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Ciliary band gene expression patterns in the embryo and trochophore larva of an indirectly developing polychaete
Gene Expression Patterns 7 (2007) 544–549 www.elsevier.com/locate/modgep
Ciliary band gene expression patterns in the embryo and trochophore larva of an indirectly developing polychaete Cesar Arenas-Mena ¤, Kimberly Suk-Ying Wong, Navid Arandi-Forosani Department of Biology, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-4614, USA Received 1 November 2006; received in revised form 25 January 2007; accepted 30 January 2007 Available online 6 February 2007
Abstract The trochophore larvae of indirectly developing spiralians have ciliary bands with motor and feeding functions. The preoral prototroch ciliary band is the Wrst diVerentiating organ in annelid and mollusk embryos. Here we report the expression of several ciliary band markers during embryogenesis and early larval stages of the indirectly developing polychaete Hydroides elegans. Genes with similarity to caveolin, -tubulin, -tubulin, and tektin are expressed in the eight primary prototroch precursors, 1q221 and 1q212. Blastomeres 1q221 and 1q212 locate at the same equatorial latitude after the complementary asymmetric division of their 1q22 and 1q21 precursors. In addition, caveolin and -tubulin are expressed in the metatroch and adoral ciliary zone. Caveolin is expressed in foregut ciliated cells, and -tubulin is expressed in apical tuft ciliated cells. The expression of a -thymosin homolog is restricted to 1q122 and 1q121 blastomeres, which locate just above and in close association with the eight primary prototroch cells 1q221 and 1q212. In addition, the -thymosin homolog has a transient expression in the hindgut and apical zone. The expression of all these genes provides a landmark for the early speciWcation of ciliary bands and other ciliated organs. © 2007 Elsevier B.V. All rights reserved. Keywords: Trochophore; Spiralian; Annelid; Embryo; Ciliary band; -Thymosin; Tubulin; Tektin; Caveolin; Polychaete; Serpulid; Lophotrochozoan; Bilaterian; Evolution; Prototroch; Metatroch; Neurotroch; Gastrotroch; Apical organ; Spiral cleavage; Equal cleavage; Trochoblast
1. Results and discussion Embryogenesis in multitude of indirectly developing bilaterians generates a free-swimming ciliated larva in charge of nourishing the formation of the adult. The signiWcance of ciliated larvae is at the core of centenary developmental-evolution discussions about bilaterian origins (Erwin and Davidson, 2002; Sly et al., 2003). Trochophores are planktotrophic cilated larvae of various spiralian groups (Nielsen, 2005). The embryogenesis of the polychaete annelid Hydroides elegans generates a trochophore in about 14 h. H. elegans has an equal-size spiral-cleaving embryo because the Wrst four blastomeres are of equal size. Nevertheless, the developmental potential among quadrant precursors (A, B, C and D)
*
Corresponding author. Tel.: +1 619 594 0717. E-mail address: [email protected] (C. Arenas-Mena).
1567-133X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2007.01.007
has not been tested in Hydroides. Orientative early cleavage diagrams based on observations in this and other equal-size cleaving polychaetes are currently available (Arenas-Mena, 2006), and the complete cleavage sequence has been recently characterized in H. elegans up to the 80-cell stage (our unpublished observations). The trochophore is endowed with one-cell thick ectoderm and endoderm epithelia and relatively simple mesodermal structures (Arenas-Mena, 2006; Hatschek, 1885; Shearer, 1911). The prototroch ciliary band derives from trochoblast precursors, which are the Wrst blastomeres that cease their proliferation and engage in diVerentiation; equivalent blastomeres also lead trochophore diVerentiation in the marine mollusk Patella vulgata (Damen and Dictus, 1994). Experimentally isolated trochoblast precursors divide a predetermined number of times and diVerentiate into ciliated cells (Wilson, 1904); this autonomous blastomere fate realization has been assumed to depend on the segregation of maternal determinants asymmetrically
C. Arenas-Mena et al. / Gene Expression Patterns 7 (2007) 544–549
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Fig. 1. Caveolin expression during embryogenesis. (a) Animal view of a 44-cell embryo. Scale bar for this and subsequent panels. (b) Animal view of a 64cell embryo. (c) Nuclear staining of the embryo in (b). (d) Serial section of the embryo in (c) at the level of 1q221 and 1q212. (e) Side view of a 64-cell embryo. (f) Nuclear staining of the embryo in (g) indicating also neighboring blastomeres. (g) Optical midsection of a 64-cell embryo. (h) Intermediate gastrula embryo. (i) Optical section at the level of the prototroch of a gastrulating embryo. Cells in the prospective foregut area start to express the gene, arrow. The ciliary band gap becomes evident, arrowhead. (j) Optical section of a late gastrula embryo with expression in cells posterior to the prototroch, i.e., prospective adoral ciliary zone and metatroch double arrowhead. (k) Lateral view of late gastrula embryo. Prospective adoral ciliary zone and metatroch indicated by double-head arrow. (l) Midsection of the embryo in (k) exhibits expression in the forming foregut. Arrowhead points to prospective gastrotroch. (m) Sagittal optical section of a 24-h trochophore exhibits the contrasted expression in the foregut. Arrowhead points to midgut-hindgut boundary. (n) Oral view of a trochophore larva. (o) Equatorial optical section at the prototroch level. Arrowhead points to dorsal gap. an, animal; at, apical tuft; acz, adoral ciliary zone; av, anal vesicle; bp, blastopore; c, cilia; fg, foregut; gt, gastrotroch; hg, hindgut; L, left; m, mouth; mg, midgut; mt, metatroch; o, oral; pt, prototroch; R, right.
