Expression in Embryos of a Larvacean Urochordate, Oikopleura

Home , Larvacea, Oikopleura, Oikopleura dioica

Developmental Biology 220, 322–332 (2000) doi:10.1006/dbio.2000.9647, available online at http://www.idealibrary.com on

View metadata, citation and similar papers at core.ac.uk brought to you by CORE Brachyury (T) Expression in Embryos of a Larvaceanprovided by Elsevier - Publisher Connector Urochordate, Oikopleura dioica, and the Ancestral Role of T

Susan Bassham and John Postlethwait Institute of Neuroscience, 1254 University of Oregon, Eugene, Oregon 97403-1254

The Brachyury, or T, gene is required for notochord development in animals occupying all three chordate subphyla and probably also had this role in the last common ancestor of the chordate lineages. In two chordate subphyla (vertebrates and cephalochordates), T is also expressed during gastrulation in involuting endodermal and mesodermal cells, and in vertebrates at least, this expression domain is required for proper development. In the basally diverging chordate subphylum Urochordata, animals in the class Ascidiacea do not employ T during gastrulation in endodermal or nonaxial mesodermal cells, and it has been suggested that nonnotochordal roles for T were acquired in the cephalochordate–vertebrate lineage after it split with Urochordata. To test this hypothesis, we cloned T from Oikopleura dioica, a member of the urochordate class Appendicularia (or Larvacea), which diverged basally in the subphylum. Investigation of the expression pattern in developing Oikopleura embryos showed early expression in presumptive notochord precursor cells, in the notochord, and in parts of the developing gut and cells of the endodermal strand. We conclude that the ancestral role of T likely included expression in the developing gut and became necessary in chordates for construction of the notochord. © 2000 Academic Press Key Words: Tunicata; Chordata; evolution of development.

INTRODUCTION though, expression of the ascidian T ortholog was found only in cells of notochord lineage and not broadly in involuting endoderm and mesoderm or in mesoderm lateral The phylum Chordata gets its name from the notochord, to the presumptive notochord (Yasuo and Satoh, 1993, but the evolutionary origin of this mesodermal rod is as yet 1994; Corbo et al., 1997). What was the original role of T in unclear (Peterson et al., 1999; Harada et al., 1995). The chordate development, and how was it recruited to its development of the notochord in vertebrate embryos re- current roles in vertebrate embryos? quires the action of the Brachyury gene (or T), as mutations in this gene in mouse (Herrmann et al., 1990) and in At least two hypotheses have been suggested to explain zebrafish (Schulte-Merker et al., 1994) block or reduce the the difference in T expression between representatives of development of the notochord. Besides its role in notochord the cephalochordate–vertebrate clade and ascidians in its development, in vertebrate embryos, T is also required for urochordate sister group. In one model, the primary ances- correct internalization and migration of posterior meso- tral function of the T gene in the chordates was in noto- derm during gastrulation (Wilson et al., 1995). Vertebrate chord specification (Yasuo and Satoh, 1994; Holland et al., embryos express T transiently around the blastopore in 1995). This hypothesis implies that a function for T in presumptive endoderm and mesoderm cells as they invo- gastrulation and paraxial mesoderm formation was ac- lute and migrate, and more persistently in the notochord quired, presumably by the evolution of positive regulatory (e.g., Wilkinson et al., 1990; Schulte-Merker et al., 1992). elements driving T expression, in the cephalochordate– Similarly, in cephalochordates, expression of at least one of vertebrate clade after its divergence from the urochordates. two genes homologous to T (Ambra-1 or -2) occurs in a ring Alternatively, both functions of T (mesendoderm formation around the blastopore during gastrulation, in the presomitic and notochord specification) may have been present in the mesoderm, and in the notochord (Holland et al., 1995). In ancestral chordate, and ascidians retained only the noto- ascidian urochordates, the role of T in notochord develop- chord function. Consistent with this proposal is the iden- ment is conserved (Yasuo and Satoh, 1998). In contrast, tification of cis-acting regulatory regions that suppress the

