Embryonic Development in the Primitive Bilaterian Neochildia Fusca: Normal Morphogenesis and Isolation of POU Genes Brn-1 and Brn-3

Dev Genes Evol (2002) 212:55Ð69 DOI 10.1007/s00427-001-0207-y

ORIGINAL ARTICLE

Nallur B. Ramachandra á Ruth D. Gates Peter Ladurner á David K. Jacobs Volker Hartenstein Embryonic development in the primitive bilaterian Neochildia fusca: normal morphogenesis and isolation of POU genes Brn-1 and Brn-3

Received: 16 October 2001 / Accepted: 26 November 2001 / Published online: 21 February 2002 © Springer-Verlag 2002

Abstract Neochildia fusca is a member of the taxon thin layer underneath the epidermis. In late embryos Acoela, a group of flatworms that, according to some and juveniles of Neochildia, the brain is formed by a recent molecular phylogenetic analyses, are distinct from 3Ð4 cell-diameter-thick layer of neurons forming a cor- other flatworms and constitute a basal branch with a tex surrounding a neuropile that is relatively free of cell sister taxon relationship to the rest of the Bilateria. In bodies. A highly regular “orthogonal” array of muscle fi- this paper, we analyze early neural development in this bers penetrates the brain. We have isolated and partially species and report the sequence and expression of two sequenced homologs of the vertebrate Brn-1 and Brn-3 Pit-Oct-Unc (POU) genes, NeocBrn-1 and NeocBrn-3. genes, which we call NeocBrn-1 and NeocBrn-3, respec- Homologs of these highly conserved genes play a role in tively. These sequences contain and span portions of the neural fate determination in vertebrates, Drosophila and POU-specific domain and a homeodomain, and are se- Caenorhabditis elegans. Acoels, including Neochildia, quence similar to their respective homologs in verte- have a unique invariant pattern of early cleavage called brates and Drosophila. RT-PCR reveals that NeocBrn-1 duet spiral cleavage. In subsequent cell divisions descen- and NeocBrn-3 are expressed from mid-embryonic to dants of the first three micromere duets form an outer adult stages. Whole-mount in situ hybridization shows layer of epidermal and neural progenitors surrounding expression of both genes in distinct subsets of nerve cells the meso/endoderm progenitors, which are themselves in juvenile and adult worms. NeocBrn-1 also appears in a descended from the macromere duet 4A, B and the subset of intra-epidermal gland cells. These observations micromere duet 4a, b. Organ formation begins at mid- are an initial step towards reconstructing the neural de- embryonic stages with the epidermal primordium adopt- velopment of a key group of bilaterians, the Acoela. ing a ciliated epithelial shape. Sub-epidermally, a bilater- These flatworms, by virtue of their distinct morphology, ally symmetric brain primordium can be seen at the ante- development and phylogenetically basal placement, are rior pole. Laterally and posteriorly, myoblasts form a likely to provide key insights into the interpretation of the evolution of metazoan neural architecture. Edited by J. Campos-Ortega Keywords Acoel á Neochildia á Nervous system á N.B. Ramachandra á V. Hartenstein (✉) Development á POU gene Department of Molecular, Cell, and Developmental Biology, University of California at Los Angeles, 621 Charles E. Young Drive South, Los Angeles, CA 90095Ð1606, USA Introduction e-mail: [email protected] Tel.: +1-310-2067523, Fax: +1-310-2063987 Genes with developmental functions are remarkably con- R.D. Gates á D.K. Jacobs served across a wide range of animals, a feature that has Department of Organismic Biology, Ecology and Evolution, proven broadly useful in comparing developmental sys- University of California at Los Angeles, 621 Charles E. Young Drive South, Los Angeles, tems in distantly related taxa. This, in turn, has renewed CA 90095Ð1606, USA interest in comparative embryology as a basis for discus- P. Ladurner sion of homologies between cells, tissues, and organs, Institut of Zoology and Limnology, University of Innsbruck, and as a means of reconstructing the evolution of devel- Technikerstrasse 25, A-6020, Innsbruck, Austria opmental systems. Here, we focus on generating a better Present address: understanding of neural development in acoel flatworms, N.B. Ramachandra, Department of Zoology, a group that appears to have a primitive form of neural University of Mysore, Manasagangotri, Mysore 570006, India development relative to other Bilateria. 56 To reconstruct the evolution of specific molecular net- terminal region of 75Ð82 amino acids (POU-specific do- works and morphogenetic events in neurogenesis re- main) and a similarly conserved carboxyl-terminal region quires a detailed knowledge of neural development in of 60 amino acids (POU homeodomain). These conserved taxa that span the phylogenetic tree. In this context, domains are separated by a less conserved linker region. members of the phylum Platyhelminthes (flatworms) are Based on the sequences of the linker region and the main of special interest, as flatworms have often been recon- basic cluster at the amino terminus of the POU homeodo- structed as basal in bilaterian phylogeny and may have main, the POU-domain family can be divided into six retained primitive features relative to their more evolved classes, IÐVI (Wegner et al. 1993). bilaterian relatives (Hyman 1940; Ehlers 1985; Ax The role of the POU genes in ontogenetic develop- 1996). Potentially primitive or ancestral features include ment and cellular differentiation has been investigated in a simple gut with a single opening, an epidermal layer of a variety of organisms, including vertebrates and Dro- multi-ciliated cells that form the main organ of locomo- sophila. Many members of the group, in particular those tion, and a compact anterior brain. In addition, platyhel- belonging to classes III and IV, are expressed in specific minths represent the simplest group of animals possess- subsets of neurons within embryonic and adult neural ing a central nervous system. tissues and have been shown to play important roles in Recent analyses of 18 s rDNA have placed the flat- the specification of these neuronal cell types (reviewed worms in alliance with spiralian phyla such as molluscs in Ryan and Rosenfeld 1997). In this paper, we report the and annelids, as well as lophophorates, in the clade cloning and expression of two POU domain-containing Lophotrochozoa within the protostome Bilateria (Adoutte genes, Brn-1 and Brn-3, in the acoel Neochildia. Both et al. 1999; Ruiz-Trillo et al. 1999). In addition Acoela, a genes show a high degree of sequence similarity with taxon classically assigned to the platyhelminths, may their vertebrate and Drosophila counterparts, and are ex- have branched off the metazoan tree earlier than other pressed in a subset of nerve cells in the brain. In order to flatworms; thus Acoela potentially represent a sister interpret the expression pattern of the POU genes, and group to higher bilaterians (“eubilaterians”). Both the other developmentally relevant genes, we have analyzed placements of non-acoel flatworms within the Loph- the normal development of Neochildia embryos using a otrochozoa (Giribet et al. 2000) and the concept that the combination of live observations, whole-mount prepara- Acoela are the basal branch of living Bilateria (Berney et tions, and histology. We introduce a series of stages, de- al. 2000; Littlewood et al. 2001) continue to be contro- fined by easily recognizable morphological criteria, versial. However, structural differences between the which will facilitate comparing the development of acoels and the other platyhelminths are consistent with acoels with that of other flatworm taxa. Knowledge of their basal placement (review in Smith and Tyler 1985). the Neochildia POU genes will help clarifying the ances- Acoels have a gut syncytium, rather than a multicellular try and evolution of the POU family of transcription reg- epithelial gut, and they lack a paired protonephridial ex- ulators; in addition, it will add a tool with which to ana- cretory system. The organization of the nervous system in lyze neural development in this