May the best cluster win: BMP signaling directs organizer formation in mouse neural tube organoids

Summary by Micaela Murphy: Krammer, T., Stuart, H.T., Gromberg, E., Ishihara, K., Cislo, D., Melchionda, M., Becerril Perez, F., Wang, J., Costantini, E., Lehr, S., Arbanas, L., Hörmann, A., Neumüller, R. A., Elvassore, N., Siggia, E., Briscoe, J., Kicheva, A., Tanaka, E. M. (2024). Mouse neural tube organoids self-organize floorplate through BMP-mediated cluster competition. Dev Cell 59, 1940-1953 e1910. 10.1016/j.devcel.2024.04.021

Image credit: Micaela Murphy

Tissue and organ development occur through proper specification of cell types and functions [1]. Accordingly, cell differentiation must be highly coordinated and controlled during embryogenesis. Organizers serve as control centers, directing cell fate decisions in surrounding tissues through secreted molecules [2,3]. In vivo, maternal factors often orchestrate early polarization events that precede organizer development [2]; however, organoid models, where one mouse embryonic stem cell (mESC) differentiates in 3D culture [4], have shown that organizers can form without such cues via self-organization5.

The ventral floorplate is a neural tube organizer that induces dorsal/ventral patterning of neural precursor cells [4]. Notably, the floorplate’s anterior region forms without early notochord signaling by an unknown mechanism [5]. To understand the spatiotemporal factors involved in floorplate organization, Krammer et al. modeled the process using neural tube organoids (NTOs) derived from mESCs [6]. Following a retinoic acid pulse, NTOs develop floorplates composed of FOXA2+ cells… but how do these cells organize prior to inducing neural progenitor specification?

Using a fluorescent reporter at the endogenous FoxA2 locus, the authors live imaged NTOs to observe FOXA2+ cell clustering dynamics. FOXA2+ cells are initially dispersed in a ‘salt-and-pepper’ pattern before coalescing at the ventral pole. The researchers observed two mechanisms for cluster selection: extinction occurs when one of multiple FOXA2+ cell clusters remains (as others disappear), while sorting converges two FOXA2+ cell clusters into one. More NTOs exhibited cluster extinction than sorting, convincing them to investigate how one cluster predominates over another.

Prior to cluster extinction, the authors measured both the average FOXA2 intensity and size of FOXA2+ clusters. Larger clusters with higher FOXA2 intensity were more likely to persist, so Krammer et al. performed an RNAseq experiment to analyze differences in signaling pathway gene expression between FOXA2+ and FOXA2- cells. Following cluster selection, various BMP ligand and inhibitor transcripts were upregulated in FOXA2+ but not FOXA2- cells, while FGF and WNT-specific genes did not exhibit unidirectional changes in expression between the two cell populations. An immunofluorescence staining time course experiment also revealed that only BMP effectors pSMAD1/5/9 levels increased during cell clustering. Furthermore, pSMAD1/5/9 staining colocalized with FOXA2- cells, suggesting an inverse relationship between FOXA2 and pSMAD1/5/9 levels.

To discern how BMP signaling orchestrates cluster selection, the authors stained for FOXA2 in NTOs treated with BMP4 or the BMP inhibitor NOGGIN. Notably, increased BMP4 signaling reduced FOXA2 expression, while reducing BMP signaling via NOGGIN increased the number of FOXA2+ clusters. In a Noggin knockout, NTOs lost most FOXA2+ clusters, suggesting that NOGGIN serves as a protective factor during cluster elimination. Moreover, FOXA2+ cells had upregulated Noggin transcripts following BMP4 exposure.

The authors propose a BMP-mediated feedback mechanism that drives cluster competition: FOXA2+ cells express BMP ligands and NOGGIN to suppress FOXA2 in other cells and protect themselves against BMP-mediated suppression, respectively. In vivo, Noggin mutant mice had smaller anterior floorplates with reduced FOXA2 expression, while notochord-dependent floorplate regions were unaffected. Future experiments could investigate the downstream effects of a minimized anterior floorplate to determine if certain neural progenitor identities are stereotypically lost without NOGGIN expression.

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