Specialized signaling centers direct cell fate and spatial organization in a limb organoid model

Background: 

The process of early mammalian development is incredibly complex, and can be thought of as building a 1000(+) piece jigsaw puzzle. Each piece must be placed in the right spot, at the right time, and in co-ordination with others already placed on the board for the final picture to emerge correctly. If one piece is misplaced, even just slightly out of alignment, it can disrupt the entire process. To complicate things further, each puzzle piece is, in its own right, its own jigsaw puzzle that must be correctly put together to fit into the larger picture.

The limb bud can be thought of as one of these pieces, which can be further built upon to eventually form the limbs of the organism (so arguably a very important piece!). Limb buds first appear as a bump in the lateral plate mesoderm (a precursor for the skeleton of the arms/legs) covered by surface ectoderm (a precursor for the epidermis). During development, the ectoderm thickens to become a structure known as the apical-ectodermal ridge (AER), a specialised signaling centre which is critical for bud extension, the next step in limb generation (this process is shown in Figure 1). Although very elaborate cross-talk between all these cell types must take place, it can be extremely difficult to investigate this in vivo.

 

Figure 1: Development of the limb bud (adapted from the preprint)

 

In vitro models of this process exist; however, these only include limb bud mesoderm. Models that investigate the interplay between limb bud mesoderm and the ectoderm are lacking. This is important as limb generation requires co-ordination between ectoderm (cells that will become the epidermis) and mesoderm (cells that will become connective tissue i.e. cartilage, bone, and tendons). Thus, the goal of this preprint is to develop an organoid model of limb bud generation that captures these diverse cell lineages. This will allow for the investigation of limb generation and morphogenesis at a single-cell level.

 

Main Findings:

Culturing a heterogenous population of limb bud cell identities

Using a published protocol, researchers induced surface ectoderm-like cells (AER precursors) from mouse embryonic stem cells. Surprisingly, following the addition of AER-inducing factors, flow cytometric analysis showed that there was a population of cells that expressed mesodermal-related genes, as well as a population that adopted a fibroblast-like morphology, suggesting the formation of both epithelial and mesenchymal populations. Further single-cell sequencing analyses indicated that there were populations within the organoids that resembled the AER, surface ectoderm, lateral plate mesoderm, and limb bud mesoderm identities.

 

Cells self-organise in adherent and free-floating cultures 

The researchers then discovered that the cells within organoids cultured on an adherent surface were able to self-organise into dome-like structures. These structures consisted of AER-like cells at the surface of the dome and an interior core of mesodermal-like cells, similar to what is observed in vivo.

The team then further investigated this self-organisation by dissociating established cultures and re-seeding them as free-floating cultures. They found that organoids developed into spherical aggregates and, with ectodermal signals, were able to break symmetry and elongate to form an anterior-posterior axis in what the researchers coined ‘budoids’.

 

Symmetry breaking leads to distinct polarised domains within the organoids

Single-cell analysis of budoids showed the emergence of a range of different cell fates, including the emergence of a chondrocyte (cartilage cell) population, which was also visualized as a polarized SOX9+ domain. The researchers showed that this chrondogenesis – cell differentiation from limb mesoderm to chondrocyte – mediated the symmetry breaking.

Next, the research team used flow cytometry to separate cell populations within the budoids. Recombinant budoids were generated by combining equal numbers of mesodermal and AER-like cells. They showed substantial symmetry breaking and elongation compared to recombinant budoids composed of mesodermal and surface ectoderm cells.

It was also these structures that allowed the researchers to dissect cell-cell interactions that are difficult to investigate in vivo. They could for example show that the AER-like recombinant budoids include distinct, polarised regions of SOX9+ cells (indicating the occurrence of chrondogenesis) and AER-like cells (as shown in Figure 2). This suggests that AER-like cells suppress chrondogenesis in cells within a close vicinity, and thus have the ability to influence tissue organisation.

 

Figure 2: Budoids self-organise into distinct polarised regions | Figure shows confocal images of recombinant budoids. SOX9+ chondrocyte/mesodermal domain is shown in magenta, ectodermal (apical ectodermal ridge-like) cells are shown in green, and circled in red dashed line.

 

Why I am highlighting this preprint

In my PhD project, I am interested in how organoids can self-organise to develop a complex 3D shape, and so this pre-print really caught my eye. I thought seeing how the organoids were able to develop into complex structures was fascinating. I thought it was also cool to see how recombinant organoids made up of different cell types had such an impact on the budoids’ ability to break symmetry, and then elongate into polarised structures with distinct domains.

This preprint has established a model that can be used in the future to gain new insights on limb bud generation, and I think the team highlight the potential of this model for quantitative analysis at a single cell level. I don’t think organoids have always been used to their full potential to quantitively analyse the processes they are recapitulating, so it was interesting to see how this was accomplished by the team here.

 

Questions for the authors:

1. These organoids were grown from mouse ESCs. How applicable is mouse limb bud generation to human limb bud generation? Do you think this protocol could be used with human ESCs and lead to similar results?
2. Which of the organoid models is more representative of in vivo limb bud generation and development: the adherent or free-floating model?
3. You stated that the focus of current efforts to regenerate mammalian limbs is to transplant only limb bud mesoderm. Do you think budoids could be applied to limb regeneration research with a higher level of success due to their heterogenous nature? How do you imagine this would happen?

Tags: 3d shape emergence, limb bud, limb bud organoid, mouse embryonic stem cells, organoid, self-organisation, stem cells

doi: https://doi.org/10.1242/prelights.38317

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