Molecular patterning during the development of Phoronopsis harmeri reveals similarities to rhynchonelliform brachiopods

Abstract

Background: Phoronids, rhynchonelliform and linguliform brachiopods show striking similarities in their embryonic fate maps, in particular in their axis specification and regionalization. However, although brachiopod development has been studied in detail and demonstrated embryonic patterning as a causal factor of the gastrulation mode (protostomy vs deuterostomy), molecular descriptions are still missing in phoronids. To understand whether phoronids display underlying embryonic molecular mechanisms similar to those of brachiopods, here we report the expression patterns of anterior (otx, gsc, six3/6, nk2.1), posterior (cdx, bra) and endomesodermal (foxA, gata4/5/6, twist) markers during the development of the protostomic phoronid Phoronopsis harmeri.

Results: The transcription factors foxA, gata4/5/6 and cdx show conserved expression in patterning the development and regionalization of the phoronid embryonic gut, with foxA expressed in the presumptive foregut, gata4/5/6 demarcating the midgut and cdx confined to the hindgut. Furthermore, six3/6, usually a well-conserved anterior marker, shows a remarkably dynamic expression, demarcating not only the apical organ and the oral ectoderm, but also clusters of cells of the developing midgut and the anterior mesoderm, similar to what has been reported for brachiopods, bryozoans and some deuterostome Bilateria. Surprisingly, brachyury, a transcription factor often associated with gastrulation movements and mouth and hindgut development, seems not to be involved with these patterning events in phoronids.

Conclusions: Our description and comparison of gene expression patterns with other studied Bilateria reveals that the timing of axis determination and cell fate distribution of the phoronid shows highest similarity to that of rhynchonelliform brachiopods, which is likely related to their shared protostomic mode of development. Despite these similarities, the phoronid Ph. harmeri also shows particularities in its development, which hint to divergences in the arrangement of gene regulatory networks responsible for germ layer formation and axis specification.

Keywords: Embryogenesis; Evolution; Gene expression; Lophophorates; Phoronid.

Fig. 1

Gross morphology and phylogenetic position of Phoronopsis harmeri. a Phylogenetic position of phoronids and Phoronopsis harmeri [11]. b Characteristic actinotroch larva with 12 tentacles. c Anterior region of Phoronopsis harmeri with a terminal anterior lophophore, used for collection of food particles and respiration, and a posterior trunk. d Ampulla of a mature female animal with visible oocytes. Anterior is to the top

Fig. 2

The embryonic development of Ph. harmeri. Nomarski images of living embryos of Ph. harmeri at representative stages of development: cleavage (a–e), blastula (f–g), gastrula (h–i) and larva (j–k). The egg undergoes its first radial holoblastic cleavage at 2 hpf (a) and forms a hatching blastula around 6–10 hpf (f). Gastrulation starts at 20 hpf (h) at the vegetal pole of the embryo and results in the flattening of the vegetal surface. At late gastrula stage (30 hpf) (i), the apical organ shifts anteriorly, the archenteron elongates posteriorly and the anterior–posterior axis becomes oblique. At early larva stage (40 hpf) (j), the embryo begins to elongate along the anterior–posterior axis and the blastopore becomes the mouth of the future larva. A thick tissue is formed at the dorsal ectoderm and around the mouth that will form the future pre-oral lobe. The bilateral symmetry is evident. The pre-tentacle actinotroch larva is formed around 60 hpf (k), with a prominent pre-oral lobe, a fully compartmentalized, functional gut and evident tentacle bulbs. h–k depict embryos in lateral view and h′–k′ show embryos in vegetal view. Insets show different focal planes of the embryos. In all panels, anterior is to the left

Fig. 3

Immunohistochemistry on blastula (a), gastrula (b–c) and larva stages (d–f) of Ph. harmeri. Immunohistochemistry on blastula, gastrula and larva stages labeled against acetylated tubulin (gray) and DAPI (blue). b–f Depict embryos in lateral view (lv) and b′–f′ show embryos in vegetal view (vv). In panels depicting gastrulae and larvae stages, anterior is to the left. am, anterior mesoderm; an, anus; ar, archenteron; at, apical organ; bc, blastocoel; bp, blastopore; es, esophagus; in, intestine; mo, mouth; ne, nephridium; np, nephridial primordium; pl, pre-oral lobe; pm, posterior mesoderm; st, stomach; tb, tentacle bulb; te, tentacle; tr, tentacular ridge; tt, telotroch; vp, vegetal plate. Scale bar: 25 µm

