Downloaded from http://cshperspectives.cshlp.org/ on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Discovering New Progenitor Cell Populations through Lineage Tracing and In Vivo Imaging Rudra Nayan Das and Karina Yaniv Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel Correspondence: [email protected]; [email protected] Identification of progenitor cells that generate differentiated cell types during development, regeneration, and disease states is central to understanding the mechanisms governing such transitions. For more than a century, different lineage-tracing strategies have been developed, which helped disentangle the complex relationship between progenitor cells and their prog- enies. In this review, we discuss how lineage-tracing analyses have evolved alongside tech- nological advances, and how this approach has contributed to the identification of progenitor cells in different contexts of cell differentiation. We also highlight a few examples in which lineage-tracing experiments have been instrumental for resolving long-standing debates and for identifying unexpected cellular origins. This discussion emphasizes how this century-old quest to delineate cellular lineage relationships is still active, and new discoveries are being made with the development of newer methodologies. evelopment of multicellular organisms progenitors from which different cell types Dfrom a single-cell zygote is a complex pro- originate, thereby establishing lineage relation- cess involving highly synchronized events of ships between different cells. Over the last cen- cellular proliferation, differentiation, and pat- tury, a large number of techniques and model terning. For development to proceed in a regu- systems have been developed for efficient and lated manner, mechanisms exist that properly reliable “lineage tracing”—the identification of regulate the generation of cell progenies from progenies generated from a progenitor cell type specific progenitor populations. These progeni- (reviewed by Kretzschmar and Watt 2012; tor cells traverse a defined developmental trajec- Woodworth et al. 2017). tory involving progressive fate decision steps, The importance of lineage tracing lies not each refining their identity, until they reach their only in revealing the identity of progenitor cells, functional end state (Weinreb et al. 2018). Pro- but is also central to the quest for understanding genitor cells also play important roles in the how the different fate decisions are made and mature animal, by serving as reservoirs of new how different cell types can be produced from cell types in contexts of normal tissue turnover, the same progenitors. The identification of cel- damage, and regeneration (Pellettieri and Alva- lular origins for differentiated cell types also rado 2007). Concordantly, a major endeavor of have important functional implications, espe- developmental biology has been to identify the cially in cases where seemingly identical cell Editors: Cristina Lo Celso, Kristy Red-Horse, and Fiona M. Watt Additional Perspectives on Stem Cells: From Biological Principles to Regenerative Medicine available at www.cshperspectives.org Copyright © 2020 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a035618 Cite this article as Cold Spring Harb Perspect Biol 2020;12:a035618 1 Downloaded from http://cshperspectives.cshlp.org/ on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press R.N. Das and K. Yaniv types are derived from ontologically distinct sub- ing specimens. As a result, the cell theory, rec- sets. A growing number of studies have described ognizing that living organisms are made of cells, how similar cell types, derived from ontologically and that cells derive from preexisting cells (for distinct subsets, can show different properties reviews, see Wolpert 1995; Mazzarello 1999), (Topouzis and Majesky 1996; Cheung et al. 2012; was formally formulated in 1838–1839. This Plein et al. 2018; Sande-Melón et al. 2019). In- new insight led to a fundamental question—“If deed, the importance of “lineage history” of a cell all cells come from preexisting cells, then how is is underlined by the recent proposal that a full the whole array of spectacular cell diversity with- characterization of cellular identity requires un- in an organism generated?” The quest for under- derstanding of three essential aspects: lineage, cel- standing the mechanisms underlying cell-type lular function, and state (Morris 2019). diversification, has led to an interesting path of Identification of progenitors of differentiat- discoveries that remains active to this day. ed cells is not a straightforward process. Because Since the beginning, one of the biggest chal- differentiation