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Embryonic expression of festina lente (fel), a novel maternal gene, in the oligochaete annelid Tubifex tubifex
Gene Expression Patterns 25-26 (2017) 29e35
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Embryonic expression of festina lente (fel), a novel maternal gene, in the oligochaete annelid Tubifex tubifex Takuma Nakamura a, Inori Shiomi a, Takashi Shimizu a, b, * a b
Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2016 Received in revised form 27 April 2017 Accepted 3 May 2017 Available online 4 May 2017
We have cloned and characterized the expression of a novel maternal gene festina lente (designated Ttufel) from the clitellate annelid Tubifex tubifex. Northern blot analyses have shown that Ttu-fel mRNA is approximately 8 kbp in length and that its expression is restricted to oocytes undergoing maturation division and early embryos up to 22-cell stage. Maternal transcripts of Ttu-fel are first detected in oocytes in the ovary of young adults (ca. 40 days after hatching); its expression continues in growing oocytes in the ovisac. Ttu-fel mRNA is distributed broadly throughout the egg undergoing maturation divisions. During the process of ooplasmic segregation that results in the pole plasm formation, Ttu-fel mRNA becomes concentrated to the animal and vegetal poles. The RNA in the animal hemisphere is distributed in a gradient with highest concentration in the cortical region. During the first two cleavages, Ttu-fel mRNA is segregated to CD cell then to D cell; it is subsequently inherited by the three D quadtrant micromeres, 1d, 2d and 3d. Around the time of transition to 22-cell stage, Ttu-fel mRNA becomes undetectable throughout the embryo. © 2017 Elsevier B.V. All rights reserved.
Keywords: Novel maternal gene Clitellate annelid Oligochaete Tubifex tubifex 2d micromere Early cleavage Spiralian Lophotrochozoan Maternal transcript degradation
Early development in the animal embryo is driven by maternally provided gene products such as mRNAs and proteins, though at some point during development there is a transition to control by zygotic gene products (Davidson, 1986). Recent studies on model organisms such as nematode, fruit fly and mouse have demonstrated that the first event of the maternal-to-zygotic transition (MZT) is the elimination of maternal transcripts and that 30e35% of maternal mRNAs are destabilized during the period of this transition in these animals (Schier, 2007; Tadros and Lipshitz, 2009). Given that the MZT occurs in all animal embryos, it is highly possible that similar maternal mRNA elimination takes place during early development in all metazoans. Although there have been few systematic studies on the events occurring during the MZT in spiralian lophotrochozoans, it has been known that in the clitellate annelid (leech) Helobdella, zygotic transcripts are needed for early cleavages (Bissen and Weisblat, 1991; Bissen and Smith, 1996; Schmerer et al., 2013). Similar early requirement for zygotically produced mRNAs has recently been
* Corresponding author. Kitano 6-1-8-17, Kiyota-ku, Sapporo 004-0866, Japan. E-mail address: [email protected] (T. Shimizu). http://dx.doi.org/10.1016/j.gep.2017.05.001 1567-133X/© 2017 Elsevier B.V. All rights reserved.
suggested in unequal cleavage of the teloblast precursor cell 2d in another clitellate annelid (oligochaete) Tubifex (M. Aoki and T. Shimizu, unpublished data). On the other hand, in these annelids, not a few maternal genes have been cloned and examined for their expression patterns, including Tubifex (Ttu) homologues of dorsal (dl), nanos (nos), p68 and vasa (vas) (Matsuo et al., 2005; Oyama and Shimizu, 2007; Oyama et al., 2008; Mohri et al., 2016) and Helobdella (He) homologues of hunchback, msx, nos, piwi, twist, vas and wnt (Kostriken and Weisblat, 1992; Master et al., 1996; Savage and Shankland, 1996; Soto et al., 1997; Kang et al., 2002; Cho et al., 2014). Semi-quantitative analyses have shown that maternally supplied transcripts of Ttu-nos, He-nos, He-vas and He-piwi decrease in amount during early cleavage stages and that this decrease is followed by upregulation of their expression that occurs during mid development (Kang et al., 2002; Cho et al., 2014; Mohri et al., 2016). In contrast, maternal transcripts of Ttu-p68 and Ttu-vas seem not to decrease in amount significantly during early and mid development, but do so during late development (Oyama and Shimizu, 2007; Oyama et al., 2008). In this study, we have isolated a novel maternal gene named festina lente (fel) from Tubifex tubifex. Compared with other maternal genes described so far, this gene is apparently unique in
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that its maternal mRNA is detected exclusively during the period of early cleavage stages and that it is inherited by the second micromere (2d) but not by the fourth micromere (4d) of the D quadrant. 1. Results and discussion 1.1. Summary of early development of T. tubife A brief review of Tubifex development is presented here as a background for the observations described below (for details, see Shimizu, 1982). Tubifex zyogtes, which are oviposited at metaphase I, form two polar bodies and then enter the first mitosis. Before the first cleavage, yolk-deficient cytoplasm called pole plasm accumulates at both poles of the egg (Fig. 1A). The early development of Tubifex consists of a stereotyped sequence of cell divisions. The first cleavage of the Tubifex egg is unequal and meridional, and produces a smaller AB cell and a larger CD cell (Fig. 1B). The second cleavage is also meridional and yields cells A, B, C and D: the CD cell divides into a smaller C cell and a larger D cell while the AB cell separates into cells A and B of various sizes (Fig. 1C). From the third cleavage on, the quadrants A, B and C repeat unequal divisions three times, and the D quadrant four times, producing micromeres at the animal side and macromeres at the vegetal side. The D quadrant micromeres are designated 1d, 2d, 3d and 4d (Fig. 1DeG). Cells 2d and 4d are much larger than cells 1d and 3d. During cleavages, the pole plasms are inherited by the D lineage cells and finally partitioned into 2d and 4d. At 22-cell stage, 2d11 (derived from 2d), 4d and 4D (sister cell of 4d) all come to lie in the future midline of the embryo (Fig. 1G). These three cells undergo bilateral cell division in the next cleavage: 4d divides equally yielding the left and right mesoteloblasts M (Fig. 1H); 4D divides equally into endodermal precursors E (Fig. 1I); and 2d111 (derived from 2d11) divides into a bilateral pair of ectoteloblast precursors NOPQ (Fig. 1J). The quadrant cells A, B and C also divide equally at the sixth cleavage. 1.2. Cloning of a “2d-cell-specific” gene, festina lente, from T. tubifex During the course of preliminary whole-mount in situ hybridization (WISH) using riboprobes that had been synthesized from 'positive' clones resulting from screening of a 1-cell-stage cDNA
library (see Experimental procedures), we found that a riboprobe synthesized from clone 4038 stained 2d cell but not 4d cell of a 22cell embryo. (Those from the remaining clones stained both 2d and 4d of the same embryos.) This preliminary observation suggests that clone 4038 contains a Tubifex gene whose transcripts are segregated preferentially to 2d cell. As described later, this gene is a novel maternal gene from spiralian lophotrochozoans, and its expression is restricted to early cleavage stage embryos. Hereafter we will name this gene festina lente (fel). Clone 4038 was found to contain a 1852-bp cDNA sequence which includes a 40-bp poly-A tail at the 30 end (DDBJ/GenBank accession number LC140925). To examine whether the 1812-bp sequence represents full length of Ttu-fel transcripts, we performed a Northern blot analysis using RNA isolated from various stages of Tubifex development. We found that “intact” Ttu-fel transcripts were approximately 8 kbp in length (Fig. 2). This
Fig. 2. Expression of Ttu-fel mRNA. Northern blot analysis of total RNA (10 mg each) of (A) embryos at stages 1, 2e8, 9e13, 14e18, and Juv (10-day-old juveniles) or (B) embryos at stages 1e2, 4e6, 7, 8, and 9e10. Total RNA was prepared and electrophoresed as described in Experimental procedures. The membrane was probed with DIG-labeled RNA. For stages 1e11, see Fig. 1. Stage 12, ectodermal teloblastogenesis; stages 13e15, germ band formation; stages 16e18, body elongation accompanied by formation of segmental ectoderm and mesoderm (for details of developmental stages, see Shimizu, 1982).