distributed in a mosaic fashion (Wilson, 1904). Very little is known about trochoblast gene expression among trochozoans except for - and -tubulin genes, which are expressed during the early development of the marine mollusk Patella vulgata (Damen and Dictus, 1994). The ciliary band represents an early morphological landmark at the boundary of the animal-vegetal hemisphere and, therefore, serves as a
reference for other embryonic gene expression patterns. Here we report the ciliary band related expression of several genes in the indirectly developing polychaete H. elegans. Gene homologs of these ciliary band markers may have similar expression in other trochozoans, a group that includes mollusks, annelids, sipunculids, echiurids, kamptozoans and apparently nemerteans (Maslakova et al., 2004). In addition,
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C. Arenas-Mena et al. / Gene Expression Patterns 7 (2007) 544–549
Fig. 2. -Thymosin mRNA staining. (a) Animal view of 72-cell embryo. Scale bar for this and subsequent panels. (b) Nuclear staining of the embryo in a. (c) Animal view at the level of 1a1122 after its ingression. (d) Nuclear staining of the embryo in (c). (e) Lateral view when the expression is strong in 8 large cells just above the primary trochoblasts. (f) Animal view when the expression aVects 7 large cells. (g) Serial section of the embryo in (f) to indicate the orientation of the slit-shaped blastopore relative to the expression pattern. Oral side to the top deduced from serial sections not shown. (h) Detail of the ciliary band gap, arrowhead. (i) Optical section of a late gastrula embryo. (j) Lateral view of embryo just about to form its anus. (k) Optical section just above the prototroch. (l) Lateral view of a trochophore larva at the level of the prototroch. (m) Nuclear staining of the embryo in (l). (n) Frontal optical section of a 24-h trochophore. (o) Transversal optical section of a 24-h trochophore at the level of the foregut and hindgut to the bottom. (p) Sagittal optical section of a 24-h trochophore. an, animal; at, apical tuft; bp, blastopore; c, cilia; fg, foregut; hg, hindgut; L, left; m, mouth; mg, midgut; o, oral; pt, prototroch; R, right.
the characterization of some of these genes in echinoderm and hemichordate larvae may provide some input to bilaterian larval origin evolutionary scenarios. 1.1. Caveolin expression Caveolins are involved in endocytosis and signal transduction silencing (Krajewska and Maslowska, 2004;). In mice, caveolins have been associated with signal transduction silencing in sensory cilia of the olfactory epithelium (Kobayakawa et al., 2002; Schreiber et al., 2000). The H. elegans caveolin homolog isolated (Blast E value: 3e¡21) is detected in trochoblast precursors 1q221 and 1q212 after the
division of 1q12 into 1q121 and 1q122 (Fig. 1a–g). In this embryo, the quadrant (q) identity (a, b, c, d) is unknown until 2d2 divides asymmetrically in 2d22 and 2d21 in 60-cell stage embryos (our unpublished observations). The complementary asymmetric division of sister blastomeres 1q22 and 1q21 results in the equal latitudinal position of the four pairs 1q221–1q212 (Fig. 1d–f). Just above each 1q221–1q212 pair there is the corresponding quadrant 1q211 blastomere, and just underneath each 1q221–1q212 quadrant pair there is the corresponding 1q222 blastomere (Fig. 1e–f). The caveolin expressing primary trochoblast precursors locate at the approximate boundary of the animal and vegetal embryo hemispheres (Fig. 1g, arrowheads). Caveolin
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Fig. 3. Expression of -tubulin (a–c), -tubulin (d–h), and tektin (i–k). (a) Optical section at the level of the prototroch. Scale bar for this and subsequent panels. (b) Lateral view of mid-gastrula embryo. (c) Sagittal optical section of late gastrula embryo. (d) Equatorial view of early gastrula embryo just after the four primary trochoblast pairs join to form a circle. (e) Midsection of an embryo with approximately 64 cells, animal side to the top. (f) Detail of primary trochoblast expressing -tubulin and having cilia. (g) Mid-gastrula embryo. (h) Sagittal section of a 24-h trochophore larva. Expression remains longer in the oral side. (i) Early gastrula embryo. (j) Late gastrula embryo. (k) 24-h trochophore. at, apical tuft; bp, blastopore; c, cilia; fg, foregut; hg, hindgut; pn, protonephridium precursor.
