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TEMPO: A system to sequentially label and genetically manipulate vertebrate cell lineages

Isabel Espinosa-Medina, Daniel Feliciano, Carla Belmonte-Mateos, Jorge García-Marqués, Benjamin Foster, Rosa Linda Miyares, Cristina Pujades, Minoru Koyama, Tzumin Lee

Preprint posted on October 28, 2021 https://www.biorxiv.org/content/10.1101/2021.10.27.466134v1

Keeping TEMPO with cell fate transitions – a new technique allows for the temporal labeling and manipulation of cell lineages in vertebrate models.

Selected by Julia Grzymkowski

Background
Most developing tissues begin with loosely specified progenitor cells that depend on an appropriate sequence of proliferative and transcriptional events which dictate their fate specification. In the central nervous system and pancreas, for example, cell-fate regulatory genes are expressed in progenitor cells in a specific order to allow for the progressive differentiation of that tissue’s necessary cell types (1, 2). Live imaging and retrospective studies have helped determine the transcriptional timing of these regulatory genes but defining the full temporal boundaries in which a gene can promote differentiation is difficult to do with current methods. Therefore, there is a need for a robust technique that allows us to both study and manipulate the spatio-temporal differentiation of diverse cell types in a developing tissue at high-resolution. To this end, Espinosa-Media et al. developed TEMPO, a method based on imaging and CRISPR/Cas9 which allows for the temporal labeling and manipulation of cell lineages in vertebrate models.

Key Findings
To design the TEMPO system, the authors utilized two types of genetic switches: CaSSA (3) and gRNA switches. CaSSA switches are disrupted reporter genes that become activated after CRISPR/Cas9 mediated double-stranded breaks followed by single-strand annealing based repair. Multiple CaSSA fluorescent reporters were integrated into different coding frames, with one reporter preactivated. After some time, a specific gRNA (plus Cas9) activates the upstream reporter, creating a frame shift that inactivates the downstream reporter. The first gRNA (plus Cas9) then goes on to activate the next gRNA in the cascade which activates the next upstream reporter and so on. The final form of TEMPO was a compact tri-cistronic transgene containing three sequentially activated fluorescent reporters (CFP, RFP, and YFP) (Fig. 1).

 

Figure 1. A) The TEMPO tri-cistronic transgene allows for the sequential activation of fluorescent reporter expression by a conditional gRNA cascade, resulting in the temporal labeling of cell lineages (B).

The authors first tested their switch system by injecting zebrafish at the 1-cell stage with ubiquitous TEMPO and Cas9, so that they were expressed in all tissues, with or without the required gRNA for activation. This allowed for the measurement of efficiency and leakiness of the reporters, with the ability to then optimize their system. Validation of the full TEMPO cascade involved injecting 1-cell stage zebrafish with two ubiquitous constructs: one containing TEMPO and the first gRNA and the other containing Cas9 and the gRNA switch. By observing fluorescence at different time points, the authors were able to see that the labeled cells changed color over time. At 8 hours post fertilization (hpf), most TEMPO+/Cas9+ cells expressed CFP (the first reporter), and later, at 24 and 48 hpf, cells expressed RFP and YFP (the second and last reporter), respectively, and these color transitions were limited to dividing cells.
TEMPO was designed to assess the spatio-temporal dynamics of cell differentiation in developing tissues, so the authors next validated their full system in neural progenitors of the zebrafish hindbrain by injecting TEMPO constructs in transgenic animals. This limited expression of TEMPO and Cas9 to specific neural progenitors. With this system the authors were able to recapitulate prior studies which found differences in the mitotic index or birthdate of progenitors along the dorso-ventral and medio-lateral axis, by finding cells expressing CFP, RFP, or YFP in different regions of the hindbrain over time. For example, early born CFP+ atoh1a+ neurons occupied the lateral most regions of the hindbrain, while later born YFP+ atoh1a+ neurons occupy medial positions. Also, later born YFP+ vsx2+ neurons occupied dorsal positions with respect to earlier born RFP+ and CFP+ in rhombomeres 2-5.
While zebrafish were imaged live for the first few experiments, TEMPO does not require live imaging, so the authors next tested their technique in mice, a vertebrate model with poor imaging accessibility. The zebrafish constructs were modified to be applicable in mouse, and the resulting constructs were electroporated into embryonic mouse cortices. Mouse brains from E13.5 to E17.5 were fixed and TEMPO+ cells were assessed. Again, the authors were able to recapitulate the results from previous studies, finding that early born CFP+ progenitors occupied deep layers within the subventricular zone, and late born RFP+ and YFP+ neurons migrate past them into the cortical plate. In addition, after the completion of neurogenesis, most YFP+ cells were found to be astrocytes, consistent with the known transition of these neurons to a gliogenic mode.
The final experiments utilized TEMPO-2.0, where the activation or inactivation of the fluorescent reporters was coupled to manipulation of genes known to regulate cell fate determination. By overexpressing the cell cycle gene cyclin D1 (important for G1/S transition) at later stages, when its expression would normally be reduced, most RFP+ and YFP+ cells were still within the deeper neocortical layers. This suggests that cyclin D1 promotes a cell fate reminiscent of those found in deeper brain regions. In contrast, overexpression of cyclin B1 (important for the G2/M transition) led to a shift from neurogenesis to gliogenesis, with an increase in CFP+ and RFP+ astrocytes.