primitive Bilaterian. adult acoels lacks a compact brain and neuropile. Nerve fibers leaving the brain form an irregular nerve net-like plexus with no straight connectives or commissures Materials and methods (Kotikova 1986; Reuter and Gustafsson 1995; Reuter et Animals al. 1998), an organization that is reminiscent of the nerve net typical of cnidarians and ctenophores. Live adult Neochildia were obtained from the Marine Biological Given the basal phylogenetic position of acoels, an Laboratory, Woods Hole, Massachusetts, and cultured for 2Ð 6 weeks at 20¡C. Eggs produced during this time were collected analysis of the function of developmentally relevant and aged. genes in these animals will likely reveal important information regarding the evolution of fundamental aspects of bilaterian neural organization. To initiate such an investi- Fixation gation, we have begun to screen for homologs of highly To remove mucus prior to fixation, Neochildia embryos and adults conserved genes involved in acoel neurogenesis. In this were treated with 2% HCl in PBS for 5 min, then fixed in a 1:1 so- paper we report the partial sequence and expression of lution of Carnoy’s solution and 4% formaldehyde for 3 h and de- two Pit-Oct-Unc (POU) genes, Brn-1 and Brn-3. hydrated through a graded ethanol series. To remove the dark pig- mentation of Neochildia, fixed specimens were incubated in 6% The POU family of transcription factors was originally H2O in methanol for 12 h under a bright light at room temperature defined on the sequence homology of approximately (Agata et al. 1998). Bleached, fixed and dehydrated embryos were 150Ð160 amino acids that was identified in the mammalian stored in methanol at Ð20¡C until used. transcription factors, Pit-1, Oct-1 and Oct-2 and the nematode factor UNC-86 (Herr et al. 1988; Ryan and Rosenfeld Electron microscopy and histology 1997; Latchman 1999). Since then, a large family of POU- domain containing transcription factors, which form a Following the fixation described above, embryos were post fixed in a mixture of 1% osmium tetroxide and 2% glutaraldehyde in unique subfamily of homeodomain proteins, has been 0.15 M cacodylate buffer for 10 min at 4¡C, washed several times characterized in a variety of species. The POU domain is in PBS and dehydrated through a graded ethanol series and ace- bipartite in nature, consisting of a highly conserved amino- tone (all steps at 4¡C). Preparations were embedded by infiltrating 57 the specimens in a 1:1 mixture of Epon and acetone overnight and Table 1 Oligonucleotide primer sequences and melting tempera- in Epon alone for 5Ð10 h. The infiltrated specimens were trans- tures ferred to molds, oriented, and placed at 60¡C for 24 h to polymer- ′ ′ ize the Epon. Alternating 1-µm semi-thin sections and sets of Primer Primer sequence 5 Ð 3 Tm 80 nm (silver) ultra-thin sections were taken with an LKB ultra- name tome. Ultra-thin sections were mounted on net grids (Ted Pella) and treated with uranyl acetate and lead citrate. Brn-F1 CAA GCA GMG RMG VAT MAA RYT RGG 57.8 Brn-R2 RTT RCA RAA CCA SAC BCK MAC MAC 56.0 SH-1 T17 CTC ATT CCT GTT AAG CTT ACC T17 62.0 Fuchsin labeling of whole-mounts SH-1 CTC ATT CCT GTT AAG CTT ACC 58.6 ACDOB3F2 CAT AAC AAC ATG GTG GCA C 58.1 The whole-mount technique employed here was adapted from AC-B1-F1 TTC ACT CAG GCT GAC GTT G 60.2 Ashburner (1989) and has been used extensively by us, and others, AC-B1-R1 CCT TGA GCC GCA ATT TTA TC 58.4 to label nucleic acids in whole embryos of insects and other inver- AC-B3-F1 AAG CTG ACG TTG GAA ACT C 58.0 tebrates. Briefly, following a fixation in 4% PBS-buffered formal- AC-B3-R1 TCA ACA CCC GAC AAA CAG 57.6 dehyde, 20Ð50 embryos were placed in small wire mesh baskets, washed in three changes of 70% ethanol and once in distilled water for 5 min each. The embryos were incubated in 2 N HCl for 10 min at 60¡C to denature the DNA. Following one wash in distilled water and two washes in 5% acetic acid for 5 min each, em- Primer design bryos were stained in 2% filtered basic fuchsin in 5% acetic acid for 15 min. Embryos were destained in 5% acetic acid to remove cytoplasmic fuchsin labeling, dehydrated through a graded ethanol To design degenerate oligonucleotide primers that would recog- series, transferred to Epon, and individually mounted on slides. nize the POU family of genes, representatives of published vertebrate and invertebrate POU genes were identified using GenBank. The amino acid translations of these genes were manually aligned Immunohistochemistry in PAUP, highly conserved areas of the gene identified, and the nucleic acid alignments for these conserved regions were used as a Neurons were visualized in whole-mount embryos using an anti- template with which to design the forward and reverse primers acetylated tubulin monoclonal antibody (acTub, 1:1,000; Sigma) Brn-F1 and Brn-R2 (Table 1). and cells in metaphase traced with anti-phosphorylated H3 histone (anti-H3, 1:300; Upstate Biotechnology). Mixed age embryos fixed in 4% PBS-buffered formaldehyde were washed in from PCR amplification of genomic DNA three to five changes of PBT (PBS plus 0.3% Triton X-100) over a 10 min period and incubated overnight in PBT containing the pri- PCR was carried out using 1 µl genomic DNA in a total volume of mary antibody at the appropriate dilution. Following a wash in 50 µl containing 1.5 mM MgCl2, 10% DMSO, 0.5 µM each primer PBT the embryos were incubated in PBT containing the secondary and 1 U AmpliTaq Gold DNA polymerase (Applied Biosystems). antibody Ð peroxidase-conjugated rabbit anti-mouse immunoglob- Gene fragments were amplified in two rounds of PCR. The prima- ulin (Jackson Labs), at a dilution of 1:800 for 4 h. To initiate the ry amplification consisted of an initial 12-min incubation required color reaction embryos were briefly rinsed in, then incubated with, to activate the polymerase, followed by 35 cycles of 94¡C for 30 s, 0.1% diamino-benzidine (DAB, Sigma) in 0.1 M phosphate buffer 45¡C for 45 s and 72¡C for 1 min and a final elongation at 72¡C (pH 7.3) containing 0.006% hydrogen peroxide. The reaction was for 7 min. Secondary amplifications were carried out using 1 µl of stopped after 5Ð10 min by diluting the substrate with 0.1 M phos- a 1:20 dilution of the primary amplification product (including the phate buffer. Preparations were dehydrated through a graded etha- negative control) employing the cycling parameters described nol series and acetone, and infiltrated with 1:1 Epon:acetone over- above, but increasing the annealing temperature from 45¡C to night. Embryos were individually mounted in a drop of fresh 53¡C. Epon, covered with a coverslip and analyzed and photographed with a Zeiss Axiophot photomicroscope. Cloning and sequencing of PCR products Cloning of POU genes Amplified PCR products were cloned using the TOPO TA clon- DNA extraction ing kit (Qiagen) and the cloned plasmid DNAs isolated using a Flexiprep kit (Pharmacia). Both procedures were carried out ac- Neochildia genomic DNA was isolated from 10-day-starved indi- cording to the manufacturers’ instructions. All plasmid DNAs viduals using a CTAB protocol (Clark 1992). In brief, flatworms were manually sequenced using M13F(Ð20) to identify clones were incubated in 500 µl 2× CTAB containing 5 µl proteinase K, containing gene fragments belonging to the POU family. These overnight at 45¡C. DNA was sequentially extracted and purified clones were fully sequenced in both directions using M13F(Ð20) using phenol and chlorofom, then ethanol precipitated and re-sus- and M13R at the DNA Sequencing Facility at California State pended in TE buffer. University, Northridge. The resulting sequences were aligned against other POU gene family members and the relationship between the Neochildea gene fragments and the different clades of RNA extraction POU genes determined using parsimony analysis and bootstrap- ping. RNA was extracted from whole embryos or the dissected anterior ends of Neochildia worms using TRIZOL reagent (GibcoBRL) or an RNeasy kit (Qiagen) according to the manufacturers instructions. Elongating the gene fragments