expression is maintained in diVerentiating trochoblasts during gastrulation stages (Fig. 1h–l), but no expression is detected in the ciliary band gap located in the prospective dorsal side of the adult (Fig. 1i, arrowhead). The oral and aboral designations do not imply any homology with deuterostome larvae and provide more precise designations for the blastopore during gastrulation in this embryo; the dorsal–ventral designation overlaps with the oral–aboral designation but provides distinct nomenclature for the future dorsal side of the ectoderm (see summary Wgures). Cells located in prospective foregut areas at the oral side of
the invaginating blastopore also express the caveolin gene (Fig. 1i, arrow). Transient and low expression is detected in ciliated apical tuft cells (Fig. 1j). During late gastrulation stages, caveolin expression is detected in cells posterior to the prototroch Fig. 1j–l); these cells likely correspond to the adoral ciliary zone, metatroch and gastrotroch precursors (Nielsen, 2005). The emerging foregut also exhibits caveolin expression (Fig. 1l), which is continuous with ventral midline expression in likely gastrotroch precursors (Figs. 1l, arrowhead). Caveolin expression continues in prototroch, metatroch, adhoral ciliary zone, and gastrotroch
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Fig. 4. Gene expression summary. (a) From left to right. Drawing of the embryo in Fig. 1b. Animal cap view. Primary prototroch precursors indicated in green. Gastrula embryos in frontal and transversal views; prototroch indicated in green. Lateral view of a trochophore; prototroch indicated in green. Transcripts of -tubulin, and tektin are detected in primary prototroch precursors and the prototroch. (b) Summary of Caveolin expression pattern indicated in red. The expression of -tubulin is similar except that no -tubulin foregut expression is detected. (c) Summary of -thymosin expression. The transcripts are detected in the animal cap cells indicated in red and later in a ring of 7 cells tightly associated with the prototroch. In trochophore larvae the transcripts are also detected in the hindgut. an, animal; at, apical tuft; ab, aboral; acz, adoral ciliary zone; bp, blastopore; d, dorsal; fg, foregut; gt, gastrotroch; hg, hindgut; L, left; m, mouth; mg, midgut; mt, metatroch; o, oral; pt, prototroch; R, right; v, ventral.
ciliated cells of 24-h trochophores (Figs. 1m-o). No expression is detected in the dorsal gap of the prototroch (Fig. 1 o, arrowhead) and the dorsal gap of the metatroch (not shown). The ciliated foregut also expresses the caveolin gene, in sharp contrast to the midgut (Fig. 1m–o). 1.2. -Thymosin mRNA expression -Thymosin binds monomeric actin and inhibits its polymerization (HuV et al., 2001; Safer and Chowrashi, 1997). Several homologs are developmentally regulated during the formation of neural, mesodermal, and endodermal organs (Huang et al., 2005). The expression of a -thymosin homolog (Blast E value: 9e¡04) was characterized during the development of H. elegans. Expression is detected after the animal hemisphere reaches 38 cells (i.e., after the division of 1q112) in a circle that includes 1q1121, 1q1122, 1q211, 1q121, and
1q122 (Fig. 2a and b). Thus the apical quartet (1q111) and the primary trochoblasts (1q221–1q212) do not express the -thymosin gene (Fig. 2a). When the small 1q1122 blastomeres “sink” among neighboring blastomeres (Fig. 2c), the expression declines in 1q1121 and 1q121, i.e., it declines in the animal pole side of the expressing ring at the same time that expression is enhanced in 1q122 and 1q211 (Fig. 2c and d), which are located immediately above the primary trochoblasts (Fig. 2e). During gastrulation, the expression declines in one of the eight 1q122 or 1q121 blastomeres (Fig. 2f). The spiral cleavage relations are less evident during these gastrulation stages, but we suspect that the blastomere loosing the expression is 1d122 (Fig. 2f) because it was the one previously located by the prospective dorsal ciliary band gap (Fig. 1c). Eventually, the ciliary band gap becomes evident (Fig. 2h). It appears that cells squeeze through the gap towards subtrochal territories; a similar migration was described during
C. Arenas-Mena et al. / Gene Expression Patterns 7 (2007) 544–549
