Lineage tracing and fate mapping - 2 BSE652 Lecture th and 14 th Jan 2016 Inter-species transplant Early stage embryonic blastomeres are pluripotent However the plane of constriction is critically important Lasso made of baby hair Early stage embryonic blastomeres are pluripotent However the plane of constriction is critically important The half with gray crescent develops into a full fledged embryo Conditional Vs Autonomous development The host and the transplant had differences in pigmentation to enable tracing the fate of the transplanted cells. Chick-quail chimera Quail cells can be detected due to the presence of heterochromatin around their nuclei OR more definitively by the presence of QCPN antigen. Chick-chick chimera Transplantation of trunk neural crest cells from a pigmented chick to the similar region of an unpigmented chick embryonic host demonstrated that neural crest cells contribute to pigmentation. Similar experiments demonstrated that retinal pigment originate from within the retina and NOT from the neural crest cells. Combination of retroviral infection and transplantation The enteric nervous system developed from the graft, by extension from the neural crest cells Genetic mosaic in mice etc. Sox9-null mutant neural crest cells can migrate to appropriate locations Please note that LacZ is knocked in the Sox9 locus Mori-Akiyama Y et al. PNAS 2003;100: 2003 by National Academy of Sciences Chimeric mice have also been generated to ask lineage related questions. These mice are typically generated through aggregate chimeras of early stage embryos. Cell marking by genetic recombination UAS-GAL4 system Cre-LoxP system FLP-FRT system Photo-activable markers Intersectional fate mapping Split GAL4 system Cre-LoxP and FLP-FRT intersection Hafezi, Yassi; and Nystul, Todd G (March 2012) Advanced Techniques for Cell Lineage Labelling in Drosophila. In: eLS. John Wiley & Sons, Ltd: Chichester. DOI: / a The Gal4 binary expression system expands levels of control. (A) The genetic components of the Gal4-UAS system are present in two distinct transgenic fly lines so that leaky expression and potential genetic selection are avoided. The driver and target UAS fly lines are crossed, and the full complement of genetic elements comes together exclusively in the progeny genome. (B) In the first phase of binary expression, the Gal4 promoter determines the time and cell specificity of transcription. After translation, Gal4 protein binds to UAS sequences placed upstream of any and all cds, causing their coordinate expression, so a single UAS- marker reveals the specific expression pattern of all UAS-linked sequences. The box above the colorless cell shows UAS-target sequences inserted into the background. (C) A tertiary level of control is possible through the use of a specific Gal4 repressor, Gal80 TS, which is temperature sensitive. At lower temperatures, the repressor is active, and there is no Gal4 transcription; at higher temperatures, the repressor cannot function, and the active repressor pool becomes depleted. Gal4 transcription progressively resumes, and binary expression is reactivated. Mitosis Mitotic recombination Lineage Tracing through Genetic Recombination Cell 148, January 20, 2012 2012 Elsevier Inc. The FLP-FRT system The positively marked mutant lineage (PMML) technique The Flp-FRT method and the PMML strategy. (A) The yeast recombinase Flippase is under the control of the Hsp70 promoter in hs-Flp. Heat shock induces Flp expression, which activates recombination specifically at target FRT sites previously inserted into the genome. (B) In the PMML strategy, FRT sites are differentially flanked by a 5-tubulin promoter on one homologous chromosome and a 3-Lac-Z on the other. (C) Heat- shock induces mitotic recombination, creating a mosaic animal with one unmarked twin spot and one twin spot in which a functional fused tubulin promoter-LacZ cds leads to expression of -galactosidase. Genetic odyssey to generate marked clones in Drosophila mosaics Ruth GriffinRuth Griffin, a,b,c Richard Binari, d,e and Norbert Perrimon d,e,1Richard BinariNorbert Perrimon Proc Natl Acad Sci U S A. Apr 1, 2014; 111(13): 47564763. TSG differential labeling of twin spots. (A) TSG is a Flp-FRTbased mitotic recombination (MR) strategy, except that the FRT site, located in an intron, is flanked with complementary GFP and RFP sequences to create TSG hybrid cassettes. (B) Heat shock-induced MR generates full-length but interrupted cds for GFP on one recombined chromosome and for RFP on the other. Splicing removes the FRT site along with the rest of the intron to reconstruct continuous full-length GFP or RFP cds. In a heterozygote, placing the wild-type allele distal to the 3 RFP and the mutant allele distal to the 3 GFP enables direct identification of both the red wild-type control and green mutant twins. (C) TSG provides a direct readout of toxicity. In control experiments in which both twin spots are homozygous wild type, statistical analysis showed equal numbers of cells present in the red and green twin spots, as expected and confirming that ectopic expression of GFP or RFP had no apparent toxic effects. The Cre-LoxP system Cell 148, January 20, 2012 2012 Elsevier Inc. Advanced Techniques for Cell Lineage Labelling in Drosophila Yassi Hafezi, University of California, San Francisco, California, USA Todd G Nystul, University of California, San Francisco, California, USA eLS. John Wiley & Sons, Ltd: Chichester. DOI: / a002253 Lineage Tracing through Genetic Recombination Isl1 marks a subset of progenitors in heart and limb. Yang L et al. Development 2006;133: Intersectional Genetic Fate Mapping Please refer to the article for legends to this figure Mapping Cell Fate and Function Using Recombinase-Based Intersectional Strategies Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA Methods in Enzymology, Volume 477 Susan M. Dymecki, Russell S. Ray, and Jun C. Kim Intersectional Genetic Fate Mapping Please refer to the article for legends to this figure Mapping Cell Fate and Function Using Recombinase-Based Intersectional Strategies Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA Methods in Enzymology, Volume 477 Susan M. Dymecki, Russell S. Ray, and Jun C. Kim An in vivo comparison of photoactivatable fluorescent proteins in an avian embryo model Developmental Dynamics Volume 236, Issue 6, pages , 7 MAY 2007 DOI: /dvdy Volume 236, Issue 6,