Fig. 1. Summary of Tubifex early development. Diagrammatic illustration of stages 1e11 of embryonic development (stage 3 is omitted). (AeC) Animal pole view of embryos at stages 1-cell (A), 2-cell (B) and 4-cell (C). pp, pole plasm. (DeG) Animal pole view of embryos undergoing the formation of the D quadrant micromeres 1d (D), 2d (E), 3d (F) and 4d (G). (HeJ) Posterior view of embryos undergoing bilateral cell division in 2d111 (derived from 2d11), 4d and 4D. (H) 4d divides into a pair of mesoteloblasts (M). (I) 4D divides into a pair of endodermal precursors (E). (J) 2d111 divides into a pair of ectoteloblast precursors (NOPQ) (Redrawn from Shimizu, 1982).
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suggests that the 1812-bp sequence (LC140925) represents only one fifth of the complete sequence. We have tried to obtain additional sequence of Ttu-fel by using commercially available 50 RACE PCR kits, but all attempts so far have been unsuccessful. This sequence does not have a significant BLAST hit when compared against sequences from three spiralian genomes (i.e., owl limpet Lottia gigantea, marine polychaete Capitella teleta and freshwater leech Helobdella robusta; see Simakov et al., 2013). The failure to find convincing orthologues in other invertebrates would not be too surprising if this sequence is some type of non-coding transcript or is derived entirely from 30 -UTR (untranslated region). On the other hand, it should be mentioned that we obtained an ORF (open reading frame) of 275 amino acids within the 1821-bp sequence by using the ORF Finder tool at NCBI (Fig. 3A). This putative protein lacks obvious orthologues by BLAST in invertebrate genomes though its internal portion showed 33e35% amino acid identity to myosin molecules of some spiralians (Fig. 3B). This may suggest a possibility that the cDNA represents an incompletely spliced transcript. At present it is equally possible that the ORF is only a “fluke”, though. 1.3. Temporal expression pattern of Ttu-fel The temporal expression profile was analyzed by Northern blot. The results are shown in Fig. 2. Ttu-fel mRNA was present at detectable level exclusively in early embryos undergoing first five cleavage divisions (Fig. 2A). The level of Ttu-fel expression appeared not to alter significantly before its abrupt decrease that occurs at the time of transition to stage 8 (Fig. 2B). Close examination of the blots also showed that Ttu-fel mRNA reduces its size by approximately 300 bp in length during the period of these cleavages. 1.4. Spatial expression pattern of Ttu-fel Whole-mount in situ hybridization (WISH) staining using antisense probes revealed the presence of easily detectable levels of Ttu-fel mRNA exclusively in early cleavage stage embryos (i.e.,
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stages 1e8; Fig. 1AeG). In embryos processed for in situ hybridization with sense probe no staining was detected from stage 1 to stage 18 (completion of embryogenesis; data not shown). During maturation division, the mRNA was present throughout the egg though the animal pole with the meiotic spindle exhibited higher level of expression than any other region of the egg (Fig. 4A). When pole plasm (i.e., yolk-deficient cytoplasm) began to form at both poles (see Fig. 1A), the mRNA was present in a thin layer along the surface of both poles (Fig. 4B). As pole plasm formation proceeded, Ttu-fel mRNA accumulated to the pole plasm domains (Fig. 4C and D) and concentrated to the poles (Fig. 4E and F). Ttu-fel transcripts in the animal hemisphere were distributed at higher density in the cortex than in more inner regions (Fig. 4D). By contrast, Ttu-fel transcripts in the vegetal hemisphere were distributed rather uniformly (Fig. 4D). In Tubifex, when pole plasm formation completes, the animal pole region protrudes outward (Shimizu, 1986). If one sees such a protruding animal pole region end-on with the animalvegetal axis, it is found that Ttu-fel mRNA is distributed in a ring (Fig. 4E, inset). Although the existence of maternal transcripts in the pole plasm domain has also been reported for some genes such as Ttu-dl, Ttu-p68, Ttu-vas and Ttu-nos (Matsuo et al., 2005; Oyama and Shimizu, 2007; Oyama et al., 2008; Mohri et al., 2016), transcripts of these genes appear to be distributed rather uniformly even in the animal pole plasm domain. During the first two cleavages, Ttu-fel mRNA is segregated to CD cell and then to D cell (Fig. 5A and B). Animal and vegetal pools of transcripts, which remained to be separate from each other shortly after the second division, were unified at the animal pole upon the beginning of the third division (data not shown). It is known that pole plasms are unified at the animal pole prior to the onset of the third cleavage (Shimizu, 1989). When the third cleavage completed, Ttu-fel mRNA was found to be distributed to both 1d and 1D (Fig. 5C and D). Subsequently a large portion of the RNA was segregated to 2d cell at the fourth cleavage (Fig. 5E and F). The bulk of Ttu-fel RNA left in 2D cell was finally inherited by 3d cell at the fifth cleavage (Fig. 5G). When 3D divided into 4d and 4D, there was no longer any sign of the mRNA in these cells even if stained embryos were
Fig. 3. Characterization of Ttu-fel, a novel gene from Tubifex tubifex. (A) Amino acid sequence of a putative Ttu-fel protein. The portion underlined was used for alignment (B) Alignment of the putative Ttu-fel protein with myosin molecules from two spiralian species. The portion underlined in (A) was used for alignment. Asterisks represent amino acid identity. Numbers in parentheses indicate the percentage amino acid identity with Ttu-fel. The following sequences were used: Cgi-myosin (accession number K1RSS3), Pdu-myosin (KJ405466), and Ttu-fel (LC140925). Species abbreviations: Cgi Crassostrea gigas (mollusc); Pdu Platynereis dumerilii (annelid); Ttu Tubifex tubifex (annelid).
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Fig. 4. Expression of Ttu-fel detected by in situ hybridization with an antisense riboprobe. (A)e(E), uncleared specimens viewed with incident light; (F), cleared specimen viewed with transmitted light. (A) Animal pole view of metaphase II egg. The arrow indicates the animal pole showing Ttu-fel staining. (B)e(F) Eggs at 40 (B), 60 (C), 80 (D), and 100 (E and F) min after the second polar body formation. (B) Animal pole view. Note Ttu-fel staining around the animal pole. The inset shows a section of bisected egg. Note a layer of Ttu-fel staining along the animal pole surface. (C) and (D) Sections of bisected eggs. AP, animal pole; VP, vegetal pole. (E) Animal pole view. Note accumulation of Ttu-fel RNA to the animal pole (AP). The inset shows the animal pole region viewed end on with the animal-vegetal axis. (F) Same egg as shown in (E). Note the accumulation of Ttu-fel RNA to both poles. AP, animal pole; VP, vegetal pole. Scale bar in (A): (AeF) 100 mm.
cleared and observed with transmitted light (data not shown). In contrast, four small cells (i.e., 1d, 2d2, 2d12 and 3d) of the same embryo still exhibited staining signal though much weaker than before (Fig. 5H and I). Around the time when 2d111 (derived from 2d11 of stage 8 embryo; Fig. 1G) divided into a bilateral pair of NOPQ cells (see Fig. 1J), Ttu-fel mRNA became undetectable throughout the embryo (Fig. 5J). Consistent with the result of Northern blot analysis (Fig. 2A), Ttu-fel mRNA was undetectable during the rest of embryogenesis, either (data not shown). As described above, deposited eggs (oocytes) in the cocoon (Fig. 4A) contain Ttu-fel mRNA, which is to be used as maternal RNA during early cleavage stages. This implies that Ttu-fel mRNA must be transcribed at a certain stage of oogenesis. To find out when transcription of maternal Ttu-fel mRNA begins, we examined juveniles and adults for Ttu-fel expression. It is known that juveniles of T. tubifex grow up to adulthood within 40 days after hatching under laboratory conditions (at 22 C; Shiomi, 2014). We reared juveniles for 5, 14, 28 or 40 days before fixation and processed them for Ttu-fel staining (see Experimental procedures). We found that positive signals of Ttu-fel staining were detected exclusively in 40day-old specimens (i.e., adults). Ttu-fel mRNA was localized in ovaries in segment XI and oocytes in the ovisac in segments XII and XIII (Fig. 6A). Ttu-fel-positive cells were seen in the dorsal portion of the ovary (Fig. 6B). In contrast, the ventral portion of the ovary appeared to be devoid of positive signals of Ttu-fel staining (data not shown). In Tubifex, oogonia (in the multiplicative phase) occupy the most ventral portion of the ovary and extend toward the dorsal side generating oocytes (see Fig. 6C; for details, see Hirao, 1964). (Usually, 7e8 oocytes are released from the dorsal portion of the ovary to the ovisac, where the oocytes undergo yolk deposition and maturation; see Hirao, 1964) Judging from their location in the ovary, it is likely that Ttu-fel is expressed in oocytes but not in oogonia.
