Session 2 – Moderated by Elif Eroğlu

Ji-Feng Fei (Online)

10.15 – 10.45

Applying stereo-seq technology to dissect the mechanisms of brain regeneration

Promoting brain regeneration is challenging due to low inherent regenerative capacity of the mammalian brain and limited understanding of cellular and molecular mechanisms guiding this process. Here, by analyzing the highly regenerative axolotl brain with Stereo-seq, we reconstructed its telencephalon architecture with spatially resolved gene expression profiles at single-cell resolution, and fine cell dynamic maps throughout development and regeneration. We demarcated major mammalian types of excitatory, inhibitory neurons, and ependymoglial cells (EGCs) in axolotl brain. Intriguingly, we identified a regeneration-unique EGC cluster, named as reactive EGC (reaEGC) that is rapidly induced upon injury in the wound area. It appears to be the major source participating in regeneration to give rise to lost neurons, via intermediate progenitors, which largely resembles the development process. Furthermore, we uncover an injury-induced nerve response in regeneration. Upon injury, spatial-defined neurons at lesion respond to wounding and switch to an immature-neuron-like status determined by their transcriptome signature. Altogether, our work resembles the first spatial-temporal single cell brain atlas of anamniote tetrapod, decodes the complex cellular and molecular dynamics of axolotl telencephalon in development and regeneration. It lays the foundation for mechanistic studies of brain regeneration and evolution in future.

Takashi Takeuchi (Online)


Multiple disruption of newt Hox13 paralogs revealed that Hoxa13 has an essential and predominant role in digit formation during limb development and regeneration

The 5’Hox genes play crucial roles in limb development and specify regions in the proximal-distal axis of limbs. However, there is no direct genetic evidence that Hox genes are essential for limb development in non-mammalian tetrapods or for limb regeneration. Here, we produced single to quadruple Hox13 paralog mutants using the CRISPR/Cas9 system along with germline mutants in newts (Pleurodeles waltl), which have strong regenerative capacities. We showed that Hox13 is essential for digit formation in development, as it is in mice. In addition, Hoxa13 has a predominant role in digit formation, unlike in mice. The predominance is probably due to the restricted expression pattern of Hoxd13 in limb buds and the strong dependence of Hoxd13 expression on Hoxa13. Finally, we indicated that Hox13 is also necessary for digit formation in limb regeneration. Our findings reveal that the general function of Hox13 is conserved between limb development and regeneration, and across taxa. The predominance of Hoxa13 function both in newt limbs and fish fins, but not in mouse limbs, suggests a potential contribution of Hoxa13 function in fin-to-limb transition.

Anastasia Polikarpova (Online)


Cellular and molecular profile of axolotl large bone fractures

Understanding of the cellular and molecular mechanisms of fracture callus formation and bone healing is needed to treat bone fracture non-unions. Axolotls cannot heal bone critical-size defects (CSD, >30% of the bone length), but are able to regenerate the amputated limb by producing blastema, a transient progenitor cell mass. A substantial part of blastema is formed by the soft connective tissue (SCT) cells which build the distal part of the skeleton in the regenerate. Restricted ability of SCT cells to contribute to bone callus and lack of blastema factors may cause bone non-union in CSD. Aim: We aim to understand if SCT cells migrate to CSD and identify the factors, inducing SCT to contribute to bone. Material and methods: We use polyolefin tube-stabilized femur fracture model in combination with CT cell tracing, bulk and single-cell RNA sequencing, EdU labelling, immunofluorescence and histological stainings. Results: Whereas in small fracture (SF) fracture bridging by cartilaginous callus took place at 3-6 weeks, and bone formed at 12 weeks post-surgery, CSD gap persisted at 12 weeks post-surgery. We found Prrx1+ progenitor cells accumulating in the CSD gap, but no callus formed and proliferation was diminished in CSD in comparison to blastema. To trace SCT cells, skin, bones or muscles from axolotls with fluorescently labelled SCT cells were transplanted to wildtype animals. The labelled SCT cells were migrating to the fracture site. RNAseq analysis shows changes in proliferation- and blastema-specific gene expression. scRNAseq analysis showed similar connective tissue cell phenotype at the early stages of CSD and BL healing (3-5 dpi). However, at 8-11 dpi, CSD and BL CT cells differ in their gene expression, in particular, CSD cells are less de-differentiated and proliferative. This could be also influenced by differences in wound epidermis and macrophage profiles. Conclusions and outlook: SCT cells are able to migrate to the CSD, indicating potency to heal the fracture, however the signals inducing blastema-like condition or cartilage formation are absent in fracture, resulting in CSD non-union. We suggest to provide the proliferation- and de-differentiation-promoting factors to CSD to induce blastema-like phenotype to promote bone bridging.