Generating cDNA The Neochildia Brn-3-specific forward ACDOB3F2 and SH-1 primer (Table 1) were used to PCR amplify the 3′ region of the RNA was reverse transcribed into a cDNA using the poly-T gene from an adult cDNA in a classic 3′ RACE protocol (Rapid primer SH-1 T17 and SuperScript II according to the manufac- Amplification of cDNA Ends) using the cycling parameters turers instructions (GibcoBRL). described above with an annealing temperature of 56¡C. 58 Temporal expression of genes the first three duets form an outer layer of blastomeres that surrounds an inner cluster formed by the macromere RT-PCR duet 4A, B (near vegetal pole) and the micromere duet The specific primers AC-B1-F1, AC-B1-R1, AC-B3-F1 and 4a, b (Fig. 1A). These inner cells are the only cells that AC-B3-R1 (Table 1) were designed for the NeocBrn-1 and produce the mesoderm and endoderm (Henry et al. NeocBrn-3 gene fragments respectively. These primers were used 2000). Similar to the situation noted for other flatworms to amplify a 198-bp fragment of NeocBrn-1 and a 209-bp fragment of NeocBrn-3 from cDNAs isolated from 3-day, 4-day, (reviewed in Hartenstein and Ehlers 2000), we see here a juvenile and adult Neochildia. The PCR protocol was identical to mechanism where gastrulation in the classical sense of the primary round of amplification described above but with an separating germ layers does not take place. Instead, cells annealing temperature of 56¡C. that act as stem cells for interior tissues, including digestive parenchyma, musculature, and nervous system, are In situ hybridization delivered into the interior of the embryo at an early stage by the orientation of the mitotic spindle. Whole-mount in situ hybridization was performed using a modi- During the first half of the second day the Neochildia fied Drosophila protocol (Tautz and Pfeifle 1989). DIG-labeled embryo (stage 3) can be characterized as a solid spheri- antisense and sense RNA probes were generated using linearized plasmid DNAs for NeocBrn-1 and NeocBrn-3 and a directionally cal mesenchyme of rather homogeneously sized cells appropriate RNA polymerase. The embryos and probes were hy- (Fig. 1B). Mitoses can be seen superficially and deep in bridized at a final concentration of 1 ng/µl for 100 µl at 55°C in the embryo. Towards the end of the second day (stage 4), 50% formamide, 5× SSC, 0.1% Triton X-100, 0.1 mg/ml heparin, three tissue layers crystallize. The surface layer consists 1 mg/ml yeast tRNA and 10 mg/ml salmon sperm DNA. Follow- ing a series of washes, the embryos were treated with RNase and of regularly spaced, mid-sized cells that form the epider- incubated overnight with an alkaline phosphatase-conjugated anti- mal primordium (Fig. 1C). These cells have not yet dif- DIG-antibody at 4¡C. The embryos were repeatedly washed and ferentiated, as evidenced by the presence of mitoses and the color reaction initiated by adding the enzyme substrate absence of ciliation. Subepidermally, is a 2Ð3 cell-diam- BCIP/NBT (Boehringer Mannheim). The color reaction was eter-thick layer of small, densely packed cells that com- stopped by rinsing in PBS and the embryos examined and photographed using a Zeiss Axiophot photomicroscope. prise the progenitors of muscles and brain. A solid cluster of large cells, the progenitors of the digestive syncytium, fills the core of the embryo. Results Organ formation commences during day 3 (stage 5). The epidermal primordium differentiates into a cuboidal, Normal development and staging