1.5. Ttu-fel is a novel maternal gene from the spiralian lophotrochozoans The embryonic expression pattern of Ttu-fel, revealed in this study, is summarized as follows. Maternal transcripts of Ttu-fel, which are segregated to the first three micromeres (i.e., 1d, 2d and 3d) of the D quadrant during cleavage stages, are eliminated from the embryo as late as 22-cell stage; this decay of Ttu-fel mRNA is not followed by zygotic activation of this gene, which resumes only when female germ cells enter meiosis in the ovary. To our knowledge, this is the first report of a lophotrochozoan maternal gene whose expression is restricted strictly to early cleavage stages. Compared with other Tubifex maternal genes that have so far been examined for their expression patterns, Ttu-fel appears to be unique in two respects. First, Ttu-fel mRNA is segregated only to 2d cell but not to 4d cell. Transcripts of Ttu-nos and Ttu-vas are inherited by both 2d and 4d (Oyama and Shimizu, 2007; Mohri et al., 2016); those of Ttu-p68 are segregated only to 4d cell but not to 2d cell (Oyama et al., 2008). Second, transcripts of Ttu-fel are detected only in oocytes and early embryos undergoing first five cleavages; Ttu-fel mRNA is undetectable during the rest of embryogenesis. By contrast, Ttu-nos, Ttu-p68 and Ttu-vas are expressed almost throughout embryogenesis, although their expression levels fluctuate during development. As described before, a number of maternal genes have been examined for their expression patterns in other spiralian lophotrochozoans as well (Kostriken and Weisblat, 1992; Master et al., 1996; Savage and Shankland, 1996; Soto et al., 1997; Kang et al., 2002; Cho et al., 2014), but their embryonic expression persists through to late development. This may suggest that Ttu-fel is a novel maternal gene from the spiralian lophotrochozoans. 1.6. Maternal transcript degradation The present study has shown that maternal transcripts of Ttu-fel
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Fig. 5. Expression of Ttu-fel during the period of early cleavages. All embryos except (F) were uncleared and viewed with incident light. (A) Stage 2 embryo consisted of cells AB and CD. Animal pole view. (B) Stage 3 embryo consisted of three cells, AB, C and D. Animal pole view. (C) Stage 5 embryo consisted for four macromeres (1A-1D) and four micromeres. Animal pole view. Individual micromeres are not depicted here. (D) Section of stage 5 embryo bisected along the animal-vegetal (dorsoventral) and anteroposterior axes. Anterior is to the top; dorsal is to the left. (E) Stage 6 embryo shortly after division of 1D into 2d and 2D. Animal pole view. The arrowhead indicates a micromere (out of focus here) associated with 2d. (F) Cleared stage 6 embryo viewed from the left side. Broken lines demarcate the contour of cells 2d and 2D. A pair of arrows indicate the boundary between these two cells. The arrowhead indicates a micromere exhibiting Ttu-fel staining signal. (G) Left-side view of embryo at the transition to stage 7. 2D is now forming a protrusion (arrowhead), which will be 3d micromere (see Fig. 1F). Note that Ttu-fel RNA is concentrated in this protrusion. (H) Left-side view of stage 8 embryo. Note concentration of Ttu-fel RNA in micromeres (arrowheads). (I) Dorsoanterior view of another stage 8 embryo. Arrowheads indicate micromeres located around 2d11 (derived from 2d). (J) Stage 11 embryo shortly after division of 2d111 (derived from 2d11) into a pair of NOPQ proteloblasts. Posterior view with dorsal to the top. Arrowheads indicate the position of micromeres. Note the absence of Ttu-fel staining in either NOPQ, M teloblast or micromeres. E, endodermal precursor. Scale bar in (J): (AeJ) 100 mm.
are eliminated abruptly around the time of the sixth cleavage in the D quadrant and that the level of Ttu-fel mRNA does not appear to alter significantly in developing embryos before this elimination. As to the mechanisms for maternal transcript degradation, it has recently been suggested that transcript destabilization is achieved through the combined action of at least two types of degradation activity, i.e., maternally encoded ('maternal') activity that functions upon egg activation and 'zygotic' activity that requires zygotic transcription (Tadros and Lipshitz, 2009). If these two types of degradation activity are present in Tubifex as well, it seems likely that maternal transcripts of Ttu-fel are targeted solely by the zygotic degradation pathway but not by the maternal pathway. In fact, in Tubifex, zygotic transcription occurs as late as the beginning of the sixth cleavage (Aoki and Shimizu, unpublished data). The present Northern blot analysis has also shown that Ttu-fel mRNA reduces its size by approximately 300 bp prior to its elimination. In view of the fact that mRNA degradation begins with the removal of its poly (A) tail (Wang et al., 2002; Schier, 2007; Tadros and Lipshitz, 2009), it is conceivable that the slight reduction of Ttufel mRNA size results from deadenylation that occurs soon after fertilization. If this is the case, it appears that for Ttu-fel transcripts,
deadenylation and degradation are temporally uncoupled. It is known that in the frog Xenopus laevis, fertilization-induced deadenylation does not trigger decay until after the onset of zygotic transcription that occurs during the blastula stages (Newport and Kirschner, 1982; Voeltz and Steitz, 1998). At present, it is also possible that the size reduction of Ttu-fel mRNA is ascribable to zygotic splicing, if the cDNA represents an incompletely spliced transcript. 2. Experimental procedures 2.1. Embryos, juveniles and adults Embryos of the freshwater oligochaete T. tubifex were obtained as described previously (Shimizu, 1982) and cultured at 22 C. For experiments, embryos were freed from cocoons in the culture medium (Shimizu, 1982). Newly hatched juveniles were reared to adulthood under laboratory conditions (at 22 C) as described previously (Shiomi, 2014). Briefly, they were put in a plastic petri dish containing a sand (tiny particles) layer and tap water, and fed with a small amount of yeast suspension once a week. Unless
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hybridization between two cDNA populations using a PCR-select cDNA subtraction kit (Clontech). As starting material to do so, we prepared total RNAs from 2d cells of 10-cell embryos and 4d cells of 22-cell embryos. Two cDNA populations called “tester” and “driver” were synthesized using RNAs isolated from 2d cells and 4d cells, respectively, and subtractive hybridization was done between these cDNA populations according to the manufacturer's protocols. A resulting pool of subtracted cDNA that was enriched in sequences preferentially expressed in 2d cells (which will be referred to as “2d-specific cDNA” hereafter) was amplified by PCR and labeled with digoxigenin (DIG). A T. tubifex stage 1 cDNA library that was constructed using a Creator SMART cDNA library construction kit (Clontech) was screened using DIG-labeled “2d-specific cDNA” under high stringent conditions (Sambrook and Russell, 2001). Positive clones (500) were isolated randomly, and plasmids were purified therefrom. Insert DNA of each of the clones was amplified by PCR and used as a template for synthesis of DIG-labeled RNA probes, which were used for preliminary whole-mount in situ hybridization (WISH). 2.4. Northern blotting For Northern blot analysis, 10 mg each of total RNA was separated by agarose gel electrophoresis and transferred to Hybond-Nþ membrane (Amersham Biosciences). The membrane was baked for 1 h at 80 C prior to hybridization. Hybridization was performed with DIG-labeled RNA antisense probe prepared according to Matsuo et al. (2005). Hybridization signals were detected by chromogenic reaction using BCIP and NBT (Matsuo et al., 2005). Fig. 6. Expression of Ttu-fel in genital segments of a young adult worm. Fragments containing genital segments that were dissected out from adults were processed for WISH as described in Experimental procedures. (A) Dorsal view of a fragment containing segments X-XIII, showing a spermatheca (st) in segment X and a pair of ovaries in segment XI. Anterior is to the top. Vertical lines with roman numerals indicate approximate position of segments. The square bracket indicates oocytes undergoing yolk deposition in the ovisac. Note intense Ttu-fel expression in the ovary. (B) Enlargement of the ovaries shown in (A). Square brackets indicate oocytes in the most dorsal portion of the right-hand ovary. Note that these oocytes exhibit more intense Ttu-fel expression than younger oocytes (arrows) do. (C) Diagrammatic illustration of the reproductive system in T. tubifex. Sperm funnel and vas deferens are omitted. Dorsal view of segments X-XIII is shown. Asterisks indicate sperm sac. Note spermathecae (st) in segment X and ovaries in segment XI. os, ovisac; ts, testis. After Hirao (1964) and Jaana (1982). Scale bars: (A and B) 200 mm.