the embryological characterization of another polychaete that forms a feeding trochophore (Mead, 1897; Treadwell, 1901). Nevertheless, all our observations derive from Wxed specimens and we cannot conWrm these migrations in H. elegans. These putative migrating cells express Otx (our unpublished results) and perhaps contribute to a form a posterior sensory organ in the same location to that recently described in the trochophore of another polychaete (Nezlin and Voronezhskaya, 2003); we have not conWrmed with neural markers the existence of such posterior sensory organ in H. elegans. The expression decline and gap are slightly displaced to the right side (Fig. 2f, g and k), as expected if generated by 1d122 expression decline; 4 expressing cells will remain on the left side, but only 3 will remain on the right side (1d121, 1c122 and 1c121). The -thymosin expressing cells Xatten and tightly associate with the eight primary prototroch-forming cells, which are evenly distributed in two pairs between left and right sides (Fig. 2i and m). B-thymosin transcripts are transiently detected in the hindgut of 24-h trochophores (Fig. 2o and p). 1.3. -Tubulin, -tubulin, and tektin expression Tubulin proteins are expressed in the ciliary band of the Patella vulgata trochophore (Damen and Dictus, 1994) where they contribute to cilia microtubules. Tektins are constituents of cilia, Xagella, and centrioles (Norrander et al., 1996), and are expressed in sperm, sensory, and brain tissues (Norrander et al., 1998). Homologs of -tubulin (Blast E value: 2e¡92), -tubulin (Blast E value: 6e¡29), and tektin (Blast E value: 1e¡45) are expressed in ciliated cells during the embryogenesis of H. elegans (Fig. 3). The tubulin homolog is transiently expressed in apical tuft cells, adoral ciliary zone, and metatroch (Fig. 3a–c), with a proWle similar to that previously described for caveolin (Fig. 1), except that there is no persistent expression in the larval foregut. The expression of -tubulin (Fig. 3d–h) and tektin (Fig. 3i–k) is restricted to the prototroch where they seem to persist at high levels in trochophore stages (Fig. 3h and k). 2. Experimental procedures Whole Mount In Situ (WMISH) and other histological procedures where as previously described (Arenas-Mena, 2006; Arenas-Mena et al., 2006). Probes where derived from randomly sequenced EST clones corresponding that were deposited in GeneBank with the following accession numbers: -tubulin, EF079877; -tubulin, EF079878; -thymosin, EF079879; caveolin, EF079880; tektin, EF079881 (Fig. 4).
Acknowledgement We thank Michael G. HadWeld from the Kewalo Marine Laboratory, University of Hawaii at Manoa, and Eugenio de Jesús Carpizo-Ituarte from the Instituto de Investigaci-
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ones Oceanológicas, Universidad Autónoma de Baja California for providing the cDNA library from which EST clones were randomly obtained. We also thank Tracy J. Uveges for providing the EST sequences and Ashley M. Woods for her preliminary WMISH experiments. References Arenas-Mena, C., 2006. Embryonic expression of HeFoxA1 and HeFoxA2 in an indirectly developing polychaete. Genes Dev. Evol. 216, 727–736. Arenas-Mena, C., Cameron, R.A., Davidson, E.H., 2006. Hindut speciWcation and cell-adhesion functions of spHox11/13b in the endoderm of the sea urchin embryo. Dev. Growth DiVer. 48, 463–472. Damen, P., Dictus, W., 1994. Cell lineage of the prototroch of Patella vulgata (Gastropoda, Mollusca). Dev. Biol. 162, 364–383. Erwin, D.H., Davidson, E.H., 2002. The last common bilaterian ancestor. Development 129, 3021–3032. Hatschek, B., 1885. Entwicklung der trochophora von Eupomatus uncinatus. Arbt. Zool. Inst. Wien. vi, 121. Huang, X., Zhang, W., Zhang, H., 2005. Phylogenetic analysis and developmental expression of thymosin-beta 4 gene in amphioxus. Dev. Genes. Evol. 215, 364–368. HuV, T., Muller, C., Otto, A., Netzker, R., Hannappel, E., 2001. beta-Thymosins, small acidic peptides with multiple functions. Int. J. Biochem. Cell Biol. 33, 205–220. Kobayakawa, K., Hayashi, R., Morita, K., Miyamichi, K., Oka, Y., Tsuboi, A., Sakano, H., 2002. Stomatin-related olfactory protein, SRO, speciWcally expressed in the murine olfactory sensory neurons. J. Neurosci. 22, 5931–5937. Krajewska, W., Maslowska, I., 2004. Caveolins: structure and function in signal transduction. Cell Mol. Biol. Lett. 9, 195–220. Maslakova, S., Martindale, M.Q., Norenburg, J., 2004. Vestigial prototroch in a basal nemertean, Carinoma tremaphoros (Nemertea; Palaeonemertea). Evol. Dev. 6, 219–226. Mead, A.D., 1897. The early development of marine annelids. J. Morph. 