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The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

The cell type-specific expression of key transcription factors is central to development. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three Brachyury -controlling notochord enhancers T3, C , and I in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, deletion of all three enhancers in mouse abolishes Brachyury/T expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. Sequence and functional conservation of Brachyury -driving notochord enhancers with the brachyury/tbxtb loci from diverse lineages of fishes dates their origin to the last common ancestor of jawed vertebrates. Our data define the enhancers for Brachyury/T/TBXTB notochord expression as ancient mechanism in axis development.

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[sup./] and Pax7[sup./] mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies., Author(s): Sergej Nowoshilow [1, 2, 3]; Siegfried Schloissnig (corresponding author) [4]; Ji-Feng Fei [5]; Andreas Dahl [3, 6]; Andy W. C. Pang [7]; Martin Pippel [4]; Sylke Winkler [1]; Alex [...]

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., The salamander is a powerful regeneration model because it can reconstitute a fully functional limb after injury. Amputation anywhere between the shoulder and the hand triggers the formation of a [...]

Background: The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging. Results: The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte‐filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2‐descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l‐thyroxine, increasing the rate of bone formation. Conclusions: In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth. Key Findings: The juvenile appendicular skeleton is formed mostly by Sox9/Col1a2 cartilage cells, transitioning to a slow and life‐long ossification that starts around sexual maturation.Axolotl growth is biphasic, defined by an explosive growth of axolotls in approximately the first 20 months of their life, after which growth velocity dramatically diminishes, however never reaching zero.A hybrid population of hypertrophic chondrocytes, expressing the chondrogenic marker SOX9 and the osteoblast marker OCN, resides next to the ossification center.Col1a2‐descendants contribute to bone and adipocytes during ossification. [ABSTRACT FROM AUTHOR]

A formaldehyde cross-linking study revealed that Mcm7p and Cdc6p initiation proteins are specifically associated with Saccharomyces cerevisiae DNA replication origins in G1 but not in G2. Results also show that Mcm7p is dependent on Cdc6p in terms of origin association. Furthermore, the Cdc6p occupancy and S- and M-CDKs were found to regulate the loading of Mcm proteins onto origins.

Salamanders exhibit extensive regenerative capacities and serve as a unique model in regeneration research. However, due to the lack of targeted gene knockin approaches, it has been difficult to label and manipulate some of the cell populations that are crucial for understanding the mechanisms underlying regeneration. Here we have established highly efficient gene knockin approaches in the axolotl (Ambystoma mexicanum) based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the Cherry reporter gene and a larger membrane-tagged Cherry-ERT2-Cre-ERT2 (~5-kb) cassette into axolotl Sox2 and Pax7 genomic loci. Depending on the size of the DNA fragments for integration, 5-15% of the F0 transgenic axolotl are positive for the transgene. Using these techniques, we have labeled and traced the PAX7-positive satellite cells as a major source contributing to myogenesis during axolotl limb regeneration. Our work brings a key genetic tool to molecular and cellular studies of axolotl regeneration. [ABSTRACT FROM AUTHOR]

We have identified fullback and fullback-like, two Xenopus laevis neurotrophin receptor homolog (NRH1) genes. The sequences of Fullback and Fullback-like are very similar to that of the neurotrophin receptor p75NTR, in both their extracellular and their intracellular domains. As their names imply, fullback and fullback-like are expressed in essentially identical patterns in the posterior of the embryo from the early gastrula stage onward. At tailbud and tadpole stages transcripts are also present in dorsal somites and the head, in addition to the growing tailbud. This expression pattern differs from that of p75NTR, suggesting that fullback and fullback-like have different functions from p75NTR during early development. [Copyright &y& Elsevier]