O amigo Fritz (Portuguese Edition)
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Our data indicate that dmrt2b does not seem to compensate for the lack of dmrt2a , supporting the findings of Liu et al. Therefore, to uncover if a compensatory mechanism is taking place, a genome wide approach could be performed comparing mutants and morphants.
To prove the absence of mutant proteins we need specific antibodies that are not available. Thus, in an attempt to understand the outcome of our mutations, we evaluated dmrt2a and dmrt2b transcripts in the different mutant backgrounds. In theory, if the transcripts are not functional they should be degraded.
This hypothesis should have been true at least in the case of dmrt2b mutant, due to non-sense mediated decay.
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However, we observed that dmrt2a and dmrt2b transcripts are up-regulated in the different mutants evaluated. This does not exclude the possibility of degradation of the mutant transcripts, only that the rate of transcript degradation is slower than the rate of production. Regarding future approaches to this problem, an effort should be placed in the identification of dmrt2a and dmrt2b promoter regions, in the generation of bona fide null mutants and in the production of specific antibodies.
As we have no certainty that dmrt2a mutants are nulls, we decided against using these lines in the microarray approach. Yet, we were able to take advantage of them to validate dmrt2a -MO, confirming its specificity. Therefore, we performed the microarray with Tg hsp HA- dmrt2a and validated its data using dmrt2a -MO. From the list of microarray validated genes, foxj1b [ 19 ] is a strong candidate to mediate the left-right asymmetry phenotypes of Dmrt2a, which were previously described in gain and loss-of-function approaches.
With our timely controlled dmrt2a overexpression experiments we identified a new phenotype in somite border formation, which recalls the Dmrt2 mouse mutant phenotype [ 9 ]. The defects observed in the mouse mutants were associated with a strong reduction of the levels of LAMININ-1, an extracellular matrix protein [ 9 ]. While in our pool of six validated genes, we did not identify classic extracellular matrix related genes, we found an interesting candidate, pxdc1b. Pxdc1b belongs to a family related to membrane attachment to organelles of the endocytic and secretory systems via binding of phosphoinositide lipids [ 22 ].
As extracellular matrix proteins are continuously being secreted [ 23 ], pxdc1b could establish a link between Dmrt2a, extracellular matrix deposition and somite border defects.
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Additionally, in our microarray, we identified cyp1a as a gene downstream of Dmrt2a. This cytochrome was one of the few genes up-regulated after dmrt2a overexpression and the only one that was validated by the loss-of-function approach.
Also, cyp1a is the only validated gene from the microdissected Region 2. However, Cyp1a physiological function in the embryo remains unknown therefore, its interaction with Dmrt2a will require further analysis. Interestingly, we validated three genes related to vascular development: Although we lack functional data to explain the relationship between Dmrt2a and these genes, when considering dmrt2a expression pattern within the somite, we can contemplate the hypothesis that Dmrt2a is affecting the small population of somite-derived endothelial cells [ 24 , 25 ].
As dmrt2a is also expressed in the myotome [ 2 ], and as it acts as a transcriptional repressor, we can speculate that it could restrict cxcl12b endotome expression domain. Therefore, a possible cell non-autonomous interaction between Dmrt2a and etv2 could occur at somite territory. Foxc proteins have been previously described to interact with etv2 [ 21 ] thus, it would be worthwhile to analyse if, the somite-expressed gene foxc1b , can cooperate with etv2 downstream of Dmrt2a.
Although in zebrafish, a link between Cxcl12 signalling and Foxc transcription factors has not been established, it would be interesting to evaluate if this interaction could occur. In this study, we generated and characterised an inducible dmrt2a transgenic line and mutant lines for dmrt2a and dmrt2b. Using our transgenic line we described for the first time dmrt2a overexpression phenotype. We confirmed that the mild phenotype present in dmrt2a mutant lines is not due to a compensatory mechanism mediated by dmrt2b.
Additionally, we took advantage of one of our dmrt2a mutant lines to confirm dmrt2a -MO specificity, validating this tool. Using our tools we performed and validated a microarray experiment that allowed the identification of six genes downstream of Dmrt2a: The identification of these six genes will contribute to the understanding of Dmrt2a mechanism of action during zebrafish early development.
In which embryonic domains is Dmrt2a controlling these genes, if their interaction with Dmrt2a is direct or not and, how can these genes contribute to the previously described Dmrt2a phenotypes, will be addressed in future studies. Adult zebrafish were only used as breeders. When growing a new fish line, embryos were bleached before reaching 24 hpf, as described [ 28 ].