13, 229–326. Nezlin, L.P., Voronezhskaya, E.E., 2003. Novel, posterior sensory organ in the trochophore larva of Phyllodoce maculata (Polychaeta). Proc. R. Soc. B: Biol. Sci. 270, S159–S162. Nielsen, C., 2005. Trochophora larvae: cell-lineages, ciliary bands and body regions. 2. Other groups and general discussion. J. Exp. Zool. B: Mol. Dev. Evol. 304B, 401–447. Norrander, J., Larsson, M., Stahl, S., Hoog, C., Linck, R., 1998. Expression of ciliary tektins in brain and sensory development. J. Neurosci. 18, 8912–8918. Norrander, J.M., Perrone, C.A., Amos, L.A., Linck, R.W., 1996. Structural comparison of tektins and evidence for their determination of complex spacings in Xagellar microtubules. J. Mol. Biol. 257, 385–397. Safer, D., Chowrashi, P.K., 1997. b-thymosins from marine invertebrates: primary structure and interaction with actin. Cell Motil. Cytoskeleton 38, 163–171. Schreiber, S., Fleischer, J., Breer, H., BoekhoV, I., 2000. A possible role for caveolin as a signaling organizer in olfactory sensory membranes. J. Biol. Chem. 275, 24115–24123. Shearer, C.M.A., 1911. On the development and structure of the trochophore of Hydroides (Eupomatus) uncinatus. Quart. J. micr. Sci. 56, 543–590. Sly, B.J., Snoke, M.S., RaV, R.A., 2003. Who came Wrst larvae or adults? origins of bilaterian metazoan larvae. Int. J. Dev. Biol. 47, 623–632. Treadwell, A.L., 1901. The cytogeny of Podarke obscura. J. Morph. 17, 399–486. Wilson, E.B., 1904. Experimental studies in germinal localization: II. experiments on the cleavage-mosaic in patella and dentalium. J. Exp. Zool. 1, 197–268.
Ciliary band gene expression patterns in the embryo and trochophore larva of an indirectly developing polychaete Cesar Arenas-Mena ¤, Kimberly Suk-Ying Wong, Navid Arandi-Forosani Department of Biology, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-4614, USA Received 1 November 2006; received in revised form 25 January 2007; accepted 30 January 2007 Available online 6 February 2007
Abstract The trochophore larvae of indirectly developing spiralians have ciliary bands with motor and feeding functions. The preoral prototroch ciliary band is the Wrst diVerentiating organ in annelid and mollusk embryos. Here we report the expression of several ciliary band markers during embryogenesis and early larval stages of the indirectly developing polychaete Hydroides elegans. Genes with similarity to caveolin, -tubulin, -tubulin, and tektin are expressed in the eight primary prototroch precursors, 1q221 and 1q212. Blastomeres 1q221 and 1q212 locate at the same equatorial latitude after the complementary asymmetric division of their 1q22 and 1q21 precursors. In addition, caveolin and -tubulin are expressed in the metatroch and adoral ciliary zone. Caveolin is expressed in foregut ciliated cells, and -tubulin is expressed in apical tuft ciliated cells. The expression of a -thymosin homolog is restricted to 1q122 and 1q121 blastomeres, which locate just above and in close association with the eight primary prototroch cells 1q221 and 1q212. In addition, the -thymosin homolog has a transient expression in the hindgut and apical zone. The expression of all these genes provides a landmark for the early speciWcation of ciliary bands and other ciliated organs. © 2007 Elsevier B.V. All rights reserved. Keywords: Trochophore; Spiralian; Annelid; Embryo; Ciliary band; -Thymosin; Tubulin; Tektin; Caveolin; Polychaete; Serpulid; Lophotrochozoan; Bilaterian; Evolution; Prototroch; Metatroch; Neurotroch; Gastrotroch; Apical organ; Spiral cleavage; Equal cleavage; Trochoblast
1. Results and discussion Embryogenesis in multitude of indirectly developing bilaterians generates a free-swimming ciliated larva in charge of nourishing the formation of the adult. The signiWcance of ciliated larvae is at the core of centenary developmental-evolution discussions about bilaterian origins (Erwin and Davidson, 2002; Sly et al., 2003). Trochophores are planktotrophic cilated larvae of various spiralian groups (Nielsen, 2005). The embryogenesis of the polychaete annelid Hydroides elegans generates a trochophore in about 14 h. H. elegans has an equal-size spiral-cleaving embryo because the Wrst four blastomeres are of equal size. Nevertheless, the developmental potential among quadrant precursors (A, B, C and D)
*
Corresponding author. Tel.: +1 619 594 0717. E-mail address: [email protected] (C. Arenas-Mena).