otherwise stated, all experiments were carried out at room temperature (20e22 C). 2.2. RNA isolation and cDNA synthesis Total RNA was isolated from Tubifex embryos by using ISOGEN (Nippon Gene) according to the manufacturer's recommendations. Poly (A)þ RNA was isolated from total RNA using Oligotex-MAG (TaKaRa) according to the manufacturer's instructions. Isolated total RNA and poly (A)þRNA were dissolved in RNase-free sterilized H2O and stored at 80 C until use. First-strand cDNA was synthesized with a NotI-dT18 primer using Time Saver cDNA Synthesis Kit (Amersham Pharmacia Biotech) according to the manufacturer's protocols. 2.3. Subtraction hybridization and library screening To identify maternal transcripts which are preferentially segregated to 2d cells but not to 4d cells, we performed subtraction
2.5. Whole-mount in situ hybridization (WISH) DIG-labeled RNA probes were prepared according to the protocols described by Matsuo et al. (2005). An antisense and sense riboprobes were synthesized with T7 and T3 RNA polymerases, respectively. Embryos, juveniles and young adults that were to be processed for WISH were fixed with 4% formaldehyde according to the methods described by Matsuo et al. (2005) and Mohri et al. (2016). After a brief rinse in PBST (PBS plus 0.1% Tween-20), embryos were freed from vitelline membrane with fine forceps. Fixed juveniles and adults were transferred to PBST, and cut into short fragments comprising five to six segments by means of razor blade; fragments containing genital segments (X and XI) were collected, and with fine forceps, not only an epithelial layer but also a muscle layer were torn off from the dorsal half of each fragment. This treatment was expected to facilitate penetration of proteinase K and riboprobes toward the mesodermal tissues of the embryo. Embryos and fragments (of juveniles and adults) thus obtained were dehydrated with a graded series of methanol, and stored at 20 C in methanol until use. For WISH, embryos, juveniles and adults that had been stored in methanol were rehydrated and processed according to the protocols described by Matsuo et al. (2005), except that juveniles and adults were treated with proteinase K (2 mg/ml) for 20 min and that hybridization (24 h) and subsequent wash were both performed at 60 C. Hybridization signals were detected by chromogenic reaction using BCIP and NBT. Whole-mount processed (stained) embryos were fixed with 3.5% formaldehyde in phosphate buffer (pH 7.4) for 12 h, mounted in PBST, and observed under incident light. Some stained embryos were cleared according to the method described by Matsuo and Shimizu (2006) and observed with transmitted light.
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2.6. Bisection of stained embryos Some of the embryos that had been stained for Ttu-fel as described above were bisected with razor blade in PBST according to the method described by Shimizu (1988). The plane of bisection included both the animal-vegetal and anteroposterior axes of the embryo. Sections of the bisected embryos were observed under incident light. Acknowledgements We thank the Shimizu lab for helpful advice. We are also grateful to Dr. Ayaki Nakamoto (Tohoku University) for his help in our access to key references. This work was supported by the Department of Biological Sciences. References Bissen, S.T., Weisblat, D.A., 1991. Transcription in leech: mRNA synthesis is required for early cleavages in Helobdella embryos. Dev. Biol. 146, 12e23. Bissen, S.T., Smith, C.M., 1996. Unequal cleavage in leech embryos: zygotic transcription is required for correct spindle orientation in a subset of early blastomeres. Development 122, 599e606. s, Y., Weisblat, D.A., 2014. Differential expression of conserved germ Cho, S., Valle line markers and delayed segregation of male and female primordial germ cells in a hermaphrodite, the leech Helobdella. Mol. Biol. Evol. 31, 341e354. Davidson, E.H., 1986. Gene Activity in Early Development. Academic Press, Orland. Hirao, Y., 1964. Reproductive system and oogenesis in the freshwater oligochaete, Tubifex hattai. J. Fac. Sci. Hokkaido Univ. Ser. VI, Zool. 15, 439e448. Jaana, H., 1982. The ultrastructure of the epithelial lining of the male genital tract and its role in spermatozeugma formation in Tubifex hattai Nomura (Annelida, Oligochaeta). Zool. Anz. (Jena) 209, 159e176. Kang, D., Pilon, M., Weisblat, D.A., 2002. Maternal and zygotic expression of a nanosclass gene in the leech Helobdella robusta: primordial germ cells arise from segmental mesoderm. Dev. Biol. 245, 28e41. Kostriken, R., Weisblat, D.A., 1992. Expression of a wnt gene in embryonic epithelium of the leech. Dev. Biol. 151, 225e241. Master, V.A., Kourakis, M.J., Martindale, M.Q., 1996. Isolation, characterization, and expression of Le-msx, a maternally expressed member of the msx gene family from the glossiphoniid leech, Helobdella. Dev. Dyn. 207, 404e419. Matsuo, K., Shimizu, T., 2006. Embryonic expression of a decapentaplegic genes in the oligochaete annelid Tubifex tubifex. Gene Expr. Patterns 6, 800e806. Matsuo, K., Yoshida, H., Shimizu, T., 2005. Differential expression of caudal and dorsal genes in the teloblast lineages of the oligochaete annelid Tubifex tubifex.