HA- dmrt2a and validated its data using dmrt2a -MO. Concerto para Violoncelo e Orquestra. In this work, we generated and validated several genetic tools that allowed us to identify six genes downstream of Dmrt2a during early embryonic development: University of San Francisco. The latter often can have obscene connotations, but it is generally not considered profane. Cosalia — a collection of things, especially useless things — is a less common placeholder.
The number of juvenile fish per litre decreased gradually until reaching adulthood. Zebrafish were raised and maintained in a six-rack recirculating system from Tecniplast supplied by a reverse osmosis unit. Additionally, adult fish ate a meal of decapsulated Artemia ZM Systems per day. All experiments were performed with embryos obtained from an outcross of homozygous progenitors, in the case of transgenic fish, and from an incross of two homozygous progenitors, in the case of mutant fish. To generate the Tg hsp The transgenic construct was generated using a set of PCR reactions: Southern blot analysis was performed as described [ 29 ].
Briefly, F1 heterozygous adult transgenic fish were anaesthetised using Tricaine Sigma-Aldrich. The tail fin was cut, and genomic DNA was extracted. Afterwards, the gel was stained and washed, and the DNA transferred to a nylon membrane positively charged Roche.
At bud-stage, embryos were placed in embryo medium 5. HA- dmrt2a and control embryos were heat-shocked with the same procedure. The G substitute chosen was NN. The upstream base chosen to each monomer was T. TALEN binding sites are in bold caps, and the spacer region is underlined. Paired Target Finder was used to check for off-targets, the Score Cut-off chosen was 3.
The primers used for this method were: In the morpholino validation experiments, we injected dmrt2a morpholino in the mutant background, in the same days and using the same settings as in the wildtype counterparts, to ensure a controlled procedure. Single whole-mount in situ hybridisation was performed as described [ 31 ]. F-actin and nuclei were detected with Alexa Fluor Phalloidin 1: The following procedure was performed as described [ 45 ].
For HA-tag detection, the membranes were incubated overnight with rabbit anti-HA antibody 1: For beta-actin, we used a rabbit anti-beta-actin 1: HA- dmrt2a and wildtype embryos were microdissected as described [ 46 ]. Tissues from Region 1 and Region 2 Fig. We obtained a pool of pieces from Region 1 and pieces from Region 2, from each condition, per replicate.
Three biological replicates for each condition were used. All samples were analysed with Affymetrix Zebrafish Gene 1. For the gain-of-function experiment, we collected embryos from the Tg hsp HA- dmrt2a line and wildtype embryos, and performed a heat-shock Fig. All embryos were microdissected in Region 1 and Region 2 Fig. From this approach, we obtained a pool of 60 pieces from Region 1 and a pool of 60 pieces from Region 2, from each condition experimental and control , per replicate.
For the loss-of-function experiment, we injected wildtype embryos with dmrt2a -MO or control-MO, and microdissected them in Region 1 and Region 2 Fig.
From this approach, we obtained a pool of 46 pieces from Region 1 and a pool of 46 pieces from Region 2, from each condition experimental and control , per replicate. Three biological replicates were used in each experiment. We evaluated each gene on an individual basis, between experimental and control samples. Data were analysed using a two-tailed t -test. All experiments were performed with at least three biological replicates, collected from different breeders.
Buy O amigo Fritz (Portuguese Edition): Read Kindle Store Reviews - Amazon. com. Os Mistérios do seu Gato (Portuguese Edition) [Dick Wolfsie e Gary SINOPSE Conheça Fritz, o gato que preferia fazer as suas necessidades no fato de flanela diz respeito a definir um comportamento apropriado para o seu melhor amigo.
P values of less than 0. The start codon is underlined, and the spacer region is in lowercase. HA-Dmrt2a dynamics after heat-shock. Immunostaining showing the expression of the HA-Dmrt2a fusion protein at different time-points post-heat-shock. The HA-Dmrt2a protein is depicted in red, and the nucleus is depicted in blue. Genes related to cardiac and vascular development are underlined. Gene Ontology GO analysis of the microarray data.
No misexpression was found in the validated genes after dmrt2a gain and loss-of-function experiments.