Why I chose this preprint
This preprint offers a highly innovative new technique for the spatio-temporal visualization and manipulation of cell lineages. I appreciated the digestible explanation of the system, proof of concept experiments, and the fact that the system was robustly tested in not one, but two vertebrate model organisms. In addition, this is now the first time in which CaSSA reporters and gRNA switches were successfully utilized in mice, showing that TEMPO could improve mouse studies within this area of research.

Questions for the authors

1. Do you think this method could be used successfully with whole mouse embryonic tissues which are cleared prior to imaging?
2. In your opinion, how many different reporters could be utilized in one TEMPO system? Could you have more than three or would that interfere with genome integration and germline transmission?
3. What are your thoughts on the utility of TEMPO to study how environmental contaminants/perturbations affect cell fate determination and even define specific temporal windows in which cells are uniquely vulnerable? Is this method limited to genetic manipulation?

References
1. T. Isshiki, B. Pearson, S. Holbrook, C. Q. Doe, Drosophila neuroblasts sequentially express transcription factors which specify the temporal identity of their neuronal progeny. Cell. 106, 511–521 (2001).
2. M. E. Wilson, D. Scheel, M. S. German, Gene expression cascades in pancreatic development. Mech. Dev. 120, 65–80 (2003).
3. Garcia-Marques J, Yang CP, Espinosa-Medina I, Mok K, Koyama M, Lee T. Unlimited Genetic Switches for Cell-Type-Specific Manipulation. Neuron. 2019 Oct 23;104(2):227-238.e7. doi: 10.1016/j.neuron.2019.07.005. Epub 2019 Aug 5. PMID: 31395429.

Tags: brain, crispr, fish, fluorescent reporter, mouse

Posted on: 17th November 2021

doi: https://doi.org/10.1242/prelights.31056

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Author's response

Isabel Espinosa-Medina shared

1. Do you think this method could be used successfully with whole mouse embryonic tissues which are cleared prior to imaging?

Yes, but to preserve the endogenous fluorescence of the reporters it would be better to avoid the use of Methanol. For references on this, see the latest clearing protocol (Wan Pet et al. FDISCO+: a clearing method for robust fluorescence preservation of cleared samples. Neurophotonics. 2021 Jul;8(3):035007. doi: 10.1117/1.NPh.8.3.035007.Epub 2021 Sep 9). Another possibility is to perform immunofluorescence to detect and amplify the TEMPO reporters (V5- tagged CFP, mCherry or mGFP antibodies can be used) prior to clearing.

2. In your opinion, how many different reporters could be utilized in one TEMPO system? Could you have more than three or would that interfere with genome integration and germline transmission?

The number of reporters could be increased, as long as they can be simultaneously imaged and distinguished (increasing the number of fluorescent reporters could require spectral unmixing strategies).
With the current TEMPO system, we could add 2 to 3 additional reporters in the same construct which currently contains Cas9, so that we limit the total number of constructs to 2 (TEMPO + Cas9). In that case genome integration / germline transmission should not be an issue. Importantly, we found that different CaSSA or gRNA switches have similar repair efficiencies, and this is crucial for the cascade to proceed even if we add more sequential reporters in the future.

3. What are your thoughts on the utility of TEMPO to study how environmental contaminants/perturbations affect cell fate determination and even define specific temporal windows in which cells are uniquely vulnerable? Is this method limited to genetic manipulation?

This is an interesting application indeed! TEMPO is not limited to genetic manipulation. One could study how different cell generations labelled by TEMPO respond to environmental changes (contaminants, nutritional changes, light/dark, temperature, etc) and eventually define which molecular factors underlie differences in cell susceptibility. Moreover, we could deploy constitutive metabolite (e.g., glucose, fatty acids) sensors, which if co-expressed with TEMPO, could reveal how changes in metabolic activity influence different vertebrate cell generations.

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