Willey, 1894) argued that a free-swimming morphology like that of larvaceans, cephalochordates, and vertebrates was the primitive state of the chordate ancestor and that the radical metamorphosis of ascidians is evolutionarily derived. Recent phylogenetic analyses of rRNA sequences agree with this view, suggesting that the larvacean lineage diverged earliest within the urochordates (Christen and Braconnot, 1998; Wada, 1998; Wada and Satoh, 1994). To test whether the notochord-restricted expression of T is a pattern that is generalizable to the urochordate subphylum, and therefore to address the hypothesis that notochord specification is the ancestral function for T in the chordates, we cloned a T homolog from the larvacean Oiko- pleura dioica and analyzed its expression during embryogenesis. In this first investigation of the molecular embryology of any larvacean, we found that during gastrulation of O. dioica embryos T is expressed in a pattern similar to that in ascidians: in cells of the notochord lineage but not in mesoderm with lateral fates. When formation and differentiation of the larvacean tail is nearly complete, however, T is employed in nonnotochordal expression domains, nota- bly in regions of the forming gut and cells of the endodermal strand. This may reflect a deeper metazoan ancestry of this gene’s function in the patterning of posterior endoderm and suggests that in the earliest chordate T was employed in FIG. 1. Phylogenetic relationships among chordates and the other deuterostomes, with a protostome outgroup. This tree topology, both notochord and hindgut development. which shows larvaceans branching basally in the urochordates, is modeled on rDNA phylogenies from Wada and Satoh (1994). MATERIALS AND METHODS Additional studies (Wada, 1998) suggest that Ascidiacea may be paraphyletic, in which case ascidian and thaliacean subsets group together to the exclusion of pleurogonid ascidians; the basal Animal culture and embryo collection. Adult and juvenile O. position of larvaceans, however, is unchanged. The hemichordates dioica were collected from the plankton in coastal waters near and echinoderms are drawn as sister groups in accordance with Coos Bay, Oregon, sorted into 12–14°C seawater in glass jars gene trees from Wada and Satoh (1994) and Lynch (1999). (2-liter), and fed until mature on a diet of natural phytoplankton and cultured algal strains (Dunaliella tertiolecta, Isochrysis gal- bana, Rhodomonas lens, Nanochloropsis sp., and Micromonas sp. (strain Dw-8)). Sperm was used immediately upon spawning or was activation of T in nonnotochordal cell lineages, specifically collected by rupturing ripe males by pipet and stored undiluted at in mesenchyme and muscle of ascidian embryos (Corbo et 12°C for up to 24 h. Females were transferred into small finger- al., 1997); this may indicate an evolutionary gain of func- bowls to spawn and eggs were collected for synchronous fertiliza- ␮ tion in the ascidian lineage which eliminates T expression tion in 0.45- m-filtered seawater. Embryos were kept at 12°C. Cloning and phylogenetic analysis. mRNA extracted from in those tissues in ascidians compared to an ancestral state. pooled embryos ranging from two-celled to late tailbud stages was Because only one of the four classes of the subphylum converted by Superscript reverse transcriptase (GIBCO/BRL) to Urochordata has been investigated with respect to T gene cDNA. A larvacean T fragment from this cDNA was amplified function, it is difficult to generalize about the role of T in with nested degenerate PCR primers corresponding to the follow- early chordates. What is needed is an investigation of T in a ing amino acid sequences: NEMIVTK and WKYVNGEW (sense) basally diverging urochordate, namely, a member of the and VTAYQNEE and NPFAKAF (antisense), corresponding to nu- urochordate class Appendicularia, also called larvaceans cleotide sequences 5Ј-AA(T/C)GA(A/G)ATGAT(T/C/A)GT(I/ (Fig. 1). Appendicularia are a sister class of the clade C)AC(I/C)AA-3Ј,5Ј-TGGAA(A/G)TA(C/T)GT(I/C)AA(T/C)GG(I/ including the sessile, filter-feeding ascidians; the abyssal, C)GA(A/G)TGG-3Ј,5Ј-TC(T/C)TC(A/G)TT(T/C)TG(A/G)TA(I/ Ј Ј ascidian-like sorberaceans; and the pelagic thaliaceans. Lar- C)GC(I/C)GT(I/C)AC-3 , and 5 -GAA(I/C)GC(T/C)TT(I/C)GC(A/ G)AA(I/C)GG(A/G)TT-3Ј, respectively. mRNA extracted from tail- vaceans may be more deeply rooted in the urochordate bud stage embryos was reverse transcribed to make pooled cDNA clade than these other classes and retain a chordate body for RACE PCR using the Marathon cDNA Amplification Kit plan throughout their life history (Holland et al., 1988). In (Clontech), and overlapping 3Ј and 5Ј RACE products of a full- contrast, metamorphosis reconfigures the ascidian into a length transcript were cloned into TA-Cloning vector pCRII (In- sessile adult that little resembles the chordate design of its vitrogen). The open-reading frame was identified by the criteria of larva. Early authors in the field of chordate evolution (e.g., Kozak (1996). The 3Ј fragment, from corresponding amino acid