After dmrt2a overexpression using Tg hsp HA- dmrt2a we did not observe misexpression of the six validated genes. Upon comparison with dmrt2a -MO injected embryos, we observed changes in the expression levels of some genes arrowheads , according to qPCR data.
Alexandre Da Costa
As depicted with asterisks, after dmrt2a -MO injection we observed only very subtle changes in the expression pattern of some genes, corresponding to the less affected genes, as quantified by qPCR. A1-C3 Lateral view, anterior to the left. We did not observe obvious differences between the three different conditions evaluated.
All embryos were collected between 3 and 4-somite stage. We are grateful to Ana Rita Grosso for her help in microarray data analysis. RAP performed all the experiments, analysed the data and wrote the manuscript. RL conceived the plan for the transgenic line. LS conceived the project, analysed the data and wrote the manuscript.
All authors read and approved the final manuscript. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rita Alexandra Pinto, Email: National Center for Biotechnology Information , U. Published online Jun Author information Article notes Copyright and License information Disclaimer. Received Oct 10; Accepted May Associated Data Supplementary Materials Additional file 1: Abstract Background Dmrt2a is a zinc finger like transcription factor with several roles during zebrafish early development: Results We generated and validated a heat-shock inducible transgenic line, to timely control dmrt2a overexpression, and dmrt2a mutant lines.
Conclusions In this work, we generated and validated several genetic tools for dmrt2a and identified six genes downstream of this transcription factor. Electronic supplementary material The online version of this article Background The DMRT Doublesex and Mab3 Related Transcription factors family of zinc finger like transcription factors has been classically associated with sexual determination and differentiation. Results Generation of a heat-shock inducible transgenic line to timely overexpress dmrt2a We generated a heat-shock inducible transgenic line Tg hsp Open in a separate window.
Transient increase of dmrt2a expression produces a similar phenotype to dmrt2a loss-of-function We used the Tg hsp A mild to negligible phenotype is observed in dmrt2a mutants In order to produce stable dmrt2a mutant lines, we used the TALEN technology Fig. Genome wide approach to identify Dmrt2a downstream genes In the context of the Tg hsp Validation of six genes downstream of Dmrt2a: Discussion In order to establish the mechanism of action of Dmrt2a, one needs to identify its downstream genes.
Conclusions In this study, we generated and characterised an inducible dmrt2a transgenic line and mutant lines for dmrt2a and dmrt2b. Transgenic line generation To generate the Tg hsp Southern blot Southern blot analysis was performed as described [ 29 ]. In situ hybridisation and immunohistochemistry Single whole-mount in situ hybridisation was performed as described [ 31 ]. Statistical analysis All experiments were performed with at least three biological replicates, collected from different breeders.
Additional files Additional file 1: Availability of data and materials The datasets supporting the conclusions of this article are available in the Gene Expression Omnibus GEO repository, with the accession number GSE https: Competing interests The authors declare that they have no competing interests. Footnotes Electronic supplementary material The online version of this article Expanding roles for the evolutionarily conserved Dmrt sex transcriptional regulators during embryogenesis.
Cell Mol Life Sci. A Drosophila doublesex-related gene, terra, is involved in somitogenesis in vertebrates. Terra is a left-right asymmetry gene required for left-right synchronisation of the segmentation clock. Left-right function of dmrt2 genes is not conserved between zebrafish and mouse. Celf1 regulation of dmrt2a is required for somite symmetry and left-right patterning during zebrafish development.
MicroRNAa regulates fast muscle differentiation by targeting dmrt2a in zebrafish embryos. Fish specific duplication of Dmrt2: Fish-specific duplicated dmrt2b contributes to a divergent function through hedgehog pathway and maintains left-right asymmetry establishment function. Targeted disruption of the DM domain containing transcription factor Dmrt2 reveals an essential role in somite patterning. Stages of embryonic development of the zebrafish. Two T-box genes play independent and cooperative roles to regulate morphogenesis of ciliated Kupffer's vesicle in zebrafish.
The nuclear localisation signal of zebrafish terra is located within the DM domain. Nodal activity in the node governs left-right asymmetry. A nodal-independent and tissue-intrinsic mechanism controls heart-looping chirality. The evolution and conservation of left-right patterning mechanisms. Sex-specific transcriptional regulation by the male and female doublesex proteins of Drosophila.
Both foxj1a and foxj1b are implicated in left-right asymmetric development in zebrafish embryos. Biochem Biophys Res Commun.
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