In a recent investigation of embryogenesis of the Fig. 1AÐF Embryonic development of Neochildia. Fuchsin-labeled rhabdocoels Mesostoma lingua, Gieysztoria superba and whole-mounts (top of each panel; embryo shown in ventral view; Craspedella pedum, and the polyclad Imogine mcgrathi anterior to the left) and schematic drawings of sagittal sections we have introduced a system of stages, based upon mor- (bottom of panels; anterior left, dorsal top) of Neochildia embryos at six different stages. A Stage 2 (late cleavage; approx. 16Ð24 h). phological criteria that can be easily distinguished in Outer layer of cells (descendants of micromeres 1Ð3a/b) form the whole-mounted material and that represent major devel- primordium of the epidermis and nervous system. Inner cells (de- opmental steps (Younossi-Hartenstein and Hartenstein scendants of micromeres 4a/b) give rise to muscle cells, digestive 2000a, b; Younossi-Hartenstein et al. 2000, 2001). In the syncytium and neoblasts. B Stage 3 (approx. 24Ð36 h). Embryo forms solid mesenchyme of homogeneously sized cells with mito- following survey of development in Neochildia we have ses (arrowheads) spread over all levels. C Stage 4 (approx. attempted to employ this system because it is possible to 36Ð48 h). Three concentric layers are distinguished by cell size recognize large-scale morphogenetic events that are sim- and shape. The outer layer forms the epidermal primordium (ep; ilar to those in the above named flatworms. blue), middle layer the muscle/brain primordium (mbp), and inner layer the digestive syncytium primordium (dsp). Cells are still Embryogenesis of Neochildia takes 4Ð5 days (18¡C). undifferentiated and show mitoses in all layers (arrowheads). Early cleavage of Neochildia and the fate of individual D Stage 5 (48Ð72 h); early organogenesis. Epidermal primordium blastomeres have been described by Bresslau (1909) and (ep) has differentiated into a cuboidal ciliated epithelium. Bilater- more recently by Henry et al. (2000). Cleavage follows ally symmetric brain primordium (br; violet) forms at the anterior a pattern called duet spiral cleavage where the pair pole. Muscle primordium (mp; green) forms a thin layer underneath the epidermis. E Stage 6 (approx. 72Ð96 h). Brain primordi- (“duet”) of blastomeres produced by the first, meridional um has condensed. Since a basement membrane is absent in division undergoes a series of four oblique divisions that acoels, differentiating muscle cells and cell bodies of epidermal give rise to four duets of micromeres (1Ð4a, b) and the cells intermingle in the body wall. F Stage 7/8 (96Ð120 h). Em- macromere duet (4A, B; Henry et al. 2000). Our whole- bryo moves by muscle contraction. Body-wall is differentiated in- to an outer, nuclear free cortex and an inner layer of densely mounts labeled with fuchsin, anti-phosphohistone and packed epidermal and muscle nuclei (epn, msn). Brain has inner anti-acetylated tubulin antibodies allowed us to follow neuropile (np) surrounded by cortex (co). The statocyst (stc) is a later stages of cleavage, and describe further advanced characteristic landmark located in the posterior cortex. Central morphogenetic events resulting in the different tissues of cells have merged into the digestive syncytium (ds). The circular opening in the body wall at a ventral-posterior location represents the juvenile worm. the simple pharynx (ph). Neoblasts (nb; turquoise) form a popula- During late cleavage (12Ð50 cells; second half of tion of large cells clustered posterior to the brain and around the day 1; stage 2 in our nomenclature) the descendants of digestive syncytium. Scale bar 20 µm 59

densely ciliated epithelium (Fig. 1D). Cilium formation approximately 250 cells on each side. Laterally and pos- can be followed with an antibody against acetylated tu- teriorly, myoblasts form a thin layer underneath the epi- bulin (Fig. 2A, B) and begins simultaneously at all posi- dermis. tions within the epidermis. Subepidermally a bilaterally The “merging” of epidermis and muscle layer, as symmetric brain primordium can be seen at the anterior well as the condensation of the brain primordium charac- pole. Neural cells are small and ovoid and amount to terizes stage 6 (day 4). Acoels lack a basement mem- 60 Fig. 2AÐF Neochildia embryos labeled with acTub antibody that recognizes cilia, gland cells and axons. A, B, D, F Parts of whole-mounted embryos, C, E show sections counter- stained with toluidine blue/ methylene blue that labels nuclei. A, B Stage 5. Epidermal cells (ep) at outer surface have started to form cilia (ci). C, D Stage 6. Cilia have length- ened and increased in density. Necks of gland cells (gl) scattered all over the epidermal layer can be recognized. D, F Stage 7/8. AcTub-positive microtubules form the so called apical web (aw) underneath the cilia. Labeled cell processes emanating from cells within the epidermal layer are interpreted as sensory axons (sn). (epn Epi- dermal cell nuclei, mn nuclei of mesenchymal cells)

