Dev Genes Evol (2000) 210:522–524
© Springer-Verlag 2000
EXPRESSION NOTE
Linda Z. Holland · Nicholas D. Holland Michael Schubert
Developmental expression of AmphiWnt1, an amphioxus gene in the Wnt1/wingless subfamily
Received: 30 March 2000 / Accepted: 30 May 2000
Abstract A full-length Wnt1 gene (AmphiWnt1) was isolated from amphioxus. Expression is first detectable in the gastrula around the lip of the blastopore. By the early neurula, transcription is in the mesendoderm near the closed blastopore, but is down-regulated in the overlying ectoderm. In the late neurula, expression is limited to the posterior wall of the neurenteric canal. Later in development, AmphiWnt1 transcripts can no longer be detected. AmphiWnt1 has no counterpart of the predominant expression domains of vertebrate Wnt1 genes in the neural tube, but its expression may be more comparable to that of wingless in the invaginating hindgut primordium of insects.
gene, AmphiWnt1, a representative of the Wnt1/wingless subfamily. To isolate AmphiWnt1 from amphioxus, we screened 60,000 clones of a gridded library from 26 h embryos (Lehrach et al. 1997) at moderately low stringency. Two probes were used, one a 360 bp piece of amphioxus Wnt4 and the second a 354 bp piece of amphioxus Wnt6;
Key words Blastopore · Cephalochordate · Lancelet · Neurenteric canal · Wnt gene
Genes of the Wnt family are numerous, with about 15 Wnt subfamilies found so far in vertebrates and many additional representatives among the invertebrates. Wnt genes encode secreted glycoproteins that play important roles in a wide variety of developmental processes (Wodarz and Nusse 1998). To date, full-length Wnt genes from four subfamilies have been studied in the Cephalochordate amphioxus, the closest living invertebrate relative of the vertebrates. Within each Wnt subfamily, each of these amphioxus genes marks the base of vertebrate diversification and is typically expressed during development in patterns comparable to those of its vertebrate homologs (Schubert et al. 2000a, b, c). The present study concerns an additional amphioxus Wnt Edited by D. Tautz L.Z. Holland · N.D. Holland · M. Schubert (✉) Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093-0202, USA e-mail:
[email protected] Tel.: +1-858-5346649, Fax: +1-858-5347313
Fig. 1 Phylogenetic tree of the Wnt1 protein subfamily. The tree is based on the most highly conserved C-terminal two-thirds of the Wnt proteins. Insect wingless sequences are referred to as Wnt1. Trees were calculated using the program PAUP (Version 3.1.1) in 100 random stepwise additions with the Caenorhabditis Lin44 protein as the outgroup. Only one most parsimonious tree was retained by the analysis (length: 788). The stability of the tree was assessed by 1000 bootstrap replicates with ten random stepwise additions per cycle. The bootstrap percentages are given in the tree above each branch. GenBank accession numbers: AmphiWnt1 (AF061974), Lin44 Caenorhabditis (2133466), Wnt1 Caenorhabditis (X72941), Wnt1 Bombyx (D14169), Wnt1 Drosophila (J03650), Wnt1 Tribolium (S41156), Wnt1 Junonia (L42142), Wnt1 sea urchin (U88625), Wnt1 Fugu (1814291), Wnt1 zebrafish (X58883), Wnt1 Xenopus (X13138), Wnt1 mouse (M11943), Wnt1 human (X03072)
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Fig. 2A–I Expression of AmphiWnt1 in whole-mounts of amphioxus embryos. In all side and dorsal views, anterior is toward the left; all scale bars 50 µm. A Side view of cup-shaped gastrula with expression around lip of blastopore. B Side view of very late gastrula with expression around blastopore. C Blastopore view of preceding embryo. D Side view of very early neurula with expression around blastopore. E Dorsal view of preceding embryo. F Blastopore view of preceding embryo. G Side view of mid-neurula with expression in mesendoderm in region of closed blastopore, but not in overlying ectoderm. H Side view of late neurula with expression in posterior wall (pw) of neurenteric canal (nec). I Dorsal view of preceding embryo with expression in posterior wall of neurenteric canal (nec)
these two clones were originally designated as Wnt-A and Wnt-B, respectively, by Holland et al. (1994). In situ hybridization of embryos and larvae was performed according to Holland et al. (1996). The phylogenetic analysis was performed with the program PAUP (Version 3.1.1) and is based on 13 taxa and 503 characters comprising the most highly conserved C-terminal two-thirds of the Wnt protein sequences. Library screening yielded a single clone of AmphiWnt1, which is 2,362 bp long, with a 126-bp 5′ UTR with two in-frame stop codons upstream from the pre-
sumed start codon (GenBank accession number AF061974). The 900 bp long 3′ UTR includes a noncanonical polyadenylation signal (AATACA) starting 20 bases upstream from the poly(A) tail (Hook and Kellems 1988). The longest open reading frame of AmphiWnt1 codes for 377 amino acids. In the phylogenetic analysis of the Wnt1 protein subfamily (Fig. 1), the AmphiWnt1 sequence groups with the deuterostome Wnt1 proteins but does not branch off at the base of vertebrate diversification, as would be expected from the phylogenetic position of amphioxus. However, the position of AmphiWnt1 in the tree is not supported by high bootstrap percentages. Thus, AmphiWnt1 is more divergent within its subfamily than any of the previously described amphioxus Wnt sequences, which typically branch off basal to the vertebrates with robust bootstrap values. The expression of AmphiWnt1 is limited to a single domain at the posterior end of the amphioxus embryo and larva. The earliest detectable expression of AmphiWnt1 begins in the cup-shaped gastrula, around the lip of the blastopore (Fig. 2A). In the very late gastrula (Fig. 2B, C) and in the very early neurula (Fig. 2D, E), expression progressively diminishes dorsally and ventrally
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while remaining strong in the lateral edges of the blastopore. At the mid-neurula stage, expression is conspicuous in the mesendoderm in the region of the closed blastopore, but not in the overlying ectoderm (Fig. 2G). In the late neurula, expression is detectable in the posterior wall of the neurenteric canal (Fig. 2H, I). It is thus possible that cells initially located laterally in the lip of the blastopore eventually give rise to the posterior wall of the neurenteric canal (however, this lineage needs to be confirmed by marking blastopore lip cells with vital dyes). No expression of AmphiWnt1 can be detected in later embryos and larvae. The restricted AmphiWnt1 expression in the posterior part of the embryo has no counterpart in vertebrates, in which Wnt1 genes are predominantly expressed in the developing neural tube (reviewed in Hollyday et al. 1995), but not around the blastopore. In contrast, the expression of amphioxus AmphiWnt1 seems to have more in common with the earliest zygotic expression of wingless (=Wnt1) in insects, which is detected at the blastoderm stage in a region associated with the posterior pole of Tribolium (Nagy and Carroll 1994) and with both the anterior and posterior poles of Drosophila (Baker 1988). Whether this represents a common evolutionary inheritance in protostomes and deuterostomes remains to be determined. Later in insect development, expression of wingless is very pleiotropic (Baker 1988; Nagy and Carroll 1994). However, the expression of several other posterior markers (e.g. caudal and brachyury) in flies and chordates has led to the suggestion that the blastopores of insects and chordates are homologous (Wu and Lengyel 1998). This idea should be tested further by studies on Wnt1 homologs from lower deuterostomes like hemichordates and tunicates. Acknowledgements J.M. Lawrence generously provided laboratory facilities in Tampa and G.D. Panopoulou made the 26 h cDNA library available. This research was supported in part by National Science Foundation grant IBN96-309938 to N.D.H. and L.Z.H.
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