1567-133X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2007.01.007
has not been tested in Hydroides. Orientative early cleavage diagrams based on observations in this and other equal-size cleaving polychaetes are currently available (Arenas-Mena, 2006), and the complete cleavage sequence has been recently characterized in H. elegans up to the 80-cell stage (our unpublished observations). The trochophore is endowed with one-cell thick ectoderm and endoderm epithelia and relatively simple mesodermal structures (Arenas-Mena, 2006; Hatschek, 1885; Shearer, 1911). The prototroch ciliary band derives from trochoblast precursors, which are the Wrst blastomeres that cease their proliferation and engage in diVerentiation; equivalent blastomeres also lead trochophore diVerentiation in the marine mollusk Patella vulgata (Damen and Dictus, 1994). Experimentally isolated trochoblast precursors divide a predetermined number of times and diVerentiate into ciliated cells (Wilson, 1904); this autonomous blastomere fate realization has been assumed to depend on the segregation of maternal determinants asymmetrically
C. Arenas-Mena et al. / Gene Expression Patterns 7 (2007) 544–549
545
Fig. 1. Caveolin expression during embryogenesis. (a) Animal view of a 44-cell embryo. Scale bar for this and subsequent panels. (b) Animal view of a 64cell embryo. (c) Nuclear staining of the embryo in (b). (d) Serial section of the embryo in (c) at the level of 1q221 and 1q212. (e) Side view of a 64-cell embryo. (f) Nuclear staining of the embryo in (g) indicating also neighboring blastomeres. (g) Optical midsection of a 64-cell embryo. (h) Intermediate gastrula embryo. (i) Optical section at the level of the prototroch of a gastrulating embryo. Cells in the prospective foregut area start to express the gene, arrow. The ciliary band gap becomes evident, arrowhead. (j) Optical section of a late gastrula embryo with expression in cells posterior to the prototroch, i.e., prospective adoral ciliary zone and metatroch double arrowhead. (k) Lateral view of late gastrula embryo. Prospective adoral ciliary zone and metatroch indicated by double-head arrow. (l) Midsection of the embryo in (k) exhibits expression in the forming foregut. Arrowhead points to prospective gastrotroch. (m) Sagittal optical section of a 24-h trochophore exhibits the contrasted expression in the foregut. Arrowhead points to midgut-hindgut boundary. (n) Oral view of a trochophore larva. (o) Equatorial optical section at the prototroch level. Arrowhead points to dorsal gap. an, animal; at, apical tuft; acz, adoral ciliary zone; av, anal vesicle; bp, blastopore; c, cilia; fg, foregut; gt, gastrotroch; hg, hindgut; L, left; m, mouth; mg, midgut; mt, metatroch; o, oral; pt, prototroch; R, right.
distributed in a mosaic fashion (Wilson, 1904). Very little is known about trochoblast gene expression among trochozoans except for - and -tubulin genes, which are expressed during the early development of the marine mollusk Patella vulgata (Damen and Dictus, 1994). The ciliary band represents an early morphological landmark at the boundary of the animal-vegetal hemisphere and, therefore, serves as a
reference for other embryonic gene expression patterns. Here we report the ciliary band related expression of several genes in the indirectly developing polychaete H. elegans. Gene homologs of these ciliary band markers may have similar expression in other trochozoans, a group that includes mollusks, annelids, sipunculids, echiurids, kamptozoans and apparently nemerteans (Maslakova et al., 2004). In addition,
546
C. Arenas-Mena et al. / Gene Expression Patterns 7 (2007) 544–549
Fig. 2. -Thymosin mRNA staining. (a) Animal view of 72-cell embryo. Scale bar for this and subsequent panels. (b) Nuclear staining of the embryo in a. (c) Animal view at the level of 1a1122 after its ingression. (d) Nuclear staining of the embryo in (c). (e) Lateral view when the expression is strong in 8 large cells just above the primary trochoblasts. (f) Animal view when the expression aVects 7 large cells. (g) Serial section of the embryo in (f) to indicate the orientation of the slit-shaped blastopore relative to the expression pattern. Oral side to the top deduced from serial sections not shown. (h) Detail of the ciliary band gap, arrowhead. (i) Optical section of a late gastrula embryo. (j) Lateral view of embryo just about to form its anus. (k) Optical section just above the prototroch. (l) Lateral view of a trochophore larva at the level of the prototroch. (m) Nuclear staining of the embryo in (l). (n) Frontal optical section of a 24-h trochophore. (o) Transversal optical section of a 24-h trochophore at the level of the foregut and hindgut to the bottom. (p) Sagittal optical section of a 24-h trochophore. an, animal; at, apical tuft; bp, blastopore; c, cilia; fg, foregut; hg, hindgut; L, left; m, mouth; mg, midgut; o, oral; pt, prototroch; R, right.