Fig. 4

Expression of endomesodermal, anterior and posterior markers during the embryonic development of Ph. harmeri. WMISH of otx, gsc, six3/6, nk2.1, cdx, bra, foxA, gata4/5/6 and twist in blastula, early gastrulae, late gastrulae, early larvae, pre-tentacle larvae and 6-tentacle larvae of Ph. harmeri. The panels of the first columns (a–bbb) depict embryos in lateral view and the panels of the second columns show embryos in vegetal view (a′–bbb′). Insets in x′, cc′, jj–jj′, nn′, pp and aaa–aaa′ show different focal planes of the embryos. Black arrow indicates the ectodermal expression of otx at the domain that gives rise to the apical organ. The inset in zz′ and pp′ shows different focal planes and higher magnification of the indicated domains. The row below the matrix depicts enlarged images of the insets in x′, cc′, jj–jj′, nn′, pp–pp′, zz′ and aaa–aaa′. In panels depicting gastrulae and larvae stages, anterior is to the left

Fig. 5

Co-expression analysis of marker genes by double fluorescent WMISH during the development of Ph. harmeri. Relative spatial expression of otx and foxA (a), otx and gsc (b, f, l), gata4/5/6 and foxA (c), foxA and twist (d), six3/6 and twist (e, h), cdx and nk2.1 (g), bra and nk2.1 (i), bra and foxA (j) and six3/6 and otx (k). Right insets in d, e, k show embryos in vegetal view. Every picture is a full projection of merged confocal stacks. Nuclei are stained blue with DAPI. Anterior is to the left

Fig. 6

Summary of gene expression during Ph. harmeri embryonic development. Schematic representation of the expression patterns of endomesodermal, anterior and posterior markers during embryonic development of Ph. harmeri. a The endodermal genes foxA and gata4/5/6 are expressed in the vegetal plate in blastula and later on are patterning the formation of the archenteron. FoxA is eventually confined in the foregut, whilst gata4/5/6 is expressed in the midgut. The mesodermal marker twist is labeling the anterior and posterior mesoderm and its derivatives. b The anterior gene six3/6 is expressed in the animal pole in blastula and at the gastrula stage is also activated in the anterior mesoderm and clusters of cells of the future midgut. At the early, pre-tentacle and six-tentacle larva stages six3/6 is restricted in the apical organ, anterior mesoderm and the oral ectoderm. Otx is expressed broadly at the blastula stage, and in gastrula it labels the anterior lip of the blastopore, adjacent to the expression of nk2.1 and gsc. At the gastrula stage, otx, nk2.1 and gsc are labeling the anterior–ventral ectoderm. Otx is also expressed in the future apical organ and the future midgut, and nk2.1 is additionally labeling the future hindgut. Later on, otx and nk2.1 are marking the ventral ectoderm of the pre-oral lobe. Otx together with gsc are demarcating the mouth. Additionally, otx labels the apical organ and nk2.1 is expressed strongly in the intestine and in the cardiac sphincter. c The posterior markers bra and cdx are expressed in the posterior lip of the blastopore at the gastrula stage. Bra is expressed in the ventral midgut, the ventral ectoderm and the posterior ciliary band, whilst cdx is confined in the intestine. At the six-tentacle larva stage, bra is activated in the intestine, the stomach diverticulum and the ventral ectoderm. The depicted expression patterns are for guidance and not necessarily represent exact expression domains. Drawings are not to scale. LV, lateral view; VV, vegetal view

Fig. 7

Comparison of embryonic gene expression patterns in representative developmental stages of Novocrania anomala, Terebratalia transversa and Phoronopsis harmeri. Schematic representation of the expression patterns of endomesodermal, anterior and posterior markers during gastrulation, axial elongation and larva formation of two members of Brachiopoda (N. anomala and T. transversa) and one member of Phoronida (Ph. harmeri). The asterisk is indicating the anterior domain. abl, anterior blastoporal lip; an, anus; bp, blastopore; mo, mouth; pbl, posterior blastoporal lip; vp, vegetal plate