Search bioRxiv⌕ Search

Biology subjects

Masselink, W.

Publications and source records attributed to Masselink, W..

4 recordsLinked to original sources

Axolotl tail regeneration emerges during a defined embryonic window

How regenerative capacity originates during development remains poorly understood, even in vertebrates with exceptional adult regenerative ability. Using the axolotl, we identify a defined embryonic window between stages 30 and 34 during which the tail region transitions from a regeneration-incompetent to a regeneration-competent state. Amputations across staged embryos reveal that earlier embryos entirely fail to regenerate, whereas later embryos regenerate functional tails. Notably, tail stumps from nonregenerating embryos can recover the ability to regenerate when reamputated at later stages, demonstrating that early regenerative failure does not permanently impair regenerative capacity. This differs from the transient refractory period described in Xenopus, where regenerative competence is lost and reacquired around the end of tail outgrowth, and indicates that staged acquisition of regenerative competence is a broadly shared but mechanistically distinct feature of amphibian development. To determine whether this transition reflects changes in progenitor composition, we analysed the single-cell transcriptional landscapes of axolotl tail buds across this window. Tail bud progenitors, including neuromesodermal progenitors, persist through the transition, indicating that the onset of regenerative competence is unlikely to be explained by the loss of embryonic progenitors. Finally, using Tbxt (Brachyury) crispant axolotls with severe axial defects, we show that tail regeneration occurs effectively despite earlier abnormal embryonic tail development, with functional uncoupling of the mechanisms of tail development and regeneration. This framework provides new opportunities for identifying the drivers of regenerative competence and understand why this capacity is lost in other vertebrate species.

developmental biology↗

Somite-independent regeneration of the axolotl primary body axis

Primary body-axis development is a highly conserved process that proceeds through somitogenesis and subsequent subdivision into dermatome, myotome, and sclerotome. Defects in somitic-clock genes such as Hes7 lead to vertebral-segmentation defects in mice and fish. Here we show that in the axolotl, although Hes7 is necessary for proper embryonic vertebral segmentation, it is-- surprisingly--dispensable during tail regeneration. We investigated the mechanism of vertebral segmentation during regeneration which initially occurs through extension of a cartilage rod ventral to the spinal cord. We find that the regenerating cartilage rod undergoes a periodic wrinkling that provides a template for vertebral segmentation. Via direct mechanical measurements and biophysical perturbations, we show that a model of compression-induced buckling instability can predict vertebral segmentation. The cartilage rod and other somitic derivatives (muscle, cartilage, tendon, fibroblasts) arise from tendon-like, Lfng+ multi-potent mesenchymal progenitors, which display a gene regulatory state distinct from somitic progenitors. In summary, we uncover a mechanism of vertebral segmentation during axolotl tail regeneration that is distinct from the somite-based developmental mechanism.

developmental biology↗

Chromatin states at homeoprotein loci distinguish axolotl limb segments prior to regeneration

The salamander limb regenerates only the missing portion. Each limb segment can only form segments equivalent to- or more distal to their own identity, relying on a property termed "positional information". How positional information is encoded in limb cells has been unknown. By cell-type-specific chromatin profiling of upper arm, lower arm, and hand, we found segment-specific levels of histone H3K27me3 at limb homeoprotein gene loci but not their upstream regulators, constituting an intrinsic segment information code. During regeneration, regeneration-specific regulatory elements became active prior to the re-appearance of developmental regulatory elements. This means that, in the hand segment, the permissive chromatin state of the hand homeoprotein gene HoxA13 engages with regeneration regulatory elements, bypassing the upper limb program.

developmental biology↗

Broad applicability of a streamlined Ethyl Cinnamate-based clearing procedure

Turbidity and opaqueness are inherent properties of tissues which limit the capacity to acquire microscopic images through large tissues. Creating a uniform refractive index, known as tissue clearing, overcomes most of these issues. These methods have enabled researchers to image large and complex 3D structures with unprecedented depth and resolution. However, tissue clearing has been adopted to a limited extent due to a combination of cost, time, complexity of existing methods and potential negative impact on fluorescence signal. Here we describe 2Eci (2nd generation Ethyl cinnamate based clearing method) which can be used to clear a wide range of tissues, including cerebral organoids, Drosophila melanogaster, zebrafish, axolotl, and Xenopus laevis in as little as 1-5 days while preserving a broad range of fluorescence proteins including GFP, mCherry, Brainbow, and alexa-fluorophores. Ethyl cinnamate is non-toxic and can easily be used in multi-user microscope facilities. This method will open up clearing to a much broader group of researchers, due to its broad applicability, ease of use, and non-toxic nature of Ethyl cinnamate.\n\nSummary statementThe non-toxic, broadly applicable, and simplified protocol of 2Eci tissue clearing makes it possible for non-specialist labs to use clearing approaches on conventional inverted microscopes.

developmental biology↗