During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine.

In eukaryotic cells, firing of DNA replication origins normally does not recur until after M phase. This characteristic is thought to be due to the properties of "initiation" proteins like Orc, Cdc6, and Mcms. Using formaldehyde cross-linking, we show that Cdc6p and Mcm7p associate specifically with replication origins during G1 but not during G2 in S. cerevisiae. Mcm7p's association with origins depends on Cdc6p. Ectopic expression of Cdc6p enables it to associate with origins during G2, but this fails to recruit Mcm7p. Our data suggest that the loading of Mcm proteins onto origins is regulated by two mechanisms: first, by Cdc6p occupancy, and second, by S- and M-CDKs, whose activity during S, G2, and M phases prevents Mcm loading.

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3 −/− and Pax7 −/− mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.

Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. Connective tissue (CT) – skeleton, periskeleton, tendon, dermis, interstitial fibroblasts – contributes the vast majority of cells that populate the blastema, however, it is unclear how individual CT cells may reprogram their fate in order to rebuild the tetrapod limb. Here we use a combination of Cre-loxP reporter lineage tracking and single-cell (sc) RNA-seq to molecularly track, for the first time, adult CT cell heterogeneity and its transition to a limb blastema state. We uncover a multi-phasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that participates in inflammation and extracellular matrix disassembly prior to proliferation, establishment of positional information, and ultimately re-differentiation. While the early regeneration transcriptome states are unique to the blastema, the later stages recapitulate embryonic limb development. Notably, we do not find evidence of a pre-existing blastema-like precursor nor limb bud-like progenitors in the uninjured adult tissue. However, we find that distinct CT subpopulations in the adult limb differentially contribute to extending bone at the amputation plane versus regenerating new segments. Together, our data illuminates molecular and cellular reprogramming during complex organ regeneration in a vertebrate.

DNA replication in budding yeast cells depends on the activation of the Cdc28 kinase (Cdk1 of Saccharomyces cerevisiae) associated with B-type cyclins Clb1 to Clb6. Activation of the kinase depends on proteolysis of the Cdk inhibitor p40SIC1 in late G 1 , which is mediated by the ubiquitin-conjugating enzyme Cdc34 and two other proteins, Cdc4 and Cdc53. Inactivation of any one of these three proteins prevents p40 SIC1 degradation and causes cells to arrest in G 1 with active Cln kinases but no Clb-associated Cdc28 kinase activity. Deletion of SIC1 allows these mutants to replicate. p40 SIC1 disappears at the G 1 /S transition and reappears only after nuclear division. Cell cycle-regulated proteolysis seems largely responsible for this pattern, but transcriptional control could also contribute; SIC1 RNA accumulates to high levels as cells exit M phase. To identify additional factors necessary for the inhibition of the Cdk1/Cdc28 kinase in G 1 , we isolated mutants that can replicate DNA in the absence of Cdc4 function. Mutations in three loci (SIC1, SWI5, and RIC3) were identified. We have shown that high SIC1 transcript levels at late M phase depend on Swi5. Swi5 accumulates in the cytoplasm during S, G 2 , and M phases of the cell cycle but enters the nuclei at late anaphase. Our data suggest that cell cycle-regulated nuclear accumulation of Swi5 is responsible for the burst of SIC1 transcription at the end of anaphase. This transcriptional control may be important for inactivation of the Clb/Cdk1 kinase in G 2 /M transition and during the subsequent G 1 period.DNA replication in budding yeast cells depends on the activation of Cdk1/Cdc28 kinase associated with six different B-type cyclins, Clb1 to Clb6. Clb-associated Cdk1 kinase is regulated by transcription of cyclin genes, by proteolysis of cyclin proteins, and by accumulation of inhibitory proteins. Due largely to the onset of CLB5 and CLB6 transcription in late G 1 (CLB1 to CLB4 RNAs do not appear until later), DNA replication is normally triggered by the appearance of active Clb5 or Clb6/Cdc28 kinases (10, 26). In the absence of Clb5 and Clb6, Clb1 to Clb4 trigger replication. Normally, DNA replication is simultaneous with bud formation, but in the clb5 clb6 mutant, DNA replication is delayed relative to bud emergence (26). Clb/Cdc28 kinase activity is also regulated by changes in cyclin stability (1) and by binding of the inhibitory protein p40 SIC1 . p40 SIC1 does not inhibit Cdc28 kinase associated with Cln cyclins which also appear in late G 1 and are necessary for activating CLB gene transcription, turning off Clb proteolysis, and triggering p40 SIC1 proteolysis (19,22,25). p40 SIC1 protein accumulates to high levels in G 1 , disappears at the G 1 /S transition, and does not reaccumulate until cells complete nuclear division and reenter G 1 . To understand how p40 SIC1 regulates the activity of cyclin B/Cdc28 kinases, we need to know how p40 SIC1 accumulation is regulated. It has been demonstrated that the appearance of Cln1 and Cln2/ Cdc28 kinases ...

Understanding how the limb blastema is established after the initial wound healing response is an important aspect of regeneration research. Here we performed parallel expression profile time courses of healing lateral wounds versus amputated limbs in axolotl. This comparison between wound healing and regeneration allowed us to identify amputation-specific genes. By clustering the expression profiles of these samples, we could detect three distinguishable phases of gene expression – early wound healing followed by a transition-phase leading to establishment of the limb development program, which correspond to the three phases of limb regeneration that had been defined by morphological criteria. By focusing on the transition-phase, we identified 93 strictly amputation-associated genes many of which are implicated in oxidative-stress response, chromatin modification, epithelial development or limb development. We further classified the genes based on whether they were or were not significantly expressed in the developing limb bud. The specific localization of 53 selected candidates within the blastema was investigated by in situ hybridization. In summary, we identified a set of genes that are expressed specifically during regeneration and are therefore, likely candidates for the regulation of blastema formation.

SUMMARYDuring salamander limb regeneration, only the structures distal to the amputation plane are regenerated, a property known as the rule of distal transformation. Multiple cell types are involved in limb regeneration; therefore, determining which cell types participate in distal transformation is important for understanding how the proximo-distal outcome of regeneration is achieved. We show that connective tissue-derived blastema cells obey the rule of distal transformation. They also have nuclear MEIS, which can act as an upper arm identity regulator, only upon upper arm amputation. By contrast, myogenic cells do not obey the rule of distal transformation and display nuclear MEIS upon amputation at any proximo-distal level. These results indicate that connective tissue cells, but not myogenic cells, are involved in establishing the proximo-distal outcome of regeneration and are likely to guide muscle patterning. Moreover, we show that, similarly to limb development, muscle patterning in regeneration is influenced by bcatenin signalling.

Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolate a pan-LPM enhancer in the zebrafish-specific draculin ( drl ) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl -based reporters captures the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncover specific activity of zebrafish-derived drl reporters in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona , and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.