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Dev. Genes Evol. 215, 238e247. Mohri, K., Nakamoto, A., Shimizu, T., 2016. The ontogeny of nanos homologue expression in the oligochaete annelid Tubifex tubifex. Gene Expr. Patterns 20, 32e41. Newport, J., Kirschner, M., 1982. A major developmental transition in early Xenopus embryos. I. Characterization and timing of cellular changes at the midblastula stage. Cell 30, 675e686. Oyama, A., Shimizu, T., 2007. Transient occurrence of vasa-expressing cells in nongenital segments during embryonic development in the oligochaete annelid Tubifex tubifex. Dev. Genes Evol. 217, 675e690. Oyama, A., Yoshida, H., Shimizu, T., 2008. Embryonic expression of p68, a DEAD-box RNA helicase, in the oligochaete annelid Tubifex tubifex. Gene Expr. Patterns 8, 464e470. Sambrook, J., Russell, D.W., 2001. Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Savage, R.M., Shankland, M., 1996. Identification and characterization of a hunchback orthologue, Lzf2, and its expression during leech embryogenesis. Dev. Biol. 175, 205e217. Schier, A.F., 2007. The maternal-zygotic transition: death and birth of RNAs. Science 316, 406e407. Schmerer, M.W., Null, R.W., Shankland, M., 2013. Developmental transition to bilaterally symmetric cell divisions is regulated by Pax-mediated transcription in embryos of the leech Helobdella austinensis. Dev. Biol. 382, 149e159. Shimizu, T., 1982. Development in the freshwater oligochaete Tubifex. In: Harrison, F.W., Cowden, R.R. (Eds.), Developmental Biology of Freshwater Invertebrates. Alan R. Liss, New York, pp. 286e316. Shimizu, T., 1986. Bipolar segregation of mitochondria, actin network, and surface in the Tubifex egg: role of cortical polarity. Dev. Biol. 116, 241e251. Shimizu, T., 1988. Localization of actin networks during early development of Tubifex embryos. Dev. Biol. 125, 321e331. Shimizu, T., 1989. Asymmetric segregation and polarized redistribution of pole plasm during early cleavages in the Tubifex embryo: role of actin networks and mitotic apparatus. Dev. Growth Differ. 31, 283e297. Shiomi, I., 2014. Experimental Studies on the Ontogeny of Germ Cells in the Oligochaete Annelid Tubifex Tubifex. Master's Thesis. Graduate School of Life Science, Hokkaido University. Simakov, O., et al., 2013. Insights into bilaterian evolution from three spiralian genomes. Nature 493, 526e531. Soto, J.G., Nelson, B.H., Weisblat, D.A., 1997. A leech homolog of twist: evidence for its inheritance as a maternal mRNA. Gene 199, 31e39. Tadros, W., Lipshitz, H.D., 2009. The maternal-to-zygotic transition: a play in two acts. Development 136, 3033e3042. Voeltz, G.K., Steitz, J.A., 1998. AUUUA sequences direct mRNA deadenylation uncoupled from decay during Xenopus early development. Mol. Cell. Biol. 18, 7537e7545. Wang, Y., Liu, C.L., Storey, J.D., Tibshirani, R.J., Herschlag, D., Brown, P.O., 2002. Precision and functional specificity in mRNA decay. Proc. Natl. Acad. Sci. U. S. A. 99, 5860e5865.
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Gene Expression Patterns journal homepage: http://www.elsevier.com/locate/gep
Embryonic expression of festina lente (fel), a novel maternal gene, in the oligochaete annelid Tubifex tubifex Takuma Nakamura a, Inori Shiomi a, Takashi Shimizu a, b, * a b
Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2016 Received in revised form 27 April 2017 Accepted 3 May 2017 Available online 4 May 2017
We have cloned and characterized the expression of a novel maternal gene festina lente (designated Ttufel) from the clitellate annelid Tubifex tubifex. Northern blot analyses have shown that Ttu-fel mRNA is approximately 8 kbp in length and that its expression is restricted to oocytes undergoing maturation division and early embryos up to 22-cell stage. Maternal transcripts of Ttu-fel are first detected in oocytes in the ovary of young adults (ca. 40 days after hatching); its expression continues in growing oocytes in the ovisac. Ttu-fel mRNA is distributed broadly throughout the egg undergoing maturation divisions. During the process of ooplasmic segregation that results in the pole plasm formation, Ttu-fel mRNA becomes concentrated to the animal and vegetal poles. The RNA in the animal hemisphere is distributed in a gradient with highest concentration in the cortical region. During the first two cleavages, Ttu-fel mRNA is segregated to CD cell then to D cell; it is subsequently inherited by the three D quadtrant micromeres, 1d, 2d and 3d. Around the time of transition to 22-cell stage, Ttu-fel mRNA becomes undetectable throughout the embryo. © 2017 Elsevier B.V. All rights reserved.
Keywords: Novel maternal gene Clitellate annelid Oligochaete Tubifex tubifex 2d micromere Early cleavage Spiralian Lophotrochozoan Maternal transcript degradation
Early development in the animal embryo is driven by maternally provided gene products such as mRNAs and proteins, though at some point during development there is a transition to control by zygotic gene products (Davidson, 1986). Recent studies on model organisms such as nematode, fruit fly and mouse have demonstrated that the first event of the maternal-to-zygotic transition (MZT) is the elimination of maternal transcripts and that 30e35% of maternal mRNAs are destabilized during the period of this transition in these animals (Schier, 2007; Tadros and Lipshitz, 2009). Given that the MZT occurs in all animal embryos, it is highly possible that similar maternal mRNA elimination takes place during early development in all metazoans. Although there have been few systematic studies on the events occurring during the MZT in spiralian lophotrochozoans, it has been known that in the clitellate annelid (leech) Helobdella, zygotic transcripts are needed for early cleavages (Bissen and Weisblat, 1991; Bissen and Smith, 1996; Schmerer et al., 2013). Similar early requirement for zygotically produced mRNAs has recently been
* Corresponding author. Kitano 6-1-8-17, Kiyota-ku, Sapporo 004-0866, Japan. E-mail address: [email protected] (T. Shimizu). http://dx.doi.org/10.1016/j.gep.2017.05.001 1567-133X/© 2017 Elsevier B.V. All rights reserved.
suggested in unequal cleavage of the teloblast precursor cell 2d in another clitellate annelid (oligochaete) Tubifex (M. Aoki and T. Shimizu, unpublished data). On the other hand, in these annelids, not a few maternal genes have been cloned and examined for their expression patterns, including Tubifex (Ttu) homologues of dorsal (dl), nanos (nos), p68 and vasa (vas) (Matsuo et al., 2005; Oyama and Shimizu, 2007; Oyama et al., 2008; Mohri et al., 2016) and Helobdella (He) homologues of hunchback, msx, nos, piwi, twist, vas and wnt (Kostriken and Weisblat, 1992; Master et al., 1996; Savage and Shankland, 1996; Soto et al., 1997; Kang et al., 2002; Cho et al., 2014). Semi-quantitative analyses have shown that maternally supplied transcripts of Ttu-nos, He-nos, He-vas and He-piwi decrease in amount during early cleavage stages and that this decrease is followed by upregulation of their expression that occurs during mid development (Kang et al., 2002; Cho et al., 2014; Mohri et al., 2016). In contrast, maternal transcripts of Ttu-p68 and Ttu-vas seem not to decrease in amount significantly during early and mid development, but do so during late development (Oyama and Shimizu, 2007; Oyama et al., 2008). In this study, we have isolated a novel maternal gene named festina lente (fel) from Tubifex tubifex. Compared with other maternal genes described so far, this gene is apparently unique in
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that its maternal mRNA is detected exclusively during the period of early cleavage stages and that it is inherited by the second micromere (2d) but not by the fourth micromere (4d) of the D quadrant. 1. Results and discussion 1.1. Summary of early development of T. tubife A brief review of Tubifex development is presented here as a background for the observations described below (for details, see Shimizu, 1982). Tubifex zyogtes, which are oviposited at metaphase I, form two polar bodies and then enter the first mitosis. Before the first cleavage, yolk-deficient cytoplasm called pole plasm accumulates at both poles of the egg (Fig. 1A). The early development of Tubifex consists of a stereotyped sequence of cell divisions. The first cleavage of the Tubifex egg is unequal and meridional, and produces a smaller AB cell and a larger CD cell (Fig. 1B). The second cleavage is also meridional and yields cells A, B, C and D: the CD cell divides into a smaller C cell and a larger D cell while the AB cell separates into cells A and B of various sizes (Fig. 1C). From the third cleavage on, the quadrants A, B and C repeat unequal divisions three times, and the D quadrant four times, producing micromeres at the animal side and macromeres at the vegetal side. The D quadrant micromeres are designated 1d, 2d, 3d and 4d (Fig. 1DeG). Cells 2d and 4d are much larger than cells 1d and 3d. During cleavages, the pole plasms are inherited by the D lineage cells and finally partitioned into 2d and 4d. At 22-cell stage, 2d11 (derived from 2d), 4d and 4D (sister cell of 4d) all come to lie in the future midline of the embryo (Fig. 1G). These three cells undergo bilateral cell division in the next cleavage: 4d divides equally yielding the left and right mesoteloblasts M (Fig. 1H); 4D divides equally into endodermal precursors E (Fig. 1I); and 2d111 (derived from 2d11) divides into a bilateral pair of ectoteloblast precursors NOPQ (Fig. 1J). The quadrant cells A, B and C also divide equally at the sixth cleavage. 1.2. Cloning of a “2d-cell-specific” gene, festina lente, from T. tubifex During the course of preliminary whole-mount in situ hybridization (WISH) using riboprobes that had been synthesized from 'positive' clones resulting from screening of a 1-cell-stage cDNA
library (see Experimental procedures), we found that a riboprobe synthesized from clone 4038 stained 2d cell but not 4d cell of a 22cell embryo. (Those from the remaining clones stained both 2d and 4d of the same embryos.) This preliminary observation suggests that clone 4038 contains a Tubifex gene whose transcripts are segregated preferentially to 2d cell. As described later, this gene is a novel maternal gene from spiralian lophotrochozoans, and its expression is restricted to early cleavage stage embryos. Hereafter we will name this gene festina lente (fel). Clone 4038 was found to contain a 1852-bp cDNA sequence which includes a 40-bp poly-A tail at the 30 end (DDBJ/GenBank accession number LC140925). To examine whether the 1812-bp sequence represents full length of Ttu-fel transcripts, we performed a Northern blot analysis using RNA isolated from various stages of Tubifex development. We found that “intact” Ttu-fel transcripts were approximately 8 kbp in length (Fig. 2). This
Fig. 2. Expression of Ttu-fel mRNA. Northern blot analysis of total RNA (10 mg each) of (A) embryos at stages 1, 2e8, 9e13, 14e18, and Juv (10-day-old juveniles) or (B) embryos at stages 1e2, 4e6, 7, 8, and 9e10. Total RNA was prepared and electrophoresed as described in Experimental procedures. The membrane was probed with DIG-labeled RNA. For stages 1e11, see Fig. 1. Stage 12, ectodermal teloblastogenesis; stages 13e15, germ band formation; stages 16e18, body elongation accompanied by formation of segmental ectoderm and mesoderm (for details of developmental stages, see Shimizu, 1982).
