High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Cell transplantation has a long and storied history as a foundational technique in developmental biology. Around the turn of the 20^th century, approaches using physical manipulations to perturb the developmental process, including transplantation, transformed embryology from an observational science into an experimental one^1,2. In one landmark experiment, Hans Spemann and Hilde Mangold ectopically transplanted the dorsal blastopore lip of a salamander embryo onto the opposite side of a host embryo, inducing the nearby tissue to form a secondary body axis³. This experiment showed that cells could induce other cells to adopt certain fates, and subsequently, transplantation developed as a powerful method for interrogating critical questions in developmental biology regarding competence and cell fate determination, cell lineage, inductive ability, plasticity, and stem cell potency^1,4,5.

More recent scientific advances have expanded the power of the transplantation approach. In 1969, Nicole Le Douarin's discovery that nucleolar staining could distinguish species of origin in quail-chick chimeras allowed for the tracking of transplanted cells and their progeny⁶. This concept was later supercharged by the advent of transgenic fluorescent markers and advanced imaging techniques⁵, and has been leveraged to track cell fate^6,7, identify stem cells and their potency^8,9, and track cell movements during brain development¹⁰. Additionally, the rise of molecular genetics facilitated transplantation between hosts and donors of distinct genotypes, supporting precise dissection of autonomous and non-autonomous functions of developmental factors¹¹.

Transplantation has also made important contributions to the study of regeneration, particularly in organisms with strong regenerative abilities such as planarians and axolotl, by elucidating the cellular identities and interactions that regulate the growth and patterning of regenerating tissues. Transplant studies have revealed principles of potency¹², spatial patterning^13,14, contributions of specific tissues^15,16, and roles for cellular memory^12,17 in regeneration.

Zebrafish are a leading vertebrate model for the study of development and regeneration, including in the nervous system, due to their conserved genetic programs, high genetic tractability, external fertilization, large clutch size, and optical clarity^18,19,20. Zebrafish are also highly amenable to transplantation at early developmental stages. The most prominent approach is the transplantation of cells from a labeled donor embryo to a host embryo at the blastula or gastrula stage to generate mosaic animals. Cells transplanted during the blastula stage will scatter and disperse as epiboly begins, producing a mosaic of labeled cells and tissues across the embryo²¹. Gastrula transplants allow for some targeting of transplanted cells according to a rough fate map as the shield forms and the A-P and D-V axes can be determined²¹. The resulting mosaics have been valuable in determining whether genes act cell autonomously, testing cell commitment, and mapping tissue movement and cell migration throughout development^5,11. Mosaic zebrafish can be generated in several ways, including electroporation²², recombination²³, and F0 transgenesis²⁴ and mutagenesis²⁵, but transplantation provides the greatest manipulability and precision in space, time, and number and types of cells. The current state of zebrafish transplantation is largely constrained to progenitor cells at early stages, with a few exceptions including transplantation of spinal motor neurons^26,27, retinal ganglion cells^28,29, and neural crest cells in the first 10-30 h post fertilization (hpf)³⁰, and of hematopoietic and tumor cells in adult zebrafish^5,31. Expanding transplantation methods to a broad range of ages, differentiation stages, and cell types would greatly enhance the power of this approach to provide insights into developmental and regenerative processes.

Here, we demonstrate a flexible and robust technique for high resolution cell transplantation effective in zebrafish embryos and larvae up to at least 7 days post fertilization. Transgenic host and donor fish expressing fluorescent proteins in target tissues can be used to extract single cells and transplant them with near-perfect spatial and temporal resolution. The optical clarity of zebrafish embryos and larvae allows for the transplanted cells to be imaged live as the host animal develops or regenerates. This approach has previously been used to examine how spatiotemporal signaling dynamics influence neuronal identity and axon guidance in the embryo³², and to examine the logic by which intrinsic and extrinsic factors promote axon guidance during regeneration in larval fish³³. While we focus here on the transplantation of differentiated neurons, our method is widely applicable to both undifferentiated and differentiated cell types across many stages and tissues to address questions in development and regeneration.