brane in between epidermis and musculature; as a result core of the embryo fuse into the digestive syncytium cell bodies of both tissues are intermingled (Fig. 1E, F). characteristic of the acoel clade. Circularly arranged Thus, whereas nuclei of epidermal cells were spaced ex- muscle fibers that appear ventrally define the pharynx. tremely regularly and lay all in the same plane during The size of the brain in juvenile Neochildia is consider- stage 5, epidermal and muscle cell nuclei form an irregu- able. Other flatworm taxa possess a ganglion-like brain lar melange at the surface of the embryo from stage 6 consisting of a central neuropile surrounded by a cortex onward. The brain becomes more compact, and a central, of cell bodies. In adults of Neochildia and other acoel cell-poor neuropile can be distinguished from an external species such a brain has been reported absent. Instead, cortex. Numerous acTub-positive processes of cells neuronal somata and neurites, together with muscle fi- located within the epidermal layer project towards bers and gland cell processes form a loose reticulated the brain primordium (Fig. 2F). These processes are mass at the anterior pole of the animal (Bedini and interpreted as sensory axons. Beside the sensory axons, Lafranchi 1991; review in Reuter and Gustafsson 1995). acTub does not label any other cells in the nervous However, we find that in late embryos and juveniles of system, indicating that no central neurons with long Neochildia, the brain is more compact. A 3Ð4 cell-diam- axons differentiate in the brain during the embryonic eter-thick layer of neurons forms a cortex surrounding a period. central neuropile that is relatively free of cell bodies. In Cell differentiation is completed during the fifth day addition, an “orthogonal” array of muscle fibers pene- of embryogenesis (stages 7 and 8; Fig. 1F). Characteris- trates the brain (Fig. 3). tic morphological criteria are the spherical statocyst that An antibody against phosphorylated H3 histone (anti- develops in the midline of the brain, the appearance in H3; Hendzel et al. 1997) allowed us to follow cell divi- the epidermis of an apical web formed by the rootlets of sion throughout embryogenesis (Fig. 4). Mitoses were epidermal cilia, and the appearance of numerous glandu- absent from all organ primordia from stage 5 onward. In lar processes penetrating the epidermal layer. Cells of the flatworms, differentiated cells have lost the ability to 61 Fig. 3AÐD Cross-sections of the body wall and brain of Neo- childia juvenile. Sections taken at different antero-posterior levels (A most anterior, D most posterior). A Ciliated epidermal cells with apical web (aw) and orthogonal system of so- matic muscle fibers (msf). Anterior brain cortex (br) with loose neuropile crossed by pre- dominantly vertical muscle fibers (msf). C Section taken slightly more posteriorly than B. Brain cortex, neuropile crossed by horizontal muscle fibers. D Posterior region of the brain. Layered arrangement of body wall [with cilia, apical web (aw), nuclei of epidermis cells (epn) and muscle cells (mn)] and brain [cortex (co), neuropile (np)]

divide. A population of totipotent stem cells called neo- Cloning of POU domain genes in Neochildia blasts adds cells during postembryonic growth. The distribution of neoblasts in several flatworm species was PCR amplification between the degenerate primers locat- recently described (Ladurner et al. 2000; Newmark and ed near the 5′ end of the POU-specific domain (Brn-F1) Sanchez-Alvarado 2000; Gschwentner et al. 2001). In and 3′ end of the homeodomain (Brn-R2 primer) resulted juvenile and adult specimens, neoblasts typically form in the isolation of a PCR fragment coding for a two POU a lateral band posterior to the brain. In Neochildia em- sequence (Fig. 5). The first of these, named NeocBrn-1, bryos, we see a bilaterally symmetric population of large is 333 bp in length (excluding the primer sequences) anti-H3-positive cells located posterior to the brain pri- and includes 51 amino acids of the POU-specific do- mordium. We interpret these cells as the embryonic main, 16 linker amino acids and 44 amino acids of the “stem neoblasts”. On average, they number approximate- homeodomain. Four independent PCR clones yielded ly 20Ð30 per individual, a number that does not increase identical sequences. This gene fragment is closest in per- from mid to late stages. This implies that the cells pro- cent amino acid identity to Drosophila drifter ventral- duced by embryonic neoblast division, rather than in- veinless (83%), followed by vertebrate Brn-1 genes creasing the number of neoblasts, add to the organ pri- (80Ð81%), Girardia tigrina GtPOU-1 (a planarian flat- mordia. worm; 72%), and C. elegans ceh-6 (70%). These search- 62

Fig. 4AÐD Labeling of Neochildia with anti-phosphohistone H3 is confined to a population of large, deeply located cells in the to visualize mitotic cells (brown). During early stages (A, B) posterior part of the animal, excluding the brain primordium (br) labeled cells can be seen scattered at the surface, as well as deep and epidermis (ep). We interpret these cells as the stem neoblasts inside the embryo. From around stage 5/6 onward (C, D), labeling (nb)

Fig. 5 Published amino acid translations of human, fly, nematode ent gaps and where relevant, the asterisks denote the end of the 3′ and flatworm homologs of Brn-3 and Brn-1, and NeocBrn-1 translated region of the gene aligned against NeocBrn-3. Dots indicate identity, dashes repres- 63