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. 324 Bassham and Postlethwait position SVYMHP through the end of the 3Ј UTR, was used to tionships. In a N-J phylogeny based on 519 unambiguously generate DIG-labeled riboprobes (Boehringer Mannheim DIG/ alignable nucleotide positions and rooted with sea urchin T, Genius RNA Labeling Kit). the tree topology better reflects expected taxonomic group- Nucleotide sequences were aligned using MegAlign software ings. OdiT groups strongly with its homologs in two (Version 3.08, DNASTAR) and trimmed to only unambiguously ascidians (Fig. 3B) (for sequence alignments, see http:// alignable regions. Gaps and any ambiguous adjacent regions were www.neuro.uoregon.edu/postle/mydoc.html). excluded. Bootstrapped phylogenetic trees of trimmed sequences were calculated by the neighbor-joining method (Saitou and Nei, 1987) using Clustal X software (http://www-igbmc.u-strasbg.fr/ Expression of OdiT from Cleaving Embryos to BioInfo/ClustalX/Top.html) and NJPLOT (http://pbil.univ- Hatchlings lyon1.fr/software/njplot.html) (Perrie`re and Gouy, 1996). In situ hybridization. Embryos were fixed in 1 mM EGTA, 2 We studied the expression of OdiT in a developmental mM MgSO4, 0.1 M MOPS, 0.5 M NaCl, and 4% paraformaldehyde series of O. dioica embryos. The 3Ј RACE product contains for1hatroom temperature before being transferred to 70% EtOH 613 bp of predicted coding sequence and 374 bp of 3Ј UTR; for 1 to 3 h. Embryos were transferred to fresh 70% EtOH for this ϳ1-kb fragment was used to make labeled riboprobes Ϫ storage at 20°C. Prior to hybridization, prehatch embryos were for whole-mount in situ hybridization (Fig. 2). Blastomeres rehydrated in PBT and manually dechorionated. A whole-mount in in O. dioica embryos do not cleave synchronously after the situ hybridization protocol was adapted from Thisse et al. (1993) with the following modifications: embryos were hybridized in a 16-cell stage, so cell numbers were ascertained by counting mixture of 50% formamide, 5ϫ SSC, 0.1 mg/ml heparin, 0.1% DAPI-stained nuclei in early embryos. OdiT expression was Tween 20, 5 mM EDTA, 1 mg/ml tRNA, and 1ϫ Denhardt’s first detected in a bilateral pair of blastomeres in 30-cell reagent at 65°C with probe. Hybridized embryos were washed for embryos (Figs. 4A and 4B). With the fifth cleavage (produc- 10 min in 50% hybridization buffer/50% (2ϫ SSC, 0.1% Tween 20) ing 30 cells), some blastomeres have already begun to at 65°C; 10 min in 2ϫ SSC/0.1% Tween 20 at 65°C; 30 min in 0.2ϫ overgrow their neighbors by epibolic divisions, marking the SSC/0.1% Tween 20 at 65°C; 30 min in 0.1ϫ SSC/0.1% Tween 20 onset of gastrulation (Delsman, 1910). The two OdiT- ϫ at 65°C; 5 min in 50% (0.1 SSC/0.1% Tween 20)/50% PBT at expressing blastomeres appear at least partly submerged room temperature. Embryos were incubated for at least 60 min at beneath superficial cells, and hence, gastrulation begins in room temperature in a mixture of PBT, 2.5 mg/ml BSA, and 2% Oikopleura before OdiT expression commences. These two goat serum. Then embryos were incubated for2hatroom temperature with presorbed alkaline-phophatase-coupled anti- blastomeres are the only cells expressing