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Prof. Lazaro CentaninClonal Analysis of post-embryonic Stem Cells

My lab is interested in understanding stem cells, a fascinating type of cell that can self-renew and is responsible to generate once and again the cells that we humans (and most other animals) lose on daily basis - skin cells, blood cells, intestine cells. In adult mammals, the number of newly generated cells match the number of lost cells, so they exhibit a fixed organismal size despite of massive stem cell proliferation.

Fish constitute a curious example in which organismal size is not fixed, but rather increases permanently even during adulthood. Permanent growth in fish depends on stem cells, and in the lab we are mainly interested in two topics: a) are the same individual stem cells driving growth and maintaining homeostasis? Or do fish have different populations for one and the other tasks?, and b) how do stem cells in different tissues coordinate their functions for the fish to maintain its shape during permanent growth?


Scaling & The Problem of Permanent Growth

We are particularly interested in learning how organs can adapt to the changing organismal size, with a focus on the molecular mechanisms for stem cell coordination within and among organs. We are tackling this topic by using organs that grow in size, like the branchia, and organs that grow in numbers, like the neuromasts of the lateral line system. We follow stem cell derived lineages in different organs to learn whether fish use the same stem cells to maintain homeostasis replacemente and to drive growth, or if they have dedicated populations for each feature. In collaboration with mathematicians, we focus on how many stem cells and stem cell types are required to setting up a functional organ during embryogenesis and in an already developed organism.


scaling fish

Life-Long Organogenesis


Since fish grow during their entire post-embryonic life, their organs have to adapt to an increasing body size. The lateral line system grows by generating new organs (so-called neuromasts) in a relatively stereotypic manner. We have generated several transgenic lines to explore how neuromasts are created after embryonic development (Development 144, 687-697), which are their cells of origin and which and how they integrate the signals from the environment to trigger organ formation. We combine these lines with a living toolkit to study stem cell lineages that relies on the generation of colourful (genetic) mosaics, and we named the toolkit Gaudi after the famous spanish architect. The Gaudi toolkit is composed of reporter lines (GaudiRSG, GaudiBBW and GaudiLxBBW) and Cre driver lines (GaudiUbiq.iCre and GaudiHsp70CRE) (Development 141, 3472-3482).


Individuals vs. Populations

Mixing up specific features of different individuals migth result in fictional, disturbing scenarios. 

Historically, lineage analysis in vertebrates was performed on entire populations of cells using either transplantation approaches or genetic tools like Cre/LoxP mediated recombination. Both methods have contributed extensively to our current knowledge on the lineage relation of differentiated cells during embryogenesis, the potential of stem cell populations to generate or re-generate missing cell types, and the cell autonomous or non cell autonomous effects of many genes during development and disease. 

A population is a group of individuals; it is not accurate to assume that each individual is capable of executing the universe of tasks performed by the population it belongs to. Numerous examples have proven that single cells would only display a subset of the properties of the population - individual potency is not necessarily collective potency. Defining the potential of individual cells is necessary to characterise the different properties that co-exist within a cell. The comparative profile of these properties defines the heterogeneity of the population, a parameter that has proven critical during homeostasis, tumor formation and growth.  

We use in the lab the Gaudi toolkit, which is largely based on brainbow constructs (Livet et al, Nature 2007) - a mutually exclusive combination of fluorescent proteins that will be stochastically assigned to each cell and inherited by its progeny. 

In our lab, following single cells is our manner to tackle heterogeneities. There is an immense functional heterogeneity among stem cell within a population, considering proliferative capacity and differentiation potential. Why do two neighbour stem cells behave so different? We are tackling functional heterogeneities on somatic stem cells by combining metabolic reporters in vivo with lineage progression. By using a quantitative 4D analysis on the stem cells we aim at linking the metabolic state of a stem cell to its future clonal progression in its intact niche.



The Gaudi Toolkit

Brainbow constructs consist of mutually exclusive combination of fluorescent proteins that will be stochastically assigned to each cell and inherited by its progeny. These were adapted to both zebrafish (Pan et al, Development 2013) and medaka (Centanin et al, Development et al 2014). We have generated a living toolkit in medaka (the so-called Gaudí toolkit) that contains several LoxP reporter lines and also universal, inducible Cre driver lines. 

.- The reporter lines were selected considering their ubiquitous expression during the entire life of the fish, which allows proper lineage analysis of many organs at a huge range of developmental and post-embryonic stages. 

.GaudiRSG: a zebrafish ubiquitin promoter followed by a floxed RPF that prevents expression of an H2B-EGFP. Default: Red; Recombined: nuclear Green

.GaudiBBW: a zebrafish ubiquitin promoter followed by a BBW2.1 inverted cassette. Default: membrane Cerulean; Recombined: nuclear Green, yellow , Tomato.

.GaudiLxBBW: a zebrafish ubiquitin promoter followed by a flowed RPF that prevents expression of a Brainbow2.1 cassette. Default: Red; Recombined: nuclear Green, yellow, membrane Cerulean, Tomato.


Driver lines:

.GaudiHsCre: This Tg line expresses a nuclear-tagged Cre recombinase under the control of the zebrafish heat-shock 70 promotor. It has a cmclc2:EGFP as an insertional control. Recombination can be induced efficiently during embryogenesis, but the activity decays during post-embryonic life. 

GaudiUbiiCre: this line contains a tamoxifen inducible Cre (ERt2:Cre) under the control of the zebrafish ubiquitin promoter. It has a cmclc2:ECFP as an insertional control. Recombination can be efficiently induced during embryonic, post-embryonic and adult stages.


All combination of Driver and Reporter lines were successfully used to trigger recombination in most of medaka tissues; the only exception that we are aware of is blood cells, which are not labelled in the Gaudi reporter lines.We have develop protocols to modulate the intensity of clone induction, which consist of different temperature shifts to the Hsp70::CreNLS line and a wide range of concentrations of trans-tamoxifen and 4-OH-tamoxifen to the  ubiquitin::ERt2Cre line. We use the recombination observed in the somites as a proxy for the recombination that occurs in the most internal organs. If you are interested in our induction protocols, please check Centanin et al 2014 or drop us an email.  If you are interest in already recombined fish, please check here and/or write to us.



Save the Fish - the 3 R's

The use of entire animals is inherent to most of our running projects. We understand the importance of an animal's life and our approaches consider the 3R principles: replace, reduce and refine. 

We have chosen to carry out all the experiments concerning stem cells in their natural niche in lower vertebrates, therefore replacing the use of warm-blooded animals. Most of our experiments consist on following homeostatic lineages, and we have checked that fish from the Gaudi toolkit do not show any behavioral phenotype when compared to wild type medaka fish.  Additionally, in the few cases in which the experiments can be done ex vivo, we are setting up collaborations to use organ culture conditions as alternatives to entire animals; when possible, we asses  stem cells at embryonic stages.


Fish are at the centre of our research and we have taken all possible measurements to reduce the number of experimental organisms. In the last years we have generated a number of transgenic lines that allow following wild type, labelled cells in each organ and tissue of the fish (the Gaudi toolkit, link). The use of these  has drastically reduced the number of experimental animals utilised by our and neighour groups. We store all organs of recombined fish that are not used for a particular experiment and these are free for other groups (or other researchers in our group) to use without performing additional animal experiments. Please check with us in case you are interested in studying the lineages of other organs in medaka!