Fig. 1. Summary of Tubifex early development. Diagrammatic illustration of stages 1e11 of embryonic development (stage 3 is omitted). (AeC) Animal pole view of embryos at stages 1-cell (A), 2-cell (B) and 4-cell (C). pp, pole plasm. (DeG) Animal pole view of embryos undergoing the formation of the D quadrant micromeres 1d (D), 2d (E), 3d (F) and 4d (G). (HeJ) Posterior view of embryos undergoing bilateral cell division in 2d111 (derived from 2d11), 4d and 4D. (H) 4d divides into a pair of mesoteloblasts (M). (I) 4D divides into a pair of endodermal precursors (E). (J) 2d111 divides into a pair of ectoteloblast precursors (NOPQ) (Redrawn from Shimizu, 1982).
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suggests that the 1812-bp sequence (LC140925) represents only one fifth of the complete sequence. We have tried to obtain additional sequence of Ttu-fel by using commercially available 50 RACE PCR kits, but all attempts so far have been unsuccessful. This sequence does not have a significant BLAST hit when compared against sequences from three spiralian genomes (i.e., owl limpet Lottia gigantea, marine polychaete Capitella teleta and freshwater leech Helobdella robusta; see Simakov et al., 2013). The failure to find convincing orthologues in other invertebrates would not be too surprising if this sequence is some type of non-coding transcript or is derived entirely from 30 -UTR (untranslated region). On the other hand, it should be mentioned that we obtained an ORF (open reading frame) of 275 amino acids within the 1821-bp sequence by using the ORF Finder tool at NCBI (Fig. 3A). This putative protein lacks obvious orthologues by BLAST in invertebrate genomes though its internal portion showed 33e35% amino acid identity to myosin molecules of some spiralians (Fig. 3B). This may suggest a possibility that the cDNA represents an incompletely spliced transcript. At present it is equally possible that the ORF is only a “fluke”, though. 1.3. Temporal expression pattern of Ttu-fel The temporal expression profile was analyzed by Northern blot. The results are shown in Fig. 2. Ttu-fel mRNA was present at detectable level exclusively in early embryos undergoing first five cleavage divisions (Fig. 2A). The level of Ttu-fel expression appeared not to alter significantly before its abrupt decrease that occurs at the time of transition to stage 8 (Fig. 2B). Close examination of the blots also showed that Ttu-fel mRNA reduces its size by approximately 300 bp in length during the period of these cleavages. 1.4. Spatial expression pattern of Ttu-fel Whole-mount in situ hybridization (WISH) staining using antisense probes revealed the presence of easily detectable levels of Ttu-fel mRNA exclusively in early cleavage stage embryos (i.e.,
31
stages 1e8; Fig. 1AeG). In embryos processed for in situ hybridization with sense probe no staining was detected from stage 1 to stage 18 (completion of embryogenesis; data not shown). During maturation division, the mRNA was present throughout the egg though the animal pole with the meiotic spindle exhibited higher level of expression than any other region of the egg (Fig. 4A). When pole plasm (i.e., yolk-deficient cytoplasm) began to form at both poles (see Fig. 1A), the mRNA was present in a thin layer along the surface of both poles (Fig. 4B). As pole plasm formation proceeded, Ttu-fel mRNA accumulated to the pole plasm domains (Fig. 4C and D) and concentrated to the poles (Fig. 4E and F). Ttu-fel transcripts in the animal hemisphere were distributed at higher density in the cortex than in more inner regions (Fig. 4D). By contrast, Ttu-fel transcripts in the vegetal hemisphere were distributed rather uniformly (Fig. 4D). In Tubifex, when pole plasm formation completes, the animal pole region protrudes outward (Shimizu, 1986). If one sees such a protruding animal pole region end-on with the animalvegetal axis, it is found that Ttu-fel mRNA is distributed in a ring (Fig. 4E, inset). Although the existence of maternal transcripts in the pole plasm domain has also been reported for some genes such as Ttu-dl, Ttu-p68, Ttu-vas and Ttu-nos (Matsuo et al., 2005; Oyama and Shimizu, 2007; Oyama et al., 2008; Mohri et al., 2016), transcripts of these genes appear to be distributed rather uniformly even in the animal pole plasm domain. During the first two cleavages, Ttu-fel mRNA is segregated to CD cell and then to D cell (Fig. 5A and B). Animal and vegetal pools of transcripts, which remained to be separate from each other shortly after the second division, were unified at the animal pole upon the beginning of the third division (data not shown). It is known that pole plasms are unified at the animal pole prior to the onset of the third cleavage (Shimizu, 1989). When the third cleavage completed, Ttu-fel mRNA was found to be distributed to both 1d and 1D (Fig. 5C and D). Subsequently a large portion of the RNA was segregated to 2d cell at the fourth cleavage (Fig. 5E and F). The bulk of Ttu-fel RNA left in 2D cell was finally inherited by 3d cell at the fifth cleavage (Fig. 5G). When 3D divided into 4d and 4D, there was no longer any sign of the mRNA in these cells even if stained embryos were
Fig. 3. Characterization of Ttu-fel, a novel gene from Tubifex tubifex. (A) Amino acid sequence of a putative Ttu-fel protein. The portion underlined was used for alignment (B) Alignment of the putative Ttu-fel protein with myosin molecules from two spiralian species. The portion underlined in (A) was used for alignment. Asterisks represent amino acid identity. Numbers in parentheses indicate the percentage amino acid identity with Ttu-fel. The following sequences were used: Cgi-myosin (accession number K1RSS3), Pdu-myosin (KJ405466), and Ttu-fel (LC140925). Species abbreviations: Cgi Crassostrea gigas (mollusc); Pdu Platynereis dumerilii (annelid); Ttu Tubifex tubifex (annelid).
