Sergej Nowoshilow

Abstract 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. However, the molecular understanding of blastema formation had previously been hampered by the inability to identify and isolate blastema precursor cells in the adult tissue. We have used a combination of Cre-loxP reporter lineage tracking and single-cell messenger RNA sequencing (scRNA-seq) to molecularly track mature connective tissue (CT) cell heterogeneity and its transition to a limb blastema state. We have uncovered a multiphasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that recapitulates an embryonic limb bud-like phenotype including multipotency within the CT lineage. Together, our data illuminate molecular and cellular reprogramming during complex organ regeneration in a vertebrate.

Abstract Lungfishes belong to lobe-fined fish (Sarcopterygii) that, in the Devonian period, ‘conquered’ the land and ultimately gave rise to all land vertebrates, including humans 1–3 . Here we determine the chromosome-quality genome of the Australian lungfish ( Neoceratodus forsteri ), which is known to have the largest genome of any animal. The vast size of this genome, which is about 14× larger than that of humans, is attributable mostly to huge intergenic regions and introns with high repeat content (around 90%), the components of which resemble those of tetrapods (comprising mainly long interspersed nuclear elements) more than they do those of ray-finned fish. The lungfish genome continues to expand independently (its transposable elements are still active), through mechanisms different to those of the enormous genomes of salamanders. The 17 fully assembled lungfish macrochromosomes maintain synteny to other vertebrate chromosomes, and all microchromosomes maintain conserved ancient homology with the ancestral vertebrate karyotype. Our phylogenomic analyses confirm previous reports that lungfish occupy a key evolutionary position as the closest living relatives to tetrapods 4,5 , underscoring the importance of lungfish for understanding innovations associated with terrestrialization. Lungfish preadaptations to living on land include the gain of limb-like expression in developmental genes such as hoxc13 and sall1 in their lobed fins. Increased rates of evolution and the duplication of genes associated with obligate air-breathing, such as lung surfactants and the expansion of odorant receptor gene families (which encode proteins involved in detecting airborne odours), contribute to the tetrapod-like biology of lungfishes. These findings advance our understanding of this major transition during vertebrate evolution.

Significance The axolotl is an important model organism because it is a tetrapod with a similar body plan to humans. Unlike humans, the axolotl regenerates limbs and other complex tissues. Therefore, the axolotl contributes to understanding evolution, development, and regeneration. With sophisticated tools for gene modification and tissue labeling, a fully assembled genome sequence was a sorely missing resource. Assembly was difficult because the genome size is 10× that of humans. Here, we use a cross-linking strategy called Hi-C to link together fragmented genome sequences to chromosome scale. We show that gene regulation occurs over very large genomic distances and that mitotic chromosomes are packaged efficiently.

Axolotls are uniquely able to mobilize neural stem cells to regenerate all missing regions of the spinal cord. How a neural stem cell under homeostasis converts after injury to a highly regenerative cell remains unknown. Here, we show that during regeneration, axolotl neural stem cells repress neurogenic genes and reactivate a transcriptional program similar to embryonic neuroepithelial cells. This dedifferentiation includes the acquisition of rapid cell cycles, the switch from neurogenic to proliferative divisions, and the re-expression of planar cell polarity (PCP) pathway components. We show that PCP induction is essential to reorient mitotic spindles along the anterior-posterior axis of elongation, and orthogonal to the cell apical-basal axis. Disruption of this property results in premature neurogenesis and halts regeneration. Our findings reveal a key role for PCP in coordinating the morphogenesis of spinal cord outgrowth with the switch from a homeostatic to a regenerative stem cell that restores missing tissue.