Lamprey Dlx genes and early vertebrate evolution

Abstract

Gnathostome vertebrates have multiple members of the Dlx family of transcription factors that are expressed during the development of several tissues considered to be vertebrate synapomorphies, including the forebrain, cranial neural crest, placodes, and pharyngeal arches. The Dlx gene family thus presents an ideal system in which to examine the relationship between gene duplication and morphological innovation during vertebrate evolution. Toward this end, we have cloned Dlx genes from the lamprey Petromyzon marinus, an agnathan vertebrate that occupies a critical phylogenetic position between cephalochordates and gnathostomes. We have identified four Dlx genes in P. marinus, whose orthology with gnathostome Dlx genes provides a model for how this gene family evolved in the vertebrate lineage. Differential expression of these lamprey Dlx genes in the forebrain, cranial neural crest, pharyngeal arches, and sensory placodes of lamprey embryos provides insight into the developmental evolution of these structures as well as a model of regulatory evolution after Dlx gene duplication events.

Figure 1

Dlx genes are associated with morphological novelty in the vertebrate lineage. A cladogram depicting hypothesized phylogenetic relationships of extant lineages within the chordates. A partial listing of morphological characters supporting this phylogenetic hypothesis is shown. Asterisked characters are those that have been shown, in gnathostomes, to be associated with Dlx gene expression. Note that some analyses (12, 13) using molecular characteristics indicate that hagfish and lampreys are monophyletic (dashed line), suggesting that modern hagfish secondarily lost certain morphological characters. The position of neural crest and placodes is speculative because hagfish embryos have not been characterized. Cladogram and character list are derived from refs. and .

Figure 2

P. marinus has four Dlx homologs. Comparative sequence alignment of the homeodomains of P. marinus DlxA, DlxB, DlxC, and DlxD with those of murine Dlx1, Dlx2, Dlx3, Dlx5, Dlx6, and Dlx7. Residues that match the consensus are boxed. Full-length cDNAs were isolated from an embryonic P. marinus cDNA library by a combination of PCR with degenerate oligonucleotides and hybridization.

Figure 3

Phylogeny of vertebrate Dlx genes. Neighbor-joining tree based on an edited clustal alignment of full-length amino acid sequences of P. marinus Dlx genes, gnathostome Dlx genes, and amphioxus Dll (see supplemental data for edited alignment), and rooted on mouse Msx1 (not shown on tree). Numbers indicate bootstrap values for selected nodes. There is no strict orthology between any lamprey Dlx gene and any gnathostome Dlx gene. Three of the P. marinus Dlx genes (DlxA, DlxB, and DlxC) group with the gnathostome Dlx2/3/5 clade, but do not group with Dlx2, Dlx3, or Dlx5 separately. Likewise, DlxD groups with the gnathostome Dlx1/6/7 clade, but does not group with Dlx1, Dlx6, or Dlx7. Amphioxus Dll is an outgroup to all vertebrate Dlx genes.

Figure 4

Lamprey Dlx gene expression in the cranial neural crest. Whole mounts and transverse sections of the head regions of 9-day-old lamprey embryos labeled with DlxA, DlxC, and DlxD riboprobes (DlxB expression is not detected at this stage). (×150.) DlxA and DlxD are both expressed in the dorsal aspect of the neural tube (D and F, arrowheads), as well as in ectomesenchyme consistent with migrating neural crest (arrows). DlxC is not detected in the neural tube, but is detected in head ectomesenchyme (E, arrows). Lines labeled by lowercase letters d, e, and f indicate sectioning planes.

Figure 5

Lamprey Dlx gene expression in the forebrain and pharyngeal arches. Whole mounts and horizontal sections of the head regions of 15-day-old lamprey embryos labeled with DlxA, DlxB, DlxC, and DlxD riboprobes. (A–D, ×150; E–K, ×75.) All four lamprey Dlx genes are expressed in each pharyngeal arch (A–D, arrows). Sections reveal that Dlx transcripts accumulate in the rostrolateral quadrant of the arch mesenchyme (F, G, I, and K, arrows), the site of future cartilage condensations. DlxA and DlxC are expressed in two bilateral pairs of transverse stripes in the ventral diencephalon and telencephalon (A, C, E, and H, arrowheads), whereas DlxD is expressed in a single pair of transverse stripes in the ventral diencephalon (D and J). DlxA, DlxB, and DlxC are also expressed in the olfactory placode (OlfP) and the otic vesicle (OtV, not shown for DlxC and DlxD). DlxB is not detected in the forebrain, olfactory placode, or otic vesicle.

Figure 6

A model for the evolution of the vertebrate Dlx gene family. This model is based on the phylogenetic tree shown in Fig. 3 and on the linkage relationships of gnathostome Dlx and Hox genes (8). We propose that a tandem duplication of an ancestral Dlx gene predated the divergence of lampreys from gnathostomes, which was followed by independent chromosomal or genome duplications and gene loss in each lineage. Dlx and Hox linkage are currently unknown in lampreys. Asterisks indicate uncertain linkage relationships.

Figure 7

A model for Dlx gene regulatory evolution. A summary of the expression of the four P. marinus Dlx genes is shown under “Modern Lampreys.” Comparisons of these expression patterns with those of several gnathostome systems (“Modern Gnathostomes”) and amphioxus (“Euchordate Ancestor”) suggest that several sites of Dlx expression evolved near the time of vertebrate origins (“Vertebrate Ancestor”). These include localized expression in the forebrain, sensory placodes, pharyngeal arches, and the dorsal fin fold. Modern gnathostomes appear to have lost Dlx expression in the somites, but co-opted Dlx function during the origin of limbs and jaws.