Fig. 7 An agarose gel of NeocBrn-1 and NeocBrn-3 RT-PCR products representing the temporal expression of these genes in Neochildia [3 3-day-old embryos (stage 5), 4 4-day-old embryos (stage 6), J juveniles, A adult, L ladder]

the POU domain, linker, homeodomain and 158 nucleo- tides of the 3′ end non-coding region. In terms of amino acid identity the NeocBrn-3 gene is closest to human and mouse Brn-3 genes (80Ð78%), followed by the Drosophila IPOU/acj6 gene (78%), Girardia tigrina (75%), and C. elegans, unc-86 (73%). These searches combined with parsimony analyses (e.g. Jacobs et al. 1998) provide strong support for the placement of Neo- cBrn-3 in a Brain 3/IPOU clade, class IV of Wegner (1993; Fig. 6). Fig. 6 Relationship of the Neochildia sequences with the Brain 1 and Brain 3 clades of POU/homeodomain genes. Taxon and gene names are followed by GenBank accession numbers. Sequences were selected for the analysis on the basis of completeness and Temporal expression of POU genes taxon distribution. Sequence from POU domain, homeodomain, intervening linker and conserved 5′ and 3′ sequence were all in- RT-PCR analysis indicates that NeocBrn-1 and NeocBrn-3 cluded in the analysis. In many cases this involves the complete are expressed at a low level in embryos and juveniles, translated product of the gene. The tree was reconstructed using a parsimony analysis implemented in PAUP. In the analysis 432 bas- and substantially increase in adults (Fig. 7). In situ hy- es of DNA with third positions removed were combined with 260 bridization of early to mid stage embryos with NeocBrn-1 amino acids (see Jacobs et al. 1998; Schubert et al. 2000 for meth- probes gives negative results, although RT-PCR shows odology). Bootstrap support is shown adjacent to each branch. expression to be present (Fig. 7). However, in a fraction Note that the major groups of genes are well supported, as are of embryos 3 days or older we detected signal in a group some of the topologies within the vertebrates. Relationships between genes in different phyla within the gene classes are less well of large internal cells which by size and position corre- supported. However due to the strong support for the major clades spond to neoblast progenitors (Fig. 8A). In late embryos of POU domains including Brain 1 and Brain 3 we can confidently and adults, NeocBrn-1 is expressed at low levels in the place one Neochildia gene in the Brain 1 group and the other in brain (Fig. 8B, C) and visualizes an interesting pattern of Brain 3 epidermal glandular cells, which, along the anterior- posterior axis, roughly coincide with the extension of the brain (Fig. 8DÐF). Given that in vertebrates and es combined with parsimony analyses (see Jacobs et al. Drosophila, several POU genes may play a specific role 1998 for analytical approach) provide strong support for in the development of ciliated sensory cells (Artinger the placement of this gene in a Brain 1/drifter clade, et al. 1998), expression in these specialized epidermal class III of Wegner (1993; Fig. 6). cells is particularly interesting. NeocBrn-3 labels a The second POU sequence generated using the degen- small and discrete population of neurons in the brain. erate PCR is 348 bp in length excluding the primer se- The cell group forms a transversally oriented “slice” quence and belongs to the Brn-3 clade (class IV POU of densely packed cells (Fig. 8G, H). No expression genes); thus, we named it NeocBrn-3. The 3′ region of could be detected by in situ hybridization at embryonic this gene was isolated using a gene-specific primer and stages. 3′ RACE. The isolated fragment encodes 99 amino acids of which 40 show overlap with the NeocBrn-3 clone and 59 are new. In all, we have a fragment of the gene en- compassing 572 bases, including the sequences encoding 64 Fig. 8 Expression pattern of NeocBrn-1 and Brn-3 in embryos (A, B) and adult (CÐH) tissues. A, C, G Whole-mounts in ventral view; B, DÐH sections. A Stage-5 embryo. Ex- pression of NeocBrn-1 appears at a low level, and only in a subset of specimens analyzed, in large, deeply situated cells that might represent stem neoblasts. B, C In late embryo and adult, NeocBrn-1 is visible in subset of neurons in the brain (br). The pharynx, located at mid-ventral level, is marked ph in C. DÐF Besides the expression in subset of nerve cells, Brn-1 also labels a ventral subpopulation of cells in the epidermal layer which we interpret as glandular cells (gl). G, H Expression pattern of NeocBrn-3 in a subset of brain neurons (br) of adult Neochildia

