Wnt Signaling Coordinates the Expression of Limb Patterning Genes During Axolotl Forelimb Development and Regeneration

Lovely, Alexander M.; Duerr, Timothy J; Qiu, Qingchao; Galvan, Santiago; Voss, S. Randal; Monaghan, James R.

doi:10.3389/fcell.2022.814250

Cited by 22 publications

(17 citation statements)

References 73 publications

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“…Axolotl AER cells had comparable signaling potential to the AER in other species (Figures 1G and S7) . Meanwhile, individual pathway examination revealed the axolotl AER differs in paralog and co-factor expressions, particularly in the FGF and WNT pathways (Figure S8) , complementing and extending previous observations 4, 7, 21 . We could not detect noticeable differences in transcriptional levels associated with DELTAs, BMPs, or TGFbs (Figure S8) .…”

Section: Introductionsupporting

confidence: 87%

“…The AEC is required to form a connective-tissue lineage-rich blastema for regeneration 11,12 , and its absence in mammals has been long-hypothesized to be one of the reasons for the regenerationincompetency 13,14 . However, previous reports provided conflicting results for the AER and AEC marker expressions 4,7,[15][16][17][18][19][20][21] , and a sub group of the axolotl connective tissue (CT) cells was suggested to express some of the AER-related genes 4,7,21,22 . Consequently, different conclusions have been drawn based either on morphological assessments or assaying the expression of a small set of specific marker genes.…”

Section: Introductionmentioning

confidence: 99%

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Multi-species atlas resolves an axolotl limb development and regeneration paradox

Zhong

Aires

Tsissios

et al. 2023

Preprint

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Humans and other tetrapods are considered to require apical-ectodermal-ridge, AER, cells for limb development, and AER-like cells are suggested to be re-formed to initiate limb regeneration. Paradoxically, the presence of AER in the axolotl, the primary regeneration model organism, remains controversial. Here, by leveraging a single-cell transcriptomics-based multi-species atlas, composed of axolotl, human, mouse, chicken, and frog cells, we first established that axolotls contain cells with AER characteristics. Surprisingly, further analyses and spatial transcriptomics revealed that axolotl limbs do not fully re-form AER cells during regeneration. Moreover, the axolotl mesoderm displays part of the AER machinery, revealing a novel program for limb (re)growth. These results clarify the debate about the axolotl AER and the extent to which the limb developmental program is recapitulated during regeneration.

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Section: Introductionsupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Multi-species atlas resolves an axolotl limb development and regeneration paradox

Zhong

Aires

Tsissios

et al. 2023

Preprint

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“…62 The same expression is seen in axolotl and mouse limb buds, and wnt5a null mice have distally truncated limbs. 63,64 The effect of Wnt5a on limb proximodistal patterning is mediated through the planar cell polarity pathway rather than canonical Wnt signaling and is dependent on AER Fgf signals. 65 Notum is a Wnt pathway antagonist, with expression highest in the distal limb bud.…”

Section: Wnt Agonists and Antagonists Are Both Proximally And Distall...mentioning

confidence: 99%

“…It is expressed in the mesoderm just under the AER, often referred to as the progress zone, in chicken limb buds (Dealy et al, 1993). The same expression is seen in axolotl and mouse limb buds, and wnt5a null mice have distally truncated limbs (Parr et al, 1993, Lovely et al, 2022. The effect of Wnt5a on limb proximodistal patterning is mediated through the planar cell polarity pathway rather than canonical Wnt signalling and is dependent on AER Fgf signals (Gao et al, 2018).…”

Section: Wnt Agonists and Antagonists Are Both Proximally And Distall...mentioning

confidence: 99%

Gene expression analysis of the Xenopus laevis early limb bud proximodistal axis

Hudson

Bromell

Day

et al. 2022

Developmental Dynamics

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Background Limb buds develop as bilateral outgrowths of the lateral plate mesoderm and are patterned along three axes. Current models of proximal to distal patterning of early amniote limb buds suggest that two signals, a distal organizing signal from the apical epithelial ridge (AER, Fgfs) and an opposing proximal (retinoic acid [RA]) act early on pattern this axis. Results Transcriptional analysis of stage 51 Xenopus laevis hindlimb buds sectioned along the proximal‐distal axis showed that the distal region is distinct from the rest of the limb. Expression of capn8.3, a novel calpain, was located in cells immediately flanking the AER. The Wnt antagonist Dkk1 was AER‐specific in Xenopus limbs. Two transcription factors, sall1 and zic5, were expressed in distal mesenchyme. Zic5 has no described association with limb development. We also describe expression of two proximal genes, gata5 and tnn, not previously associated with limb development. Differentially expressed genes were associated with Fgf, Wnt, and RA signaling as well as differential cell adhesion and proliferation. Conclusions We identify new candidate genes for early proximodistal limb patterning. Our analysis of RA‐regulated genes supports a role for transient RA gradients in early limb bud in proximal‐to‐distal patterning in this anamniote model organism.

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“…Alternatively, HCR probes may be synthesized by individual laboratories following principles described by Choi et al (2018). Some groups have generated informatics pipelines that facilitate HCR probe design (Lovely et al, 2022).…”

Section: Aliquoting Of Hcr Probes and Hairpinsmentioning

confidence: 99%

Hybridization Chain Reaction for mRNA Localization in Single Cells from Mouse and Human Cryosections

2022

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In situ hybridization has been a robust method for detection of mRNA expression in whole-mount samples or tissue sections for more than 50 years. Recent technical advances for in situ hybridization have incorporated oligobased probes that attain greater tissue penetration and signal amplification steps with restricted localization for visualization of specific mRNAs within single cells. One such method is third-generation in situ hybridization chain reaction (V3HCR). Here, we report an optimized protocol for V3HCR detection of gene expression using sectioned frozen tissues from mouse and human on microscope slides. Our methods and modifications for cryosectioning, tissue fixation, and processing over a three-day V3HCR protocol are detailed along with recommendations for aliquoting and storing V3HCR single-stranded DNA probes and hairpin amplifiers. In addition, we describe a method for blocking background signal from lipofuscin, a highly autofluorescent material that is widespread in human neurons and often complicates imaging efforts. After testing multiple strategies for reduction of lipofuscin, we determined that application of a lipofuscin quencher dye is compatible with V3HCR, in contrast to other methods like cupric sulfate quenching or Sudan Black B blocking that cause V3HCR signal loss. This adaptation enables application of V3HCR for in situ detection of gene expression in human neuronal populations that are otherwise problematic due to lipofuscin autofluorescence.

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Wnt Signaling Coordinates the Expression of Limb Patterning Genes During Axolotl Forelimb Development and Regeneration

Cited by 22 publications

References 73 publications

Multi-species atlas resolves an axolotl limb development and regeneration paradox

Multi-species atlas resolves an axolotl limb development and regeneration paradox

Gene expression analysis of the Xenopus laevis early limb bud proximodistal axis

Hybridization Chain Reaction for mRNA Localization in Single Cells from Mouse and Human Cryosections

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