The Role of Cell Tracing and Fate Mapping Experiments in Cardiac Outflow Tract Development, New Opportunities through Emerging Technologies

Abstract

Whilst knowledge regarding the pathophysiology of congenital heart disease (CHDs) has advanced greatly in recent years, the underlying developmental processes affecting the cardiac outflow tract (OFT) such as bicuspid aortic valve, tetralogy of Fallot and transposition of the great arteries remain poorly understood. Common among CHDs affecting the OFT, is a large variation in disease phenotypes. Even though the different cell lineages contributing to OFT development have been studied for many decades, it remains challenging to relate cell lineage dynamics to the morphologic variation observed in OFT pathologies. We postulate that the variation observed in cellular contribution in these congenital heart diseases might be related to underlying cell lineage dynamics of which little is known. We believe this gap in knowledge is mainly the result of technical limitations in experimental methods used for cell lineage analysis. The aim of this review is to provide an overview of historical fate mapping and cell tracing techniques used to study OFT development and introduce emerging technologies which provide new opportunities that will aid our understanding of the cellular dynamics underlying OFT pathology.

Keywords: bicuspid aortic valve; cardiac development; cell identity; congenital heart disease; developmental biology; lineage tracing; outflow tract.

Figure 1

Vital dye and viral labeling techniques. (A) Lipophilic dyes are used to visualise and track distinct cell compartments over time. (B) Alternatively, retroviral vectors can allow for expression of a reporter gene (such as LacZ) to enable cell tracing of infected cells. (C) Placing the reporter gene under the transcriptional control of a cell specific promoter allows for increased accuracy in targeting cells through gene specific viral labelling.

Figure 2

The quail-chick chimeric system. A schematic representation of heterospecific transplantation of the neural crest tissue common to chimeric lineage tracing. The dorsal part of the neural tube of the quail, containing the neural crest cells, is excised and interchanged into a chick host of a comparable embryonic stage at the same locus. 1: somite 1, 2: somite 2, OV: otic vesicle. Image obtained with permission from TP Kelder & R Vincente-Steijn et al. [48].

Figure 3

Genetic lineage tracing. (A) Genetic lineage tracing can be achieved using the Cre-LoxP system. Upon expression, the Cre recombinase specifically targets LoxP DNA motives and allows for excision of DNA elements floxed by LoxP sites. The reporter line contains a stop-cassette inhibiting expression of the reporter gene in the absence of Cre. (B) Crossing the reporter line with the Cre line results in offspring carrying both constructs. (C) Cells expressing the gene of interest will express the Cre-recombinase. Cre will then permanently remove the stop-cassette and thus allow expression of the reporter gene, resulting in cell lineage labelling. (D) Modified Cre-recombinases can be used to allow for more temporal control in genetic labelling. Cre-ER variants can prevent Cre-recombinase activity by restricting mobility to the cells cytoplasm in absence of Tamoxifen. Upon stimulation of Tamoxifen-Cre activity is restored resulting in an inducible system for genetic lineage tracing.

Figure 4

Transversal sections of immunofluorescent outflow tracts show challenges with genetic lineage tracing. (A) A Wnt1^Cre; R26R^mTmG embryo showing neural crest cell derived cells (green). Interestingly, Wnt1 derived cells with NKX2.5 stained nuclei can also be observed at the so-called flow divider [84], in front of the primitive foregut which is the central part of the second heart filed contribution (white arrowhead) as well as in the atrial myocardium (white arrow). (B) a Tie2^Cre; R26R^mTmG embryo showing endothelial derived cells (green). Endothelial cells undergoing epithelial to mesenchymal transition give rise to cells within the septal and parietal cushions. Cells derived from myeloid lineages also express Tie2 resulting in lineage positive blood cells (yellow arrowhead). (C) A Mef2c^Cre; R26R^mTmG embryo showing second heart field derived cells (green). Cre positive endothelial cells can also be found lining the heart (red arrowhead). Mef2c derived cells also contribute directly to the septal cushion (red arrow). Ra: Right atrium, Ao: Aorta, PT: Pulmonary trunk, Pc: Parietal cushion, Sc: Septal cushion. Scalebar: 50 µm.

Figure 5

Retrospective lineage tracing. (A,B) Traditional retrospective lineage tracing exploits spontaneous and rare recombination events. The nlaacZ sequence was designed to be inactive by default but increase the odds of recombination events within the region. Upon spontaneous recombination, β-galactosidase is expressed resulting in a lineage label. (C,D) Early recombination (i.e., during early stages of development) will give large clusters of cells with reporter gene expression, whilst late spontaneous recombination results in smaller clusters of LacZ positive cells. (E) Modern retrospective lineage tracing derives lineage hierarchy from single cell transcriptomics data. Principle component analysis (PC) aids in clustering groups of cells based on similarity in gene expression profiles. Follow up algorithms can then produce a phylogenetic lineage tree derived from expressional gradients among cell clusters to reconstruct cell lineage development.