Signaling centers are specialized groups of cells that release morphogens, guiding the fate and behavior of adjacent cells during morphogenesis. For instance, the apical-ectodermal-ridge (AER) in developing limbs, is crucial for coordinating cell fate and tissue morphogenesis by abundant ligand secretion. Similarly, the apical-epithelial-cap (AEC) that forms at the amputation plane in regenerating limbs is believed to perform functions analogous to those of the AER. Despite their importance, the cellular and molecular understanding of these signaling centers is limited, necessitating more quantitative research.

The AER is thought to reappear as the AEC to initiate limb regeneration. However, morphological analyses and limited gene expression studies suggest that the AER is absent in axolotls, a key model organism for regenerative research. This raises the question of which tissue drives axolotl limb regeneration if not the AER. In Chapter 2, I aimed to re-evaluate the existence of axolotl AER using high-throughput profiling. I collected publicly available single-cell RNA sequencing (scRNA-seq) datasets and established a multi-species limb atlas, incorporating cells from humans, mice, chickens, frogs, and axolotls. Using this atlas, along with quantitative staining experiments conducted by my lab members, I provided evidence that axolotls possess AER cells. Additionally, combining scRNA-seq and spatial transcriptomics, I demonstrated that axolotl AEC exhibits a distinct gene expression profile, with notably lower expression of key developmental ligands compared to the AER. These findings showcase the power of single-cell profiling and other high-throughput technologies in advancing our understanding of developmental and regenerative biology.

Next, I aimed to understand how AER, as a signaling center, influences the underlying mesoderm in Chapter 3. This work builds on a stem cell-based protocol developed by my colleagues to generate limb bud-like cells. Using scRNA-seq analysis, I found that the protocol allows simultaneous generation of AER- and limb mesoderm-like cells from mouse embryonic stem cells. These cells self-organized into dome-like structures in 2D cultures, resembling limb buds, and formed limb organoids in 3D, called budoids. Again with scRNA-seq, I characterized the cellular heterogeneity in 3D cultures and revealed that budoids show molecular and cellular aspects of limb development. To explore the molecular influence of AER, my colleagues carried out hybridization-based in situ sequencing (HybISS) experiments (Gyllborg et al., 2020), an imaging-based spatial transcriptomics technique. I implemented a pipeline to analyze the resulting data, revealing that AER cells can promote limb mesoderm and fibroblast identities in their vicinity while promoting the polarization of budoid structures.

Collectively, I applied single-cell and spatial assays to investigate the role of epidermal signaling centers in limb development and regeneration in both in vivo and in vitro systems. These findings clarified debated topics and long-standing assumptions, which significantly impact the understanding of signaling centers in limb morphogenesis.