The role of miR-133a and RARβ2 in salamander axonal regeneration after spinal cord injury and theurupeutic implications for mammalian recovery

For over a century, Mexican salamanders (also called the axolotl or Ambystoma mexicanum) have been studied as a model organism for limb and tail regeneration. Compared to the limited regenerative ability of mammals, salamanders are able to completely regrow their spinal cord after injury. By better understanding this process of successful regeneration in axolotls, new therapeutic procedures can be developed to mimic axolotl regeneration in the mammalian spinal cord, thus enhancing functional recovery in mammals. While techniques have allowed investigation into the complex network of signaling factors and pathways that are involved in this process, this review will focus on the retinoid pathway and retinoic acid receptor β2

Protocol for tail vein injection in Xenopus tropicalis tadpoles

Summary: Functional studies in post-embryonic Xenopus tadpoles are challenging because embryonic perturbations often lead to developmental consequences, such as lethality. Here, we describe a high-throughput protocol for tail vein injection to introduce fluorescent tracers into tadpoles, which we have previously used to effectively inject morpholinos and molecular antagonists. We describe steps for safely positioning tadpoles onto agarose double-coated plates, draining media, injecting into the ventral tail vein, rehydrating plates, and sorting tadpoles by fluorescence with minimal injury for high-throughput experiments.For complete details on the use and execution of this protocol, please refer to Kakebeen et al.,1 Patel et al.,2 and Patel et al.,3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

wnt16 regulates spine and muscle morphogenesis through parallel signals from notochord and dermomyotome.

Bone and muscle are coupled through developmental, mechanical, paracrine, and autocrine signals. Genetic variants at the CPED1-WNT16 locus are dually associated with bone- and muscle-related traits. While Wnt16 is necessary for bone mass and strength, this fails to explain pleiotropy at this locus. Here, we show wnt16 is required for spine and muscle morphogenesis in zebrafish. In embryos, wnt16 is expressed in dermomyotome and developing notochord, and contributes to larval myotome morphology and notochord elongation. Later, wnt16 is expressed at the ventral midline of the notochord sheath, and contributes to spine mineralization and osteoblast recruitment. Morphological changes in wnt16 mutant larvae are mirrored in adults, indicating that wnt16 impacts bone and muscle morphology throughout the lifespan. Finally, we show that wnt16 is a gene of major effect on lean mass at the CPED1-WNT16 locus. Our findings indicate that Wnt16 is secreted in structures adjacent to developing bone (notochord) and muscle (dermomyotome) where it affects the morphogenesis of each tissue, thereby rendering wnt16 expression into dual effects on bone and muscle morphology. This work expands our understanding of wnt16 in musculoskeletal development and supports the potential for variants to act through WNT16 to influence bone and muscle via parallel morphogenetic processes

<i>wnt16</i> is expressed in dermomyotome-like cells at 22 hpf.

(A) A schematic of a transverse section through the zebrafish trunk with compartments labeled and color coded. (B-C) Chromogenic in situ hybridization of an anterior (note the yolk in the ventral space) transverse section through the zebrafish trunk shows cells expressing wnt16 and pax7a are located in the external cell layer (magnified in C). (D) Transverse sections, beginning in the anterior trunk (left), and moving posteriorly (right), representing less mature somites, show variation in wnt16+ labeling.</p

<i>WNT16</i> is a gene of major effect on lean mass at the <i>CPED1-WNT16</i> locus.

(A) Schematic depicting variant associations and all genes with transcriptional start sites within ±500kb of the most significantly associated SNP at 7q31.31. (B) Z-scores for somatic mutants for tspan12, ing3, cped1, wnt16, and fam3c. (C) Segmentation of microCT images for cped1w1003 mutants for bone (top) and lean (bottom) tissue. (D) Z-scores for cped1w1003 and wnt16w1001 mutants. P-values were determined using an unpaired t-test with the number of fish per group provided in the figure. *p<0.05, **p<0.01, ***p<0.001.</p

Isolation of <i>wnt16</i> mutant alleles.

(A) Sequence and genomic location of w1001, w1008, and w1009. Grey highlight indicates gRNA target sequence used for CRISPR-based gene editing with PAM underlined. (B) Predicted effects of alleles on amino acid sequence. (C) RT-PCR assessing wnt16 transcript in wnt16w1001 mutants. No evidence of transcript reduction or alternative splicing is observed. (D) Calcein staining of 13 dpf animals show similar reductions in vertebral mineralization and post-cranial body length in w1001, w1008, and w1009 mutants. (E) Quantification of mineralized area shows similar changes in mutants for all three alleles. (TIF)</p

Zygotic and maternal zygotic <i>wnt16</i>^<i>-/-</i> mutant phenotypes.

P-values were determined using a two-way ANOVA with Fisher’s LSD post hoc test. *p (TIF)</p

Inference of myomere morphology from vertebral measures.

(A) Schematic demonstrating correlated features. (B-D) wnt16-/- mutants exhibit altered centrum length and neural arch angle. P-values were determined using an unpaired t-test. **p (TIF)</p

Data used for calculating Z-scores for Fig 9D.

(TIF)</p

RNA ISH for markers of early muscle (<i>myog</i>, <i>pax7a</i>) and notochord (<i>ntla/tbxta</i>) differentiation in <i>wnt16</i>^-/- mutant embryos at 1 dpf.

Scale bar: 200 μm. (TIF)</p