Discussion gestive cavity, and a protonephridial system, and the body wall is atypical in that a basement membrane is ab- Morphological aspects of acoel development sent. Further, axons of the trunk are not organized in a regular orthogonal system of connectives and commissu- Acoels have attracted the attention of phylogeneticists res, but rather form a variable, nerve net-like reticulum for a long time. Although they share many characteris- of fibers (reviewed in Smith and Tyler 1985; Reuter and tics with other flatworm taxa, they differ in several im- Gustafsson 1995). The latter has been attributed to the portant ways. For example, they lack both a cellular di- lack of a basement membrane, which in other animals is 65 instrumental in creating a smooth cleft between epider- ample, in polyclad embryos, the primordium of the brain mis and muscle layer in which axons can form straight and epidermal lobes surrounding the mouth opening de- fascicles. These properties have either been viewed velop at, or shift towards, the ventral side. as primitive characters, inherited from an ancestral cili- In the acoel Neochildia, the overt “ventralization” ate protozoan (Hadzi 1963; Hanson 1977) or cnidarian originating in the mesoderm primordium does not occur. (Hyman 1951; von Salvini-Plawen 1978), or as second- No asymmetries are visible among the progeny of the 4a, ary “regressive” changes (Ax 1963; Remane 1963; Rieger b micromere or the 4A, B macromere duet. Likewise, in 1985; Smith and Tyler 1985). Based on these differ- the developing epidermis or brain primordium, it is im- ences, there have been repeated suggestions that the possible to make out any dorso-ventral asymmetries on acoels be taken out of the platyhelminth phylum and the basis of morphological criteria. These asymmetries placed in a separate group representing taxa that split only become apparent at late stages when a mouth open- from the bilaterian line earlier than the “true” platyhel- ing, surrounded by circular muscle fibers, forms at minths. Recent evidence, both structural and molecular, the ventral side of the embryo. It is likely that in acoels, supports this view. The cell lineage analysis by Henry latent dorso-ventral asymmetries exist from early stages et al. (2000) shows that the pattern of cleavages and cell onward, given that there is a fixed relationship between lineage in the acoel Neochildia differs significantly from micromere identity and dorso-ventral position of their those in other flatworms. Perhaps most notable is the pe- (epidermal) progeny (Henry et al. 2000). However, the culiar type of spiral cleavage, called duet cleavage, mechanism by which the dorso-ventral axis is specified, found in the acoel, and the developmental origin of the and in particular, the role of the mesodermal lineage dur- mesendoderm. In duet spiral cleavage, the pair of equal ing this process, may be quite different when comparing sized blastomeres generated by the first cleavage (mac- acoels with typical spiralians. romere duet 1A, B) sequentially buds off three pairs of Acoels like other flatworm taxa, have neoblasts, a micromeres (1a, bÐ3a, b). The 3A, B macromere duet special cell type that serves as a totipotent stem cell pop- then sinks into the mass of surrounding micromeres and ulation. In several flatworm species, it has been shown divides one more time into 4A, B and the 4a, b micro- that once differentiated, cells of the epidermis, muscula- mere quartet. These four cells give rise to the entire en- ture, nervous system, or other organs, no longer divide doderm (i.e. the digestive syncytium) and mesoderm (i.e. (Baguna 1981; for review of classical literature on neo- musculature, neoblasts); the progeny of the 1a, bÐ3a, b blasts, see Ehlers 1985). This in itself is similar to the micromeres produce epidermis and nervous system. This situation in other systems where many differentiated cell lineage relationship between tissues and blastomeres is types also fail to divide. However, in these other sys- significantly different from that found in other flatworms tems, “dedicated” (i.e. tissue-specific) stem cell popula- and spiralians in general. In a typical spiralian, meso- tions exist that add new cells or replace sequestered derm is derived from two different sources, the 4d mi- ones. Examples are the stratum germinativum of the ver- cromere (mesoendoderm) and the micromeres (variably tebrate epidermis, or the stem cells in the intestinal mu- the first, second or third quartets; for review see Henry cosa. Such tissue-specific stem cells are not found in et al. 2000). flatworms. Instead, proliferating, motile neoblasts per- Apart from the different origin of mesoderm in vade the entire body and are able to differentiate into any acoels, the time and mechanism by which the dorso-ven- cell type needed. Little is known about the embryonic or- tral axis is established in these animals may differ funda- igin and development of neoblasts. Using BrdU or an an- mentally from what is known in other spiralians. In both tibody against phosphorylated histone, both of which acoels and typical spiralians alike, the first asymmetry label proliferating cell populations, neoblasts were visu- that becomes manifest is the animal-vegetal axis. This alized in several recent papers for postembryonic stages axis appears at the time point when the first micromeres of planarians (Newmark and Sanchez-Alvarado 2000), are formed. Micromeres take up a position at the animal Macrostomum sp. (Ladurner et al. 2000), and the acoel pole, macromeres at the vegetal pole. This animal-vege- Convolutriloba (Gschwentner et al. 2001). In these ani- tal axis, in a somewhat oversimplified manner of speak- mals they form a bilaterally symmetric band of quite ing, is translated into the antero-posterior axis of the la- densely packed cells flanking the digestive system. The ter embryo (Boyer et al. 1998; Henry et al. 2000). Thus, head region is devoid of neoblasts. descendants of the micromeres at the animal pole form Using anti-phospho histone, we have visualized in the epidermis, nerve cells and sensory receptors of the this study a population of large proliferating cells in mid head. Micromere descendants located more vegetally to late stage embryos of Neochildia that we interpret as (e.g. descendants of the third quartet/duet) give rise to the embryonic neoblasts. Their distribution in roughly posterior epidermis. Superimposed upon this a-p axis is two lateral bands in the posterior half of the embryo is the dorso-ventral axis that, in typical spiralians, is first also reminiscent of the findings in other species cited manifested in the asymmetry among the fourth micro- above. The fact that the embryonic neoblasts divide mere quartet (Henry et al. 2000). Thus, the mesodermal could be interpreted in two different ways. First, it is descendants of the 4d micromere move to one side of the possible that neoblasts add to their own number. Second- early embryo that is thereby defined as the ventral side. ly, progeny of embryonic neoblasts might differentiate Further dorso-ventral asymmetries soon follow. For ex- and contribute to developing organs. According to this 66 view, neoblasts would start to add to and/or replace cell decarboxylase, an enzyme in the metabolic pathway of populations of various organs during embryogenesis. In serotonin and dopamine (Johnson and Hirsh 1990; Yeo our study, the number of neoblasts labeled at a given et al. 1995). Beside the CNS, drifter/ventral veinless ap- stage did not increase over time making the second pos- pears in numerous non-neural tissues, including the tra- sibility more likely. As such, cells found in the various cheal system, the epidermis, hind- and foregut, and ring organs of hatching flatworms might have two quite dif- gland (Anderson et al. 1995; Billin and Poole 1995; ferent origins. Either they are formed in the “traditional” DeCelis et al. 1995) and, using a genetic approach, a role way, that is, they derive from blastomeres that divide in a for drifter/ventralveinless in tracheal and