OdiT until after digoxigenin antibody (Boehringer Mannheim) and treated to the the 44-cell stage, the end of gastrulation; they can be seen in standard detection described by Boehringer Mannheim. Embryos the 44-cell embryo entirely submerged beneath the closed were mounted in 40% glycerol with 0.6 ␮g/ml DAPI. Embryos and blastopore whose perimeter consists of 6 cells arranged as hatchlings hybridized with a labeled sense riboprobe showed no petals of a flower (Figs. 4C and 4D). At the neurula stage, staining at any stage (not shown). when the ball-shaped embryo flattens slightly (Delsman, 1910), two bilateral pairs of blastomeres are labeled by the probe (Fig. 4E). Up to this stage, expressing blastomeres RESULTS were separated from one another by two intervening midline cells. Bilateral bands of expressing cells then become Phylogenetic Relationships of OdiT contiguous across the midline and form a chevron (Fig. 4F), We cloned a fragment of T (Brachyury) from O. dioica beginning the convergence and extension of the notochord. using degenerate primers designed from aligned protein The tail of O. dioica has 20 muscle cells arranged in two sequences of chordate T homologs. Sequencing this frag- rows of 10 cells each on either side of the 20 cells of the ment provided information to design primers for the ampli- notochord. Just prior to extension of the notochordal rod, 19 fication of overlapping 3Ј and 5Ј fragments of a single gene labeled cells are lined in a staggered row at early tailbud by RACE PCR. The sequencing of this gene (Fig. 2) showed stage (Fig. 5A). Muscle cell precursors adjacent to notochord that it constitutes the 3Ј UTR and the entire coding region precursors are not labeled by the OdiT probe (Figs. 5B and of a T homolog we named O. dioica T (OdiT). In a 5C). At hatching, 19 notochordal cells are linearly arranged neighbor-joining (N-J) phylogeny of alignable T-box family (Fig. 5D) and a cell adjacent to the posteriormost cell in the protein sequences (173 amino acids), OdiT falls within a row is also detected by the probe (Fig. 5E). We interpret this strongly supported clade (bootstrap ϭ 999) including T as notochord cell 20 which has not yet intercalated into the proteins from vertebrates, cephalochordates, urochordates, notochordal axis. a hemichordate, echinoderms, and a cnidarian (Fig. 3A). In larvaceans, the tail is twisted 90° relative to the trunk, Excluded from this clade were genes representing other dorsal to the left, ventral to the right. In hatchlings whose T-box subfamilies, as well as a second ascidian T-box gene tails have just straightened, two T-expressing cells lie called As-T2 which is thought to be orthologous to verte- adjacent and to the right of the notochord (Fig. 5F). Their brate tbx6 (Mitani et al., 1999). Because the T-domain itself position and shape suggest these are the subchordal cells, represents nearly all of the alignable sequence and provides products of the caudal endoderm, which migrate back into a limited number of informative sites, the topology of the the tail after retracting into the trunk with rest of the gene phylogeny does not conform well to taxonomic rela- endodermal strand (Delsman, 1912).

