We show that after tail amputation in Ambystoma mexicanum (Axolotl) the correct number and spacing of dorsal root ganglia are regenerated. By transplantation of spinal cord tissue and nonclonal neurospheres, we show that the central spinal cord represents a source of peripheral nervous system cells. Interestingly, melanophores migrate from preexisting precursors in the skin. Finally, we demonstrate that implantation of a clonally derived spinal cord neurosphere can result in reconstitution of all examined cell types in the regenerating central spinal cord, suggesting derivation of a cell with spinal cord stem cell properties.neural stem cell | segmentation | Hox R egeneration of the central body axis occurs after tail amputation in salamander amphibians. During this process the spinal cord regrows, and the correct number of segmented vertebrae and myotomes are formed (1). Additionally, neural crest derivatives, such as melanophores, and the peripheral nervous system (PNS), including dorsal root ganglia (DRG) and Schwann cells, are regenerated (2-4).An important challenge is to define and study the stem cells that are responsible for regenerating the CNS and PNS. Previously, we used electroporation of GFP expression plasmids into the spinal cord to identify and track the radial glial cells that contribute to regenerating the spinal cord in the salamander Ambystoma mexicanum (axolotl) (5, 6). Live cell tracking showed that single cells could give rise to clones populating multiple molecular domains of the regenerating spinal cord (6). These results indicated that multipotent progenitors exist during spinal cord regeneration. Because of the transient expression of the plasmids, the long-term fate of the stem cells could not be tracked, so the origin of the PNS was not addressed.The source of neural crest derivatives in the regenerated tail has been an open question since 1885 (7). During early development the neural crest arises in the dorsal region of the neural tube, from which cells emigrate to the surroundings to form neurons and glia of PNS, smooth muscles, head skeletal elements, enteric neurons, and melanophores (8). It is not yet known conclusively, however, whether during regeneration newly regenerated neural crest structures derive from a population of neural crest-like cells that migrate out of the regenerating spinal cord or arise directly from cells in the periphery. Immunohistochemical studies using markers such as HNK1 suggested that there may be a population of cells in the lateral walls of the spinal cord with neural crest properties (4). Furthermore, morphological studies suggested that cells may migrate via the forming ventral roots to populate the spinal ganglia outside the spinal cord. Such findings could be consistent with recent findings in mouse that boundary cap cells can act as a neural crest source (9). To track the origin of neural crest structures during newt tail regeneration, Benraiss et al. (3) attempted to label spinal cord cells via biolistic transfection of a human alkaline phosph...
All mature blood cell types in the adult animal arise from hematopoietic stem and progenitor cells (HSPCs). However, the developmental cues regulating HSPC ontogeny are incompletely understood. In particular, the details surrounding a requirement for Wnt/β-catenin signaling in the development of mature HSPCs are controversial and difficult to consolidate. Using zebrafish, we demonstrate that Wnt signaling is required to direct an amplification of HSPCs in the aorta. Wnt9a is specifically required for this process and cannot be replaced by Wnt9b or Wnt3a. This proliferative event occurs independently of initial HSPC fate specification, and the Wnt9a input is required prior to aorta formation. HSPC arterial amplification occurs prior to seeding of secondary hematopoietic tissues and proceeds, in part, through the cell cycle regulator myca (c-myc). Our results support a general paradigm, in which early signaling events, including Wnt, direct later HSPC developmental processes.
Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.
SummaryIt has previously been reported that mouse epiblast stem cell (EpiSC) lines comprise heterogeneous cell populations that are functionally equivalent to cells of either early- or late-stage postimplantation development. So far, the establishment of the embryonic stem cell (ESC) pluripotency gene regulatory network through the widely known chemical inhibition of MEK and GSK3beta has been impractical in late-stage EpiSCs. Here, we show that chemical inhibition of casein kinase 1alpha (CK1alpha) induces the conversion of recalcitrant late-stage EpiSCs into ESC pluripotency. CK1alpha inhibition directly results in the simultaneous activation of the WNT signaling pathway, together with inhibition of the TGFbeta/SMAD2 signaling pathway, mediating the rewiring of the gene regulatory network in favor of an ESC-like state. Our findings uncover a molecular mechanism that links CK1alpha to ESC pluripotency through the direct modulation of WNT and TGFbeta signaling.
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