the characterization of some of these genes in echinoderm and hemichordate larvae may provide some input to bilaterian larval origin evolutionary scenarios. 1.1. Caveolin expression Caveolins are involved in endocytosis and signal transduction silencing (Krajewska and Maslowska, 2004;). In mice, caveolins have been associated with signal transduction silencing in sensory cilia of the olfactory epithelium (Kobayakawa et al., 2002; Schreiber et al., 2000). The H. elegans caveolin homolog isolated (Blast E value: 3e¡21) is detected in trochoblast precursors 1q221 and 1q212 after the
division of 1q12 into 1q121 and 1q122 (Fig. 1a–g). In this embryo, the quadrant (q) identity (a, b, c, d) is unknown until 2d2 divides asymmetrically in 2d22 and 2d21 in 60-cell stage embryos (our unpublished observations). The complementary asymmetric division of sister blastomeres 1q22 and 1q21 results in the equal latitudinal position of the four pairs 1q221–1q212 (Fig. 1d–f). Just above each 1q221–1q212 pair there is the corresponding quadrant 1q211 blastomere, and just underneath each 1q221–1q212 quadrant pair there is the corresponding 1q222 blastomere (Fig. 1e–f). The caveolin expressing primary trochoblast precursors locate at the approximate boundary of the animal and vegetal embryo hemispheres (Fig. 1g, arrowheads). Caveolin
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Fig. 3. Expression of -tubulin (a–c), -tubulin (d–h), and tektin (i–k). (a) Optical section at the level of the prototroch. Scale bar for this and subsequent panels. (b) Lateral view of mid-gastrula embryo. (c) Sagittal optical section of late gastrula embryo. (d) Equatorial view of early gastrula embryo just after the four primary trochoblast pairs join to form a circle. (e) Midsection of an embryo with approximately 64 cells, animal side to the top. (f) Detail of primary trochoblast expressing -tubulin and having cilia. (g) Mid-gastrula embryo. (h) Sagittal section of a 24-h trochophore larva. Expression remains longer in the oral side. (i) Early gastrula embryo. (j) Late gastrula embryo. (k) 24-h trochophore. at, apical tuft; bp, blastopore; c, cilia; fg, foregut; hg, hindgut; pn, protonephridium precursor.
expression is maintained in diVerentiating trochoblasts during gastrulation stages (Fig. 1h–l), but no expression is detected in the ciliary band gap located in the prospective dorsal side of the adult (Fig. 1i, arrowhead). The oral and aboral designations do not imply any homology with deuterostome larvae and provide more precise designations for the blastopore during gastrulation in this embryo; the dorsal–ventral designation overlaps with the oral–aboral designation but provides distinct nomenclature for the future dorsal side of the ectoderm (see summary Wgures). Cells located in prospective foregut areas at the oral side of
the invaginating blastopore also express the caveolin gene (Fig. 1i, arrow). Transient and low expression is detected in ciliated apical tuft cells (Fig. 1j). During late gastrulation stages, caveolin expression is detected in cells posterior to the prototroch Fig. 1j–l); these cells likely correspond to the adoral ciliary zone, metatroch and gastrotroch precursors (Nielsen, 2005). The emerging foregut also exhibits caveolin expression (Fig. 1l), which is continuous with ventral midline expression in likely gastrotroch precursors (Figs. 1l, arrowhead). Caveolin expression continues in prototroch, metatroch, adhoral ciliary zone, and gastrotroch
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Fig. 4. Gene expression summary. (a) From left to right. Drawing of the embryo in Fig. 1b. Animal cap view. Primary prototroch precursors indicated in green. Gastrula embryos in frontal and transversal views; prototroch indicated in green. Lateral view of a trochophore; prototroch indicated in green. Transcripts of -tubulin, and tektin are detected in primary prototroch precursors and the prototroch. (b) Summary of Caveolin expression pattern indicated in red. The expression of -tubulin is similar except that no -tubulin foregut expression is detected. (c) Summary of -thymosin expression. The transcripts are detected in the animal cap cells indicated in red and later in a ring of 7 cells tightly associated with the prototroch. In trochophore larvae the transcripts are also detected in the hindgut. an, animal; at, apical tuft; ab, aboral; acz, adoral ciliary zone; bp, blastopore; d, dorsal; fg, foregut; gt, gastrotroch; hg, hindgut; L, left; m, mouth; mg, midgut; mt, metatroch; o, oral; pt, prototroch; R, right; v, ventral.