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Fig. 4. Expression of Ttu-fel detected by in situ hybridization with an antisense riboprobe. (A)e(E), uncleared specimens viewed with incident light; (F), cleared specimen viewed with transmitted light. (A) Animal pole view of metaphase II egg. The arrow indicates the animal pole showing Ttu-fel staining. (B)e(F) Eggs at 40 (B), 60 (C), 80 (D), and 100 (E and F) min after the second polar body formation. (B) Animal pole view. Note Ttu-fel staining around the animal pole. The inset shows a section of bisected egg. Note a layer of Ttu-fel staining along the animal pole surface. (C) and (D) Sections of bisected eggs. AP, animal pole; VP, vegetal pole. (E) Animal pole view. Note accumulation of Ttu-fel RNA to the animal pole (AP). The inset shows the animal pole region viewed end on with the animal-vegetal axis. (F) Same egg as shown in (E). Note the accumulation of Ttu-fel RNA to both poles. AP, animal pole; VP, vegetal pole. Scale bar in (A): (AeF) 100 mm.
cleared and observed with transmitted light (data not shown). In contrast, four small cells (i.e., 1d, 2d2, 2d12 and 3d) of the same embryo still exhibited staining signal though much weaker than before (Fig. 5H and I). Around the time when 2d111 (derived from 2d11 of stage 8 embryo; Fig. 1G) divided into a bilateral pair of NOPQ cells (see Fig. 1J), Ttu-fel mRNA became undetectable throughout the embryo (Fig. 5J). Consistent with the result of Northern blot analysis (Fig. 2A), Ttu-fel mRNA was undetectable during the rest of embryogenesis, either (data not shown). As described above, deposited eggs (oocytes) in the cocoon (Fig. 4A) contain Ttu-fel mRNA, which is to be used as maternal RNA during early cleavage stages. This implies that Ttu-fel mRNA must be transcribed at a certain stage of oogenesis. To find out when transcription of maternal Ttu-fel mRNA begins, we examined juveniles and adults for Ttu-fel expression. It is known that juveniles of T. tubifex grow up to adulthood within 40 days after hatching under laboratory conditions (at 22 C; Shiomi, 2014). We reared juveniles for 5, 14, 28 or 40 days before fixation and processed them for Ttu-fel staining (see Experimental procedures). We found that positive signals of Ttu-fel staining were detected exclusively in 40day-old specimens (i.e., adults). Ttu-fel mRNA was localized in ovaries in segment XI and oocytes in the ovisac in segments XII and XIII (Fig. 6A). Ttu-fel-positive cells were seen in the dorsal portion of the ovary (Fig. 6B). In contrast, the ventral portion of the ovary appeared to be devoid of positive signals of Ttu-fel staining (data not shown). In Tubifex, oogonia (in the multiplicative phase) occupy the most ventral portion of the ovary and extend toward the dorsal side generating oocytes (see Fig. 6C; for details, see Hirao, 1964). (Usually, 7e8 oocytes are released from the dorsal portion of the ovary to the ovisac, where the oocytes undergo yolk deposition and maturation; see Hirao, 1964) Judging from their location in the ovary, it is likely that Ttu-fel is expressed in oocytes but not in oogonia.
1.5. Ttu-fel is a novel maternal gene from the spiralian lophotrochozoans The embryonic expression pattern of Ttu-fel, revealed in this study, is summarized as follows. Maternal transcripts of Ttu-fel, which are segregated to the first three micromeres (i.e., 1d, 2d and 3d) of the D quadrant during cleavage stages, are eliminated from the embryo as late as 22-cell stage; this decay of Ttu-fel mRNA is not followed by zygotic activation of this gene, which resumes only when female germ cells enter meiosis in the ovary. To our knowledge, this is the first report of a lophotrochozoan maternal gene whose expression is restricted strictly to early cleavage stages. Compared with other Tubifex maternal genes that have so far been examined for their expression patterns, Ttu-fel appears to be unique in two respects. First, Ttu-fel mRNA is segregated only to 2d cell but not to 4d cell. Transcripts of Ttu-nos and Ttu-vas are inherited by both 2d and 4d (Oyama and Shimizu, 2007; Mohri et al., 2016); those of Ttu-p68 are segregated only to 4d cell but not to 2d cell (Oyama et al., 2008). Second, transcripts of Ttu-fel are detected only in oocytes and early embryos undergoing first five cleavages; Ttu-fel mRNA is undetectable during the rest of embryogenesis. By contrast, Ttu-nos, Ttu-p68 and Ttu-vas are expressed almost throughout embryogenesis, although their expression levels fluctuate during development. As described before, a number of maternal genes have been examined for their expression patterns in other spiralian lophotrochozoans as well (Kostriken and Weisblat, 1992; Master et al., 1996; Savage and Shankland, 1996; Soto et al., 1997; Kang et al., 2002; Cho et al., 2014), but their embryonic expression persists through to late development. This may suggest that Ttu-fel is a novel maternal gene from the spiralian lophotrochozoans. 1.6. Maternal transcript degradation The present study has shown that maternal transcripts of Ttu-fel
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Fig. 5. Expression of Ttu-fel during the period of early cleavages. All embryos except (F) were uncleared and viewed with incident light. (A) Stage 2 embryo consisted of cells AB and CD. Animal pole view. (B) Stage 3 embryo consisted of three cells, AB, C and D. Animal pole view. (C) Stage 5 embryo consisted for four macromeres (1A-1D) and four micromeres. Animal pole view. Individual micromeres are not depicted here. (D) Section of stage 5 embryo bisected along the animal-vegetal (dorsoventral) and anteroposterior axes. Anterior is to the top; dorsal is to the left. (E) Stage 6 embryo shortly after division of 1D into 2d and 2D. Animal pole view. The arrowhead indicates a micromere (out of focus here) associated with 2d. (F) Cleared stage 6 embryo viewed from the left side. Broken lines demarcate the contour of cells 2d and 2D. A pair of arrows indicate the boundary between these two cells. The arrowhead indicates a micromere exhibiting Ttu-fel staining signal. (G) Left-side view of embryo at the transition to stage 7. 2D is now forming a protrusion (arrowhead), which will be 3d micromere (see Fig. 1F). Note that Ttu-fel RNA is concentrated in this protrusion. (H) Left-side view of stage 8 embryo. Note concentration of Ttu-fel RNA in micromeres (arrowheads). (I) Dorsoanterior view of another stage 8 embryo. Arrowheads indicate micromeres located around 2d11 (derived from 2d). (J) Stage 11 embryo shortly after division of 2d111 (derived from 2d11) into a pair of NOPQ proteloblasts. Posterior view with dorsal to the top. Arrowheads indicate the position of micromeres. Note the absence of Ttu-fel staining in either NOPQ, M teloblast or micromeres. E, endodermal precursor. Scale bar in (J): (AeJ) 100 mm.