epidermal pat- more or less fixed pattern; alternatively, they are pro- terning has been established. In the Crustacean, Artemia duced in an “ad hoc” manner by totipotent neoblasts that franciscana a homolog of drifter/ventralveinless is ex- form a motile cell population, circulating and probing pressed in the larval salt gland, an organ which is in- where in the embryo additional cells are needed, and volved in osmoregulation and disappears in the adult then differentiating into that cell type. (Chavez et al. 1999). In the adults of the triclad flat- The lack of acTub-positive cells in the central ner- worm, Girardia tigrina, the Brn-1 homolog, GtPOU-1 is vous system of Neochildia embryos is in contrast to find- expressed in neurons of the central and peripheral ner- ings in other flatworm species where acTub labels vous system (Munoz-Marmol et al. 1998). subsets of brain neurons and their axons (Hartenstein In the present study, the Neochildia Brn-1 homolog and Ehlers 2000; Younossi-Hartenstein and Hartenstein NeocBrn-1 is expressed in a ventral population of pre- 2000b; Younossi-Hartenstein et al. 2000, 2001). How- sumed gland cells, as well as a small subset of brain neu- ever, the lack of acTub labeling of CNS structures in rons. The expression during embryonic stages is also Neochildia is not the first instance in which acTub does confirmed by isolation of RNA and amplification of not recognize any epitope in the CNS. We have had the Brn-1 gene by RT-PCR from the 3- and 4-day embryos. same experience with various representatives of mol- The interpretation of the spatial expression pattern by luscs and annelids (V. Hartenstein, unpublished data). whole-mount in situ techniques is ambiguous. We were The lack of staining could be an artifact due to insuffi- unable to find a signal in most embryos. In a subset of cient penetration of the antibody. Alternately, acetylation embryos from day 3 onward we found the gene ex- of tubulin in axons does not take place in the embryonic pressed in a subset of large, internal cells which most period of species, such as Neochildia, where acTub picks likely represent neoblast progenitors, given their large up no central neurons. One should note that in all species size and internal position. This would point at the possi- we investigated, only a subset of axons which were rec- bility that the Brn-1-positive neurons we see in the adult ognizable by histology labeled with acTub. In some sys- brain are derived from neoblasts in the late embryo. tems, such as Drosophila melanogaster, only axons that Alternatively, neoblasts could only transiently express have reached a certain length and/or diameter are visual- the gene in the embryo, and neurons turn it on postem- ized with acTub (V. Hartenstein, unpublished). bryonically. The POU-IV class proteins have critical functions in the vertebrate CNS and sense organs. The original Brn-3 POU genes in neural development factor was isolated as a novel POU protein by He et al. (1989) by using the degenerate RT/PCR approach. Sub- POU domain genes encode a large family of transcrip- sequently, three distinct Brn-3 factors were discovered tion factors that have been identified in a basal metazoan which are encoded by different genes (Theil et al. 1994). taxa such as sponges, cnidarians and ctenophores (D.K. These are known as Brn-3a (Brn-3.0), Brn-3b (Brn3.2) Jacobs, unpublished) as well as in bilaterians, including and Brn-3c (Brn-3; for review, see Latchmann 1999). nematodes, arthropods, echinoderms and vertebrates. Brn-3a is expressed in multiple distinct nuclei of the The expression of many POU domain proteins in the mouse brainstem and spinal cord, as well as sensory neu- nervous system across evolutionary boundaries suggests rons of the cranial and dorsal root ganglia. Knockout a fundamental requirement for these genes during neuro- mice show widespread loss of sensory and motor nal development. In particular, the class III and IV POU neurons and die shortly after birth (Xiang et al. 1996). proteins are candidate regulators of CNS-specific genes Brn-3b is found in the brainstem and at high levels in (Josephson et al. 1998). Brn-1 is widely expressed in the retinal ganglion cells; mutant mice lacking Brn-3b are embryonic neuroepithelium in rat. In the cortex, Brn-1 is viable but show specific loss of retinal neurons leading strongly expressed in the ventricular zone and repressed to blindness (Gan et al. 1996, 1999). Brn-3c shows ex- when neural progenitor cells first differentiate into pression in and is absolutely required by the hair cells of neurons (Alvarez-Bolado et al. 1995). In zebrafish, the inner ear (Xiang et al. 1997a, b). Similarly, POU4F3 Brn-1 appears in the neural plate where the brain forms causes X-linked mixed deafness in humans with the (Hauptmann and Gerster 2000). deletion of eight bases at the POU homeodomain in an The Drosophila Brn-1 homolog, drifter/ventral vein- Israeli family (Vahava et al. 1998). In spite of these di- less, is expressed in several subsets of glial and nerve verse roles of Brn-3 genes in neural development a pre- cells, notably the dopaminergic and serotonergic neu- ponderance of function appears to relate to the develop- rons, where it is believed to directly upregulate dopa- ment of the sensory cells involved in vision, hearing and 67 olfaction, all of which are placoidally derived (Artinger of visual organs, neuroendocrine centers and various et al. 1998). The Drosophila Brn-3- homolog, IPOU/ brain structures are laid out in the fate map of the head acj6, encodes a 367-amino-acid protein that acts as a are conserved in phyla separated as far as chordates and transcriptional repressor in the central and peripheral arthropods (Hartenstein and Reh, in press). This indi- nervous system from late embryonic to adult stages cates that the bilaterian ancestor might have possessed a (Certel et al. 2000). Acj6 may play an important role in head in which photoreceptors, various brain structures regulating synaptic target selection by central neurons. It and neuroendocrine cells were arranged in a manner that is also noteworthy that, as in the vertebrate Brn-3, there may have been similar to the one found in present day is an association with sensory innervation including ex- taxa. To further address this hypothesis comparative pression in olfactory, optic and antennal neurons. C. ele- analyses of invertebrate neural development, using mo- gans unc-86 POU protein is required for the commitment lecular markers for defined cell types, are indispensable. of several sensory neuroblast lineages as well as for the In this paper we have initiated the developmental analy- specification and maintenance of particular neural phe- sis of the nervous system in the acoel Neochildia notypes. Mutants of Unc-86 have shown defects in che- and provided two molecular markers, NeocBrn-1 and motaxis, mechanosensation and egg laying (Finney and NeocBrn-3. We plan to expand our effort to other con- Ruvkin 1990). served genes expressed in the developing nervous In Neochildia adult brain, NeocBrn-3 is expressed in system of flatworms, and thereby hope to contribute to a fairly small cluster of seemingly contiguous cells of the the elucidation of nervous systems in simple inverte- brain. Given the paucity of specific markers for subsets brates. of neurons in flatworm brains, nothing can be stated con- cerning the identity of the NeocBrn-3-positive cells. In Acknowledgements We would like to acknowledge Vyacheslav embryos, although NeocBrn-3 RNA was detected from Palchevskiy for technical assistance, Amelia Younossi-Hartenstein for expert help and advice, and support from NSF grant 3 days onwards, a signal could not be detected by in situ IBN-01110718 to V.H., and NASA Exobiology (NAG5Ð7207) and hybridization. We were therefore unable to monitor the Astrobiology to D.J. origin of the Brn-3-positive cells. The relatively late on- set of NeocBrn-1 and 3 parallels the expression of homologs of these genes in Drosophila. 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