FIG. 2. The nucleotide and predicted amino acid sequences of the entire coding region of OdiT. The DNA-binding T-box domain is underlined. An arrow marks the 3Ј end of the clone used to generate riboprobe for in situ hybridizations.

In hatchlings 6 h postfertilization, as organogenesis is DISCUSSION being completed in the trunk, OdiT signal rapidly decreases in the notochordal cells and new expression domains appear In order to explore the evolutionary origins of develop- in dorsal and ventral cells of the posterior trunk. Initially, a mental innovations in the phylum Chordata, we have ﬂattened cell immediately rostral and ventral to the noto- begun to investigate the molecular embryology of O. di- chord and underlying the ectoderm is labeled by the probe oica. This organism occupies an important position in (Fig. 6A), as are a series of cells at a posteriodorsal position chordate phylogeny, and larvacean anatomy suggests that it in the trunk and lying just left of the midline under the may facilitate detection of homologies between urochor- epidermis (Figs. 5F and 6A); based on their positions, these dates and vertebrates on the basis of both morphological cells may be of the developing hindgut and esophagus, and molecular criteria. To investigate the ancestral func- respectively. In the latest stage hatchlings we examined tion of a gene important in the development of a novel (“Stage II”; Galt and Fenaux, 1990) the ventral expression chordate character, the notochord, we have cloned the domain was a smooth-bordered oblong lying against the larvacean ortholog (OdiT) of the vertebrate T gene and epidermis and surrounded by the expanding body lumen analyzed its expression during embryogenesis. We isolated (Fig. 6B). When fully developed, a sausage-shaped rectum the complete coding region of the OdiT message as well as lies in this position. the entire 3Ј UTR. Gene phylogenies established the orthol-

FIG. 3. Gene phylogenies indicate OdiT is a Brachyury ortholog. (A) Neighbor-joining (N-J) tree of alignable tbx family protein sequence. Included in the tree are T homologs from a broad phylogenetic range as well as representative members of the other tbx subfamilies (Wattler et al., 1998). (B) N-J tree of alignable nucleotide sequence from chordate T genes, with two deuterostome outgroups, a hemichordate and an echinoderm. Bootstrap values greater than 50% are shown. GenBank accession numbers are X91903 (Ambra-1), P80492 (Ambra-2), AB018527 (Ap-Bra), D16441 (As-T), D83265 (As-T2), AF123247 (Ciona Bra), AB001939 (Cynops brachyury), U67086 (Ch-T), U67087 (Ch-TbxT), D50332 (HpTa), AF105065 (HyBra), AB001871 (Me-Tam), Q07998 (ntl), AF204208 (OdiT), AB004912 (PfBra), NM_003181 (Homo T), X51683 (Mus T), U57331 (tbx6), NM_005149 (TBX19), P55965, S74163 (byn), and M77243 (Xbra). For alignments, see http://www.neuro.uoregon.edu/postle/mydoc.html.

ogy of OdiT to the T (Brachyury) subfamily of T-box genes eny of alignable DNA sequences using a sea urchin T as the by analysis of both predicted amino acid sequence and outgroup, OdiT falls within the chordate sequences and nucleotide sequence (Fig. 3). In a phylogeny of T-domain groups strongly with the ascidian Ts. Orthologs are genes in sequences from a broad range of T-box proteins, OdiT two species that can trace their ancestry back to a single groups unambiguously within the T clade. And in a phylog- gene in the last common ancestor of those species (Fitch,

FIG. 4. Expression of OdiT in early O. dioica embryos (cleavage stage, gastrulae, and neurulae) is detected only in notochord precursors. (A) Thirty-cell embryo. (B) Diagram of 30-cell embryo, after Delsman (1910). Predicted OdiT-expressing cells shaded. (C) Forty-four-cell embryo. Superﬁcial view of blastopore showing 2 internalized Odi-T-expressing cells. (D) Diagram of 44-cell embryo, vegetal view showing cell arrangement around blastopore, after Delsman (1910). (A–D) Vegetal pole view. (E) Early neurula. Vegetal pole at bottom. (F) Late neurula. Scale bar represents 20 ␮m.

