Molecular basis of positional memory in limb regeneration [RNA-seq]
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ABSTRACT:
PROVIDER: PRJNA1016419 | ENA |
REPOSITORIES: ENA
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Project description:Amputation of a salamander limb triggers anterior and posterior connective tissue cells to form distinct signalling centres that together fuel regeneration. Anterior and posterior identities are established during development and are thought to persist lifelong in the form of positional memory. However, neither the molecular basis of positional memory nor whether positional memory can be altered is known. Here, we identify a positive feedback loop responsible for posterior identity in the axolotl (Ambystoma mexicanum) limb. Posterior cells express residual Hand2 transcription factor from development, which primes them to form a Shh signalling centre after limb amputation. During regeneration, Shh signalling is also upstream of Hand2 expression. After regeneration, Shh is shut down but Hand2 is sustained, safeguarding posterior memory. We exploited this regeneration circuitry to convert anterior cells to a posterior cell memory state. Transient exposure of anterior cells to Shh during regeneration kick-started an ectopic Hand2-Shh loop, leading to stable Hand2 expression and a lasting competence to express Shh. Our results implicate positive feedback in the stability of positional memory and reveal that positional memory is more easily reprogrammed in one direction (anterior to posterior) than the other. Modifying positional memory in regenerative cells changes their signalling outputs, which has implications for tissue engineering.
2025-03-21 | GSE243137 | GEO
Project description:Amputation of a salamander limb triggers anterior and posterior connective tissue cells to form distinct signalling centres that together fuel regeneration. Anterior and posterior identities are established during development and are thought to persist lifelong in the form of positional memory. However, neither the molecular basis of positional memory nor whether positional memory can be altered is known. Here, we identify a positive feedback loop responsible for posterior identity in the axolotl (Ambystoma mexicanum) limb. Posterior cells express residual Hand2 transcription factor from development, which primes them to form a Shh signalling centre after limb amputation. During regeneration, Shh signalling is also upstream of Hand2 expression. After regeneration, Shh is shut down but Hand2 is sustained, safeguarding posterior memory. We exploited this regeneration circuitry to convert anterior cells to a posterior cell memory state. Transient exposure of anterior cells to Shh during regeneration kick-started an ectopic Hand2-Shh loop, leading to stable Hand2 expression and a lasting competence to express Shh. Our results implicate positive feedback in the stability of positional memory and reveal that positional memory is more easily reprogrammed in one direction (anterior to posterior) than the other. Modifying positional memory in regenerative cells changes their signalling outputs, which has implications for tissue engineering.
2025-03-21 | GSE284768 | GEO
Project description:Molecular basis of positional memory in limb regeneration [RNA-seq]
| PRJNA1200631 | ENA
Project description:Regenerating limbs retain their proximodistal (PD) positional identity following amputation. This positional identity is encoded genetically by PD patterning genes, which instruct blastema cells to regenerate the appropriate PD limb segment. Retinoic acid (RA) is known to specify proximal limb identity, but how RA concentration is established in the blastema is unknown. Here, we show that RA breakdown via CYP26B1 is essential for determining the RA concentration within blastemas. CYP26B1 inhibition molecularly reprograms distal blastemas into a proximal identity, phenocopying the effects of administering excess RA. We identify Shox as an RA responsive gene that is differentially expressed between proximally and distally amputated blastemas. Ablation of Shox results in shortened limbs with proximal skeletal elements that fail to undergo endochondral ossification. These results suggest that PD positional identity is determined by RA degradation and that targets of RA have a critical role in skeletal element formation during limb regeneration.
2024-07-22 | GSE272731 | GEO
Project description: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. Profiling chromatin accessibility (ACRs) of Axoltol limb connective tissue cells, and compare among developmental limb buds, and mature limb at different segments and regeneration time points.
2023-04-10 | GSE217592 | GEO
Project description: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. Profiling histone marks of Axoltol limb connective tissue cells, and comparing among limb different limb segments and mature vs regeneration time points.
2023-04-10 | GSE217591 | GEO
Project description:Retinoic acid breakdown is required for proximodistal positional identity during axolotl limb regeneration
| PRJNA1138466 | ENA
Project description: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. We performed Differential Gene expression analysis using data obtained from RNA-seq of two mature limb segments (mature arm vs hand). Therefore, we performed libraries from the mature arm and 5, 9, 13 dpa regenerating arm blastema.
2023-04-10 | GSE217593 | GEO
Project description:We know little about the control of positional information (PI) during axolotl limb regeneration which ensures that the limb regenerates exactly what was amputated and the work reported here investigates this phenomenon. Retinoic acid administration changes the PI in a proximal direction so that a complete limb can be regenerated from a hand. Rather that identifying all the genes altered by RA treatment of the limb, we have eliminated many off-target effects by using retinoic acid receptor selective agonists. We firstly identify the receptor involved in this respecification process as RARα and secondly identify the genes involved by RNA sequencing of the RARα-treated blastemal mesenchyme. We find 1177 up-regulated genes and 1403 down-regulated genes which could be identified using the axolotl genome. These include several genes known to be involved in retinoic acid metabolism and in patterning. Since positional in-formation is thought to be a property of the cell surface of blastemal cells when we examine our dataset with an emphasis on this aspect, we find the top canonical pathway is integrin signaling. In the extracellular matrix compartment, we find an MMP and several collagens are up-regulated and several cell membrane genes and secretory factors are also up-regulated. This provides data for future testing of the function of these candidates in the control of PI during limb regeneration.
2021-08-20 | GSE182296 | GEO
Project description:Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed ‘positional identity’. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/RARRES1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting HoxA13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.
2022-01-30 | GSE184948 | GEO