are eliminated abruptly around the time of the sixth cleavage in the D quadrant and that the level of Ttu-fel mRNA does not appear to alter significantly in developing embryos before this elimination. As to the mechanisms for maternal transcript degradation, it has recently been suggested that transcript destabilization is achieved through the combined action of at least two types of degradation activity, i.e., maternally encoded ('maternal') activity that functions upon egg activation and 'zygotic' activity that requires zygotic transcription (Tadros and Lipshitz, 2009). If these two types of degradation activity are present in Tubifex as well, it seems likely that maternal transcripts of Ttu-fel are targeted solely by the zygotic degradation pathway but not by the maternal pathway. In fact, in Tubifex, zygotic transcription occurs as late as the beginning of the sixth cleavage (Aoki and Shimizu, unpublished data). The present Northern blot analysis has also shown that Ttu-fel mRNA reduces its size by approximately 300 bp prior to its elimination. In view of the fact that mRNA degradation begins with the removal of its poly (A) tail (Wang et al., 2002; Schier, 2007; Tadros and Lipshitz, 2009), it is conceivable that the slight reduction of Ttufel mRNA size results from deadenylation that occurs soon after fertilization. If this is the case, it appears that for Ttu-fel transcripts,
deadenylation and degradation are temporally uncoupled. It is known that in the frog Xenopus laevis, fertilization-induced deadenylation does not trigger decay until after the onset of zygotic transcription that occurs during the blastula stages (Newport and Kirschner, 1982; Voeltz and Steitz, 1998). At present, it is also possible that the size reduction of Ttu-fel mRNA is ascribable to zygotic splicing, if the cDNA represents an incompletely spliced transcript. 2. Experimental procedures 2.1. Embryos, juveniles and adults Embryos of the freshwater oligochaete T. tubifex were obtained as described previously (Shimizu, 1982) and cultured at 22 C. For experiments, embryos were freed from cocoons in the culture medium (Shimizu, 1982). Newly hatched juveniles were reared to adulthood under laboratory conditions (at 22 C) as described previously (Shiomi, 2014). Briefly, they were put in a plastic petri dish containing a sand (tiny particles) layer and tap water, and fed with a small amount of yeast suspension once a week. Unless
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hybridization between two cDNA populations using a PCR-select cDNA subtraction kit (Clontech). As starting material to do so, we prepared total RNAs from 2d cells of 10-cell embryos and 4d cells of 22-cell embryos. Two cDNA populations called “tester” and “driver” were synthesized using RNAs isolated from 2d cells and 4d cells, respectively, and subtractive hybridization was done between these cDNA populations according to the manufacturer's protocols. A resulting pool of subtracted cDNA that was enriched in sequences preferentially expressed in 2d cells (which will be referred to as “2d-specific cDNA” hereafter) was amplified by PCR and labeled with digoxigenin (DIG). A T. tubifex stage 1 cDNA library that was constructed using a Creator SMART cDNA library construction kit (Clontech) was screened using DIG-labeled “2d-specific cDNA” under high stringent conditions (Sambrook and Russell, 2001). Positive clones (500) were isolated randomly, and plasmids were purified therefrom. Insert DNA of each of the clones was amplified by PCR and used as a template for synthesis of DIG-labeled RNA probes, which were used for preliminary whole-mount in situ hybridization (WISH). 2.4. Northern blotting For Northern blot analysis, 10 mg each of total RNA was separated by agarose gel electrophoresis and transferred to Hybond-Nþ membrane (Amersham Biosciences). The membrane was baked for 1 h at 80 C prior to hybridization. Hybridization was performed with DIG-labeled RNA antisense probe prepared according to Matsuo et al. (2005). Hybridization signals were detected by chromogenic reaction using BCIP and NBT (Matsuo et al., 2005). Fig. 6. Expression of Ttu-fel in genital segments of a young adult worm. Fragments containing genital segments that were dissected out from adults were processed for WISH as described in Experimental procedures. (A) Dorsal view of a fragment containing segments X-XIII, showing a spermatheca (st) in segment X and a pair of ovaries in segment XI. Anterior is to the top. Vertical lines with roman numerals indicate approximate position of segments. The square bracket indicates oocytes undergoing yolk deposition in the ovisac. Note intense Ttu-fel expression in the ovary. (B) Enlargement of the ovaries shown in (A). Square brackets indicate oocytes in the most dorsal portion of the right-hand ovary. Note that these oocytes exhibit more intense Ttu-fel expression than younger oocytes (arrows) do. (C) Diagrammatic illustration of the reproductive system in T. tubifex. Sperm funnel and vas deferens are omitted. Dorsal view of segments X-XIII is shown. Asterisks indicate sperm sac. Note spermathecae (st) in segment X and ovaries in segment XI. os, ovisac; ts, testis. After Hirao (1964) and Jaana (1982). Scale bars: (A and B) 200 mm.
otherwise stated, all experiments were carried out at room temperature (20e22 C). 2.2. RNA isolation and cDNA synthesis Total RNA was isolated from Tubifex embryos by using ISOGEN (Nippon Gene) according to the manufacturer's recommendations. Poly (A)þ RNA was isolated from total RNA using Oligotex-MAG (TaKaRa) according to the manufacturer's instructions. Isolated total RNA and poly (A)þRNA were dissolved in RNase-free sterilized H2O and stored at 80 C until use. First-strand cDNA was synthesized with a NotI-dT18 primer using Time Saver cDNA Synthesis Kit (Amersham Pharmacia Biotech) according to the manufacturer's protocols. 2.3. Subtraction hybridization and library screening To identify maternal transcripts which are preferentially segregated to 2d cells but not to 4d cells, we performed subtraction
2.5. Whole-mount in situ hybridization (WISH) DIG-labeled RNA probes were prepared according to the protocols described by Matsuo et al. (2005). An antisense and sense riboprobes were synthesized with T7 and T3 RNA polymerases, respectively. Embryos, juveniles and young adults that were to be processed for WISH were fixed with 4% formaldehyde according to the methods described by Matsuo et al. (2005) and Mohri et al. (2016). After a brief rinse in PBST (PBS plus 0.1% Tween-20), embryos were freed from vitelline membrane with fine forceps. Fixed juveniles and adults were transferred to PBST, and cut into short fragments comprising five to six segments by means of razor blade; fragments containing genital segments (X and XI) were collected, and with fine forceps, not only an epithelial layer but also a muscle layer were torn off from the dorsal half of each fragment. This treatment was expected to facilitate penetration of proteinase K and riboprobes toward the mesodermal tissues of the embryo. Embryos and fragments (of juveniles and adults) thus obtained were dehydrated with a graded series of methanol, and stored at 20 C in methanol until use. For WISH, embryos, juveniles and adults that had been stored in methanol were rehydrated and processed according to the protocols described by Matsuo et al. (2005), except that juveniles and adults were treated with proteinase K (2 mg/ml) for 20 min and that hybridization (24 h) and subsequent wash were both performed at 60 C. Hybridization signals were detected by chromogenic reaction using BCIP and NBT. Whole-mount processed (stained) embryos were fixed with 3.5% formaldehyde in phosphate buffer (pH 7.4) for 12 h, mounted in PBST, and observed under incident light. Some stained embryos were cleared according to the method described by Matsuo and Shimizu (2006) and observed with transmitted light.
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2.6. Bisection of stained embryos Some of the embryos that had been stained for Ttu-fel as described above were bisected with razor blade in PBST according to the method described by Shimizu (1988). The plane of bisection included both the animal-vegetal and anteroposterior axes of the embryo. Sections of the bisected embryos were observed under incident light. Acknowledgements We thank the Shimizu lab for helpful advice. We are also grateful to Dr. Ayaki Nakamoto (Tohoku University) for his help in our access to key references. This work was supported by the Department of Biological Sciences. References Bissen, S.T., Weisblat, D.A., 1991. Transcription in leech: mRNA synthesis is required for early cleavages in Helobdella embryos. Dev. Biol. 146, 12e23. Bissen, S.T., Smith, C.M., 1996. Unequal cleavage in leech embryos: zygotic transcription is required for correct spindle orientation in a subset of early blastomeres. Development 122, 599e606. s, Y., Weisblat, D.A., 2014. Differential expression of conserved germ Cho, S., Valle line markers and delayed segregation of male and female primordial germ cells in a hermaphrodite, the leech Helobdella. Mol. Biol. Evol. 31, 341e354. Davidson, E.H., 1986. Gene Activity in Early Development. Academic Press, Orland. Hirao, Y., 1964. Reproductive system and oogenesis in the freshwater oligochaete, Tubifex hattai. J. Fac. Sci. Hokkaido Univ. Ser. VI, Zool. 15, 439e448. Jaana, H., 1982. The ultrastructure of the epithelial lining of the male genital tract and its role in spermatozeugma formation in Tubifex hattai Nomura (Annelida, Oligochaeta). Zool. Anz. (Jena) 209, 159e176. Kang, D., Pilon, M., Weisblat, D.A., 2002. Maternal and zygotic expression of a nanosclass gene in the leech Helobdella robusta: primordial germ cells arise from segmental mesoderm. Dev. Biol. 245, 28e41. Kostriken, R., Weisblat, D.A., 1992. Expression of a wnt gene in embryonic epithelium of the leech. Dev. Biol. 151, 225e241. Master, V.A., Kourakis, M.J., Martindale, M.Q., 1996. Isolation, characterization, and expression of Le-msx, a maternally expressed member of the msx gene family from the glossiphoniid leech, Helobdella. Dev. Dyn. 207, 404e419. Matsuo, K., Shimizu, T., 2006. Embryonic expression of a decapentaplegic genes in the oligochaete annelid Tubifex tubifex. Gene Expr. Patterns 6, 800e806. Matsuo, K., Yoshida, H., Shimizu, T., 2005. Differential expression of caudal and dorsal genes in the teloblast lineages of the oligochaete annelid Tubifex tubifex.
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