1970). We conclude that OdiT is a true T ortholog and not ancestral pattern, and ascidian embryos might have lost T a different member of the T-box gene family. function in endoderm and paraxial mesoderm formation. In cephalochordates and vertebrates, T is expressed in Peterson et al. (1999) favor the notochord-plus- presomitic mesoderm, in the notochord, and in involuting surrounding-cells hypothesis and propose a parsimonious mesodermal and endodermal cells surrounding the closing model for the evolution of T function based on gene blastopore (or equivalent) (e.g., Wilkinson et al., 1990; expression in arthropods and the deuterostome phyla. Ac- Schulte-Merker et al., 1992). In previously investigated cording to this model, prior to the protostome/ basal chordates (ascidians), T is expressed in cells that have deuterostome split, T served a function in posterior gut become segregated during cleavage to a purely notochordal development. Kusch and Reuter (1999) additionally propose lineage, but not in mesoderm lateral to the presumptive that T had an ancestral role in mesoderm development in notochord or the nearby endoderm (Yasuo and Satoh, 1993, the last common ancestor of protostomes and deuteros- 1994; Corbo et al., 1997). In light of this result in ascidian tomes. The chordate lineage acquired notochord expression embryos, it has been proposed that the ancestral role for T after divergence from the echinoderm-hemichordate clade. in chordates was in notochord speciﬁcation (Yasuo and Although vertebrates and cephalochordates express T in Satoh, 1994). If the notochord-only hypothesis is true, then posterior endoderm and paraxial mesoderm early in devel- the nonnotochordal expression domains in cephalochordate opment, neither of these proposed ancestral expression and vertebrate embryos represent functions of the T gene domains has been demonstrated in the normal development that were acquired after the evolutionary split between the of ascidians. To evaluate these alternatives, we examined T urochordates and the cephalochordate–vertebrate lineage. expression in a basally diverging urochordate. Recent mo- Alternatively, the expression pattern in the lecular phylogenetic analyses have placed the class Appen- cephalochordate–vertebrate clade—in the notochord, sur- dicularia at a basal node within the urochordate clade (e.g., rounding mesoderm, and endoderm—could have been the Wada, 1998). This contradicts the prevailing hypothesis

FIG. 5. In tailbud stage embryos and hatchlings, OdiT transcripts are detected in the notochord, then also in endodermal expression domains. (A) Early tailbud stage, dorsal view, anterior at top. Posteriormost notochord cells lie in lateral positions and have not yet intercalated. (B) Mid-tailbud stage, lateral view. Arrows indicate nuclei of muscle cell precursors. (C) Late tailbud stage. Optical section through trunk and tail. Inset shows plane of optical section. (D) Early hatchling, lateral view. (E) Ventral view of early hatchling. Arrow indicates 20th notochord cell. (F) Larva 30 min after hatching. Black arrows indicate two subchordal cell precursors. Open arrow indicates dorsal trunk expression domain. Scale bar represents 10 ␮m in (A–E) and 20 ␮m in (F).

that larvaceans arose by paedomorphosis from the thali- homolog might share an expression pattern with the out- aceans (Garstang, 1928) and are the most derived of the group, the cephalochordate–vertebrate clade. In other urochordates. Instead, the molecular data support the work words, OdiT might be expressed in endoderm and nonno- of morphologists who argued that the motile body plan of tochordal mesoderm. the adult larvacean is a plesiomorphy of Chordata (e.g., Lankester, 1882; Lohmann, 1933). If the notochord- Early Expression of Larvacean T Is Restricted to restricted expression pattern of As-T is the ancestral condi- the Developing Notochord tion for Urochordata, the larvacean homolog is predicted to be expressed only in notochord lineage cells. If the ascidian Expression of OdiT was detected neither in endodermal pattern is an apomorphy of the ascidian clade and larva- cells while these were internalized by epiboly during gas- ceans are basal within the urochordates, the larvacean trulation nor in paraxial mesoderm during tail formation

FIG. 6. OdiT expression persists into late organogenesis in the trunk, after expression is no longer detected in the notochord. (A) Larva, 1 h after hatching, lateral view. Black and clear arrows indicate ventral and dorsal trunk expression domains, respectively. (B) Larva 2 h after hatching. Ventral expression in developing rectum (r). L, trunk lumen. Scale bar represents 20 ␮m in (A) and 50 ␮m in (B).

