The sea lamprey (Petromyzon marinus) serves as a comparative model for reconstructing vertebrate evolution. To enable more informed analyses, we developed a new assembly of the lamprey germline genome that integrates several complementary data sets. Analysis of this highly contiguous (chromosome-scale) assembly shows that both chromosomal and whole-genome duplications have played significant roles in the evolution of ancestral vertebrate and lamprey genomes, including chromosomes that carry the six lamprey HOX clusters. The assembly also contains several hundred genes that are reproducibly eliminated from somatic cells during early development in lamprey. Comparative analyses show that gnathostome (mouse) homologs of these genes are frequently marked by polycomb repressive complexes (PRCs) in embryonic stem cells, suggesting overlaps in the regulatory logic of somatic DNA elimination and bivalent states that are regulated by early embryonic PRCs. This new assembly will enhance diverse studies that are informed by lampreys’ unique biology and evolutionary/comparative perspective.

Nested Hox expression domains are found in jawed vertebrates and in non-vertebrate chordates, but it is unclear whether there is a link between brain regionalization and Hox expression in jawless vertebrates; here, Hox expression is shown to be integrated with hindbrain segmentation in lampreys. In the heads of developing gnathostomes (jawed vertebrates), nested Hox expression domains are associated with rhombomeres, the segments of the hindbrain. Nested Hox expression domains are also found in non-vertebreate chordates such as amphioxus, but their relationship with brain patterning is unclear as it is hard to relate regions of simple, unsegmented nervous systems to the gnathostome brain. What goes on in agnathans (jawless vertebrates) is unclear. Some have suggested that there might be no clear correspondence between brain regionation and Hox expression in these creatures but here Robb Krumlauf and colleagues not only show that Hox expression and hindbrain segmentation align in lampreys, but also that the correspondence is both deep and detailed. These results suggest that coupling of Hox expression to segmentation of the hindbrain is an ancient trait with an origin at the base of vertebrates. A defining feature governing head patterning of jawed vertebrates is a highly conserved gene regulatory network that integrates hindbrain segmentation with segmentally restricted domains of Hox gene expression. Although non-vertebrate chordates display nested domains of axial Hox expression, they lack hindbrain segmentation. The sea lamprey, a jawless fish, can provide unique insights into vertebrate origins owing to its phylogenetic position at the base of the vertebrate tree1,2,3. It has been suggested that lamprey may represent an intermediate state where nested Hox expression has not been coupled to the process of hindbrain segmentation4,5,6. However, little is known about the regulatory network underlying Hox expression in lamprey or its relationship to hindbrain segmentation. Here, using a novel tool that allows cross-species comparisons of regulatory elements between jawed and jawless vertebrates, we report deep conservation of both upstream regulators and segmental activity of enhancer elements across these distant species. Regulatory regions from diverse gnathostomes drive segmental reporter expression in the lamprey hindbrain and require the same transcriptional inputs (for example, Kreisler (also known as Mafba), Krox20 (also known as Egr2a)) in both lamprey and zebrafish. We find that lamprey hox genes display dynamic segmentally restricted domains of expression; we also isolated a conserved exonic hox2 enhancer from lamprey that drives segmental expression in rhombomeres 2 and 4. Our results show that coupling of Hox gene expression to segmentation of the hindbrain is an ancient trait with origin at the base of vertebrates that probably led to the formation of rhombomeric compartments with an underlying Hox code.

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA. Recent comparative analyses of vertebrate genomes has resulted in the identification of highly conserved non-coding sequences near genes that coordinate early development. Many of these sequences can activate gene expression and are thought to be important regulatory elements. Surprisingly, a large set of these long, near-identical sequences is found in every jawed vertebrate, including sharks, yet almost completely absent in non-vertebrates. This study looks for this set of sequences in the lamprey, a representative of our most distant vertebrate relatives, in order to determine when and how such a large set of important non-coding regulatory sequences became established in the genome. Although the lamprey divergence is only a little older than the divergence of cartilaginous fish (including sharks), relatively few, and considerably shorter, conserved non-coding sequences are identifiable. Nevertheless, these shorter lamprey sequences are capable of driving gene expression in a precise spatial pattern in zebrafish embryos in the same way as the equivalent human elements. This analysis has shed light on the emergence of these regulatory sequences during early vertebrate evolution, at a time of whole-genome duplications and considerable morphological variation, consistent with a role for these sequences in directing gene regulatory networks for vertebrate development.

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.