ciliated cells of 24-h trochophores (Figs. 1m-o). No expression is detected in the dorsal gap of the prototroch (Fig. 1 o, arrowhead) and the dorsal gap of the metatroch (not shown). The ciliated foregut also expresses the caveolin gene, in sharp contrast to the midgut (Fig. 1m–o). 1.2. -Thymosin mRNA expression -Thymosin binds monomeric actin and inhibits its polymerization (HuV et al., 2001; Safer and Chowrashi, 1997). Several homologs are developmentally regulated during the formation of neural, mesodermal, and endodermal organs (Huang et al., 2005). The expression of a -thymosin homolog (Blast E value: 9e¡04) was characterized during the development of H. elegans. Expression is detected after the animal hemisphere reaches 38 cells (i.e., after the division of 1q112) in a circle that includes 1q1121, 1q1122, 1q211, 1q121, and
1q122 (Fig. 2a and b). Thus the apical quartet (1q111) and the primary trochoblasts (1q221–1q212) do not express the -thymosin gene (Fig. 2a). When the small 1q1122 blastomeres “sink” among neighboring blastomeres (Fig. 2c), the expression declines in 1q1121 and 1q121, i.e., it declines in the animal pole side of the expressing ring at the same time that expression is enhanced in 1q122 and 1q211 (Fig. 2c and d), which are located immediately above the primary trochoblasts (Fig. 2e). During gastrulation, the expression declines in one of the eight 1q122 or 1q121 blastomeres (Fig. 2f). The spiral cleavage relations are less evident during these gastrulation stages, but we suspect that the blastomere loosing the expression is 1d122 (Fig. 2f) because it was the one previously located by the prospective dorsal ciliary band gap (Fig. 1c). Eventually, the ciliary band gap becomes evident (Fig. 2h). It appears that cells squeeze through the gap towards subtrochal territories; a similar migration was described during
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the embryological characterization of another polychaete that forms a feeding trochophore (Mead, 1897; Treadwell, 1901). Nevertheless, all our observations derive from Wxed specimens and we cannot conWrm these migrations in H. elegans. These putative migrating cells express Otx (our unpublished results) and perhaps contribute to a form a posterior sensory organ in the same location to that recently described in the trochophore of another polychaete (Nezlin and Voronezhskaya, 2003); we have not conWrmed with neural markers the existence of such posterior sensory organ in H. elegans. The expression decline and gap are slightly displaced to the right side (Fig. 2f, g and k), as expected if generated by 1d122 expression decline; 4 expressing cells will remain on the left side, but only 3 will remain on the right side (1d121, 1c122 and 1c121). The -thymosin expressing cells Xatten and tightly associate with the eight primary prototroch-forming cells, which are evenly distributed in two pairs between left and right sides (Fig. 2i and m). B-thymosin transcripts are transiently detected in the hindgut of 24-h trochophores (Fig. 2o and p). 1.3. -Tubulin, -tubulin, and tektin expression Tubulin proteins are expressed in the ciliary band of the Patella vulgata trochophore (Damen and Dictus, 1994) where they contribute to cilia microtubules. Tektins are constituents of cilia, Xagella, and centrioles (Norrander et al., 1996), and are expressed in sperm, sensory, and brain tissues (Norrander et al., 1998). Homologs of -tubulin (Blast E value: 2e¡92), -tubulin (Blast E value: 6e¡29), and tektin (Blast E value: 1e¡45) are expressed in ciliated cells during the embryogenesis of H. elegans (Fig. 3). The tubulin homolog is transiently expressed in apical tuft cells, adoral ciliary zone, and metatroch (Fig. 3a–c), with a proWle similar to that previously described for caveolin (Fig. 1), except that there is no persistent expression in the larval foregut. The expression of -tubulin (Fig. 3d–h) and tektin (Fig. 3i–k) is restricted to the prototroch where they seem to persist at high levels in trochophore stages (Fig. 3h and k). 2. Experimental procedures Whole Mount In Situ (WMISH) and other histological procedures where as previously described (Arenas-Mena, 2006; Arenas-Mena et al., 2006). Probes where derived from randomly sequenced EST clones corresponding that were deposited in GeneBank with the following accession numbers: -tubulin, EF079877; -tubulin, EF079878; -thymosin, EF079879; caveolin, EF079880; tektin, EF079881 (Fig. 4).
Acknowledgement We thank Michael G. HadWeld from the Kewalo Marine Laboratory, University of Hawaii at Manoa, and Eugenio de Jesús Carpizo-Ituarte from the Instituto de Investigaci-
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