(Figs. 4 and 5A–5E). Early OdiT expression appeared to be OdiT, however, are in the position of A21/A21, left and restricted to cells of notochordal fate, as identified by right side progeny of blastomeres A2/A2, which Delsman comparison with Delsman’s fatemap (Delsman, 1910) and (1910) predicted would give rise to the notochord (Figs. 4A as deduced by the continuity of T expression in a develop- and 4B). At early neurula stage, 4 cells are detected by the mental series that included hatchlings with differentiated probe (Fig. 3E). Two are likely to be the original A21 pair, notochords. We interpret expression in a single cell adjacent while the second pair may be progeny of B21/B21. The B21 to the end of the 19 cells lined up in the notochord as a 20th pair are proposed to correspond approximately to ascidian late interdigitating notochord cell. This would agree with B6.2/B6.2 (Delsman, 1910; Galt and Fenaux, 1990). In ascid- ascidian development where interdigitation of notochord ians, progeny of these B-line (posterior, vegetal) blastomeres cells is not synchronous (Cloney, 1964) and notochord express As-T a cleavage later than do A-line (anterior, elongation is completed in the rostral end earlier than in the vegetal) notochord precursors (Yasuo and Satoh, 1994) and caudal end of the extending tail (Miyamoto and Crowther, produce the posteriormost notochord cells (Nishida, 1987; 1985; Corbo et al., 1997). This cell, however, remains Satoh, 1994). distinctive; Galt (1972) noted a 20th cell in O. dioica that is While the fatemaps of larvaceans and ascidians are likely histologically notochord-like but it remains physically to share similarities, the two classes of urochordates differ separate from the rest of the notochord. significantly in timing and pace of gastrulation. Delsman Our results support some of the observations made by observed that the gastrulation of O. dioica is precocious and Delsman in O. dioica embryos and his predictions of cell succinct; O. dioica begins gastrulating with the 5th cleav- fate which he based on comparison with ascidian embryos age (during the transition from 16 to 30 cells) and completes (Delsman, 1910). There is no modern cell-lineage map for it by the end of the 6th wave of cleavage resulting in 44 cells this or any larvacean. The first pair of cells expressing (Delsman, 1910; Galt and Fenaux, 1990; Figs. 4A–4D). OdiT

