed from the fish and put into 1x PBS on ice. Liver samples were then transferred into 1.5 ml tubes containing 900 µl of sterile PBS and 100 µl of Collagenase I. After an incubation for 1h at 32°C at 550 rpm, 100 µl of FBS were added and tubes were placed on ice. Livers were then pushed through a 100 µm cell strainer placed on a 50 ml tube using syringe plunger. Cell strainer and plunger were rinsed twice with 1 ml of PBS each. After centrifugation at 250 x g for 5 min (4°C), the pellet was resuspended in 1 ml PBS containing 25 µl of Collagenase I and 3 µl Dispase followed by an incubation for 30 min at 37°C. Afterwards, 100 µl of FBS were added and the solution was pushed through a 40 µm cell strainer placed on a 50 ml tube. Kidney samples were directly placed on a 40 µm cell strainer, 1 ml of PBS with 1% FBS was added and then pushed through the cell strainer using a syringe plunger. Cell strainer and plunger were rinsed twice with 1 ml of PBS each.
After centrifugation at 250 x g for 5 min (4°C), the pellet was resuspended in 200 µl PBS containing 1% FBS. The cell suspension was stained with 5 nM Sytox Red Dead Cell Stain (Invitrogen) to identify dead cells and were subsequently sorted on a BD FACS AriaIIIu Cell Sorter using a 100 µm Nozzle. After doublet and dead cell exclusion, the GFP-positive cells were sorted in PBS. The GFP+ gate was set according to the negative control obtained from respective wild type tissues. Data were analyzed using FlowJo v10 Software (BD).
Immunofluorescence staining
Liver tissues were fixed overnight in 4% paraformaldehyde in PBS and subsequently washed three times in PBS-T (0.2% Tween in PBS). Tissues were placed for 5 min in 5% sucrose, then for 2h in 20% sucrose and subsequently overnight in 30% sucrose. Tissues were embedded and shock frozen in molds filled with NEG-50™ cryosection medium. The tissue was cut into 20 µm slices. After defrosting for 25 min, the sections were washed four times for 10 min at RT in PBS-T (0.2% Tween) and were permeabilized by a short washing step with permeabilization solution (0.1% Tween, 0.3% Triton X 100 in PBS). Immediately afterwards, the sections were washed again two times for 10 min at RT in PBS-T (0.2% Tween) and incubated in blocking buffer (2% BSA and 10% NGS in PBS-T (0.2% Tween)) for 1h at RT. Afterwards, the samples were incubated overnight at 4°C with an anti-GFP antibody (Thermo Fisher Scientific Inc., United States: A-11122, rabbit) diluted 1:200 in blocking buffer. Sections were then washed four times for 10 minutes at RT in PBS-T (0.2% Tween) prior incubation with bisbenzimide Hoechst 33258 and a secondary anti-rabbit Alexa Fluor® 546 antibody (Thermo Fisher Scientific Inc., United States: A-11071, goat) diluted 1:500 in blocking solution for 1h at RT. After multiple washing steps in PBS-T (0.2% Tween), the slides were mounted with 70µl ProLong® Diamond antifade reagent (Thermo Fisher Scientific Inc.). Before imaging, the samples were incubated at 4°C overnight. Image stacks were recorded as optical sections with the Axio Imager 2 equipped with an ApoTome.2 slider (Zeiss, Germany). The ZEN 3.4 software (Zeiss, Germany) was used to process the images and to create extended depth of focus projections of the acquired z-stacks.
Statistical analysis
Data were analyzed depending on the experimental setup via t-test or One-Way ANOVA followed by Tukey’s post hoc test. Equal or unequal variance was determined via F-Test followed by a Student’s or Welch’s t-test, respectively. Significant changes are indicated by * if p≤0.05, ** if p≤0.01, and *** if p≤0.001.
Author Contributions
J.K. and C.E. conceived the study. C.A. supported characterization of the senescence reporter fish. V.L.H. was instrumental in establishing HDR-based targeted insertions in the lab and performed the imaging of the senescence reporter line. J.K. performed all other experiments and data analyses. C.E. supervised the study. J.K. and C.E. wrote the manuscript.
Competing Interests statement
The authors declare no competing interests.
Acknowledgements
We thank Hanna Reuter for suggesting the name klara, Nils Hartmann for the movie on wild type killifish mating, Annekatrin Richter for providing the picture on oocyte injection and Hakar Aliyas, Caglar Avci, Michelle Burkhardt, Christina Ebert, Gabriele Günther, Maleen Hofmann, Erik Hüttenrauch and Dagmar Kruspe for technical support. We are very grateful to members of FLI’s killifish facility, most notably to Simone Dunkel, Martin Neumann, Marcus Schmidt, Uta Naumann and Beate Hoppe. We also would like to thank members of the Core Facility Flow Cytometry, namely Johanna Schleep, Simone Tänzer and Katrin Schubert for their contribution. This project was made possible by funding from the Carl Zeiss Foundation in the context of the IMPULS consortium (project number P2019-01-006) to C.E. and a fellowship from the Leibniz Graduate School on Ageing and Age-Related Diseases (LGSA) to J.K. The FLI is a member of the Leibniz Association and is financially supported by the Federal Government of Germany and the State of Thuringia.