can make progenies very distinct lenges of lineage-tracing experiments has been from their progenitors, both in morphology and finding ways to reliably track specific cells and molecular composition, clonal relationships be- their progenies within the entire population. tween cell types can only be discovered through Some of the earliest work on lineage tracing well-strategized techniques. The basis of line- took advantage of invertebrate animal models, age-tracing experiments consists in selecting a such as leech (Whitman 1887) and sea squirt reliable and specific identifier for a cell or a cell (Conklin 1905), for direct microscopic observa- population that is stable over the experimental tions of early development of the fertilized egg. time and is inherited by all its progenies through Edwin Conklin, for instance, used a natural label the subsequent rounds of cell division and dif- —an orange-yellow spot—in the cytoplasm of ferentiation (Fig. 1A). At the experimental end the one-cell-stage sea-squirt egg, that was car- point, the identifier should be readily detected in ried with the cells in subsequent divisions and the cells that inherited it (the progenies), thus could be followed up to the tadpole stage (Conk- allowing to establish lineage relationships be- lin 1905). This spot, called the “yellow crescent” tween them and the cells bearing the identifier is now known to contain lipid inclusions at the beginning of the experiment. Typically, that migrate to the vegetal hemisphere of the visual identifiers are used such as dyes or fluo- egg immediately after sperm entry (Gilbert rescent proteins (FPs), but other identifiers like 2000). Conklin was able to trace the cells for genetic barcodes, or heritable change in the ge- up to eight rounds of cell division and found nome have also been successfully used. the yellow cytoplasmic cells to generate meso- Herein, we review different lineage-tracing dermal structures. Unfortunately, natural labels techniques and discuss how they help uncover are not readily available for most model systems, novel progeny–progenitor relationships among and, thus, highlighting specific cells in the rap- various cell types and in different contexts. We idly dividing and differentiating embryo called focus primarily on model systems that were for the establishment of more sophisticated most influential in establishing vertebrate cell methodologies. lineages, including mouse, zebrafish, salaman- The use of “vital dyes” that stain living cells der, and quail-chick chimeras. without killing them became one such way to label and follow cells. The power of this ap- proach was shown by Walter Vogt, who con- TOOLS FOR LINEAGE TRACING structed a fate map of the 32-cell-stage Xenopus embryo (Vogt 1929; discussed in Anderson and A Historical Perspective Dietrich 2012). Small pieces of agar chips were As advancements were made on light microsco- saturated with vital dyes and applied to the dif- py, the 17th century saw an increased interest in ferent regions of the late-blastula embryo. Cells microscopic observation and description of liv- that absorbed the dye retained the label over 2 Cite this article as Cold Spring Harb Perspect Biol 2020;12:a035618 Downloaded from http://cshperspectives.cshlp.org/ on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Progenitor Cell Discovery through Lineage Tracing A Basic design of a lineage-tracing experiment Developmental time Growth and differentiation Introduction of visual or genetic “cell-identifier” using: Site-specific recombination/ Genetic scarring/ Transplantation/ Dyes A “cell-identifier” is used to label specific “Cell-identifier” is inherited Identification of differentiated progenies cell(s); the identifier is heritable and by progenies of (bearing the “cell-identifier”) at a desired retrievable at a later time labeled cells time point and organ B Use of Cre-lox-mediated recombination for lineage tracing loxP loxP Cell-specific Cre coding sequence Stable Transcriptional Reporter transcriptional transcriptional stop signal coding driver driver sequence Reporter protein Cre Cre-loxP-mediated progenies 1 recombination F Cell-specific expression of Cre Cell-specific expression of reporter B′ Tracing of reporter-labeled progenies (cell-identifier) Developmental time Progenies of labeled cells can be identified at different developmental stages and tissues by reporter expression Figure 1. Lineage tracing and usage of Cre-lox-mediated site-specific recombination. (A) Basic outline of lineage tracing. “Cell-identifier” of various kinds are introduced in selected cells (red), that are inherited by the daughter cells. “Cell-identifier”-tagged cells (red) at later stages of life are identified