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. 330 Bassham and Postlethwait expression is first detected at the 30-cell stage, the early (Kusch and Reuter, 1999). The cells of the endodermal gastrula (Figs. 4A and 4B). As-T expression, in contrast, is strand, including subchordal cells, are the only cells de- first detected in 64-cell ascidian embryos at least one scribed as migratory in larvacean development (Delsman, cleavage before the onset of gastrulation which begins after 1912), and T may function to alter adhesion properties for 110 cells (Yasuo and Satoh, 1994). The onset of T expression the segregation and migration of these subchordal cells in these urochordates is apparently independent of the from the endodermal strand. timing of gastrulation. In ascidians, As-T is not detected in Expression of OdiT after hatching further supports the blastomeres until they become segregated to a purely noto- hypothesis that T’s function originally included a role in chordal fate (Yasuo and Satoh, 1994). Expression of T in patterning posterior endoderm. O. dioica hatches at 6 h larvaceans is probably also constrained by the timing of after fertilization (at 12°C) while organogenesis is still in segregation of cell fates with the result that, while it is progress in the trunk and there are no open lumens in the detected at an earlier cleavage stage than in ascidians, it gut or body cavity (Galt and Fenaux, 1990). Shortly after first appears during, rather than before, gastrulation. hatching, new expression of OdiT appears in the trunk While these results, taken alone, do not contradict the while detectable transcript rapidly disappears from the hypothesis that T’s role in the ancestral chordate was in notochord. The elongate cluster of expressing cells just notochord specification, rather than in broader mesodermal rostral to the notochord is likely the developing hindgut. In domains, OdiT expression in endoderm cells (discussed the latest stage of the hatchlings we examined, the borders below) supports the hypothesis that T also performed a of this expression domain had smoothed to a sausage shape function in endoderm development in the chordate ances- apposed to the epidermis where Delsman (1912) shows that tor. the anus forms (Fig. 6B). The identity of an expressing row of cells in the posteriodorsal trunk is less certain. Given its position in the dorsal trunk slightly left of the midline, we Larvacean T Is Expressed in Posterior Endoderm interpret it as the developing esophagus which curves left to Most oikopleurids have in the tail a characteristic cell feed dorsally into the left half of the two-lobed stomach. type that varies in number between species, O. dioica Alternatively, these posteriodorsal cells may be of the having two such “subchordal cells” (Lohmann, 1933). The developing left stomach lobe itself which later is distin- subchordal cells are named for their position ventral to the guished from the right lobe by a row of gland cells extending notochord; these cells appear on the animal’s right because along the left wall to the esophagus (Fenaux, 1998). the tail is twisted 90° relative to the trunk, and thus, caudal Expression of OdiT in the larvacean hindgut provides an right corresponds to ventral. Although their function is not interesting parallel to the expression of T in an entero- understood, the subchordal cells may have a secretory role pneust hemichordate, Ptychodera flava. PfBra (a T homolog (Fredriksson and Olsson, 1991), and Delsman (1912) de- in P. flava) is expressed in the anal region of the developing scribed them as a product of the caudal endoderm, the larval hindgut and in the intestine of the metamorphic endodermal strand. This one-cell wide strand runs ventral juvenile (Tagawa et al., 1998; Peterson et al., 1999). The to the notochord in a position similar to vertebrate hypo- hemichordate expression as well as similar expression of T chord cells (Kimmel et al., 1995) which express many of the in an embryonic seastar, however, appears to be in struc- same genes expressed in notochord (e.g., Yan et al., 1995). tures derived from invaginating ectoderm, rather than in As the caudal endoderm retracts into the trunk during the endodermally derived portion of the gut (Peterson et al., extension of the tail, the presumptive subchordal cells 1999; Shoguchi et al., 1999). The expression of T in the detach themselves from the endodermal strand and migrate hindgut of larvaceans and hemichordates, therefore, can not back into the tail (Delsman, 1912). Shortly after hatching, be homologized without violating the distinction between two cells to the right of the notochord are detected with the two embryonic germ layers. Peterson et al. (1999) infer a OdiT probe (Fig. 5F). Their position relative to the noto- broader homology of T expression in posterior gut between chord, their elongate shape, and the fact that they are seen the deuterostome phyla and a protostome outgroup (Ar- sometimes at the base of the tail or sometimes further thropoda). In their model for evolution of T utilization, the caudal suggest they are the two migrating subchordal cells ancestral role for T in bilaterians was to help form the later found in the tailfin of adult O. dioica. The putative posterior gut. In arthropods, the apparent T ortholog, byn, is subchordal cells apparently express T as they migrate. What expressed in the developing hindgut (Kispert et al., 1994). could be the role of T in these cells? In mouse, T is Kispert et al. (1994) note that it is somewhat arbitrary to necessary for migration of cells through the primitive define this part of the arthropod gut as ectodermal because streak (Wilson et al., 1995), and the authors propose that this label is based on epithelial properties shared with genes downstream of T mediate cell-surface changes neces- epidermis rather than on the hindgut’s internalized position sary for migration of cells away from the midline after after gastrulation. If one regards hindgut development in ingression. Similarly, in fly embryos mutant for the T these bilaterians as homologous, the larvacean pattern can homolog, byn (brachyenteron), caudal visceral mesodermal be reconciled with that described in outgroups to the cells are retarded in their ability to migrate, a deficiency the chordates (hemichordates, seastars, and insects). Therefore, authors attribute to a defect in the cells’ adhesive properties our data support the model that T had a role in posterior gut

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