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Slit-Robo expression in the leech nervous system: insights into eyespot evolution
Cell & Bioscience volume 13, Article number: 70 (2023)
Slit and Robo are evolutionarily conserved ligand and receptor proteins, respectively, but the number of slit and robo gene paralogs varies across recent bilaterian genomes. Previous studies indicate that this ligand-receptor complex is involved in axon guidance. Given the lack of data regarding Slit/Robo in the Lophotrochozoa compared to Ecdysozoa and Deuterostomia, the present study aims to identify and characterize the expression of Slit/Robo orthologs in leech development.
We identified one slit (Hau-slit), and two robo genes (Hau-robo1 and Hau-robo2), and characterized their expression spatiotemporally during the development of the glossiphoniid leech Helobdella austinensis. Throughout segmentation and organogenesis, Hau-slit and Hau-robo1 are broadly expressed in complex and roughly complementary patterns in the ventral and dorsal midline, nerve ganglia, foregut, visceral mesoderm and/or endoderm of the crop, rectum and reproductive organs. Before yolk exhaustion, Hau-robo1 is also expressed where the pigmented eye spots will later develop, and Hau-slit is expressed in the area between these future eye spots. In contrast, Hau-robo2 expression is extremely limited, appearing first in the developing pigmented eye spots, and later in the three additional pairs of cryptic eye spots in head region that never develop pigment. Comparing the expression of robo orthologs between H. austinensis and another glossiphoniid leech, Alboglossiphonia lata allows to that robo1 and robo2 operate combinatorially to differentially specify pigmented and cryptic eyespots within the glossiphoniid leeches.
Our results support a conserved role in neurogenesis, midline formation and eye spot development for Slit/Robo in the Lophotrochozoa, and provide relevant data for evo-devo studies related to nervous system evolution.
The nervous system is a major organ system in nearly all metazoans, with the exception of sponges and placozoans . Nervous system development in animals, as divergent as flies and mice, shares many features, including the evolutionarily conserved systems of ligands and receptors, such as the Slit/Robo system, that serve as guidance cues in patterning their respective nerve cords. Such commonalities have been taken as evidence that their last common ancestor (Urbilateria) presumably already had a clearly differentiated nerve cord . On the other hand, a wide range of neural architectures presented by basally branching bilaterians—such as Xenacoelomorpha, Rotifera, Nemertea and Brachiopoda, together with diverse neural patterning processes they utilize—suggest that the apparent conservation of neural patterning processes and morphology between vertebrates and insects reflects an evolutionary convergence . In either case, studying the genetic basis underlying the development of nervous system across diverse metazoan lineages is essential to understanding their evolutionary history.
Slit is a large, secreted glycoprotein that was first discovered in Drosophila, secreted by a subset of glial cells and mediated repulsive interactions along the midline of the developing central nervous system (CNS) . It consists of four leucine-rich repeat (LRR) domains and multiple EGF repeats that mediate biding to the Immunoglobulin-like (IG) domains of Roundabout (Robo) [5,6,7,8]. The Slit/Robo interaction is of high apparent affinity and evolutionarily conserved [6, 7]. slit exists as a single copy gene in all invertebrates examined to date , whereas vertebrates have three paralogs—slit1, slit2, and slit3 . On the other hand, robo is more variable in copy number across species: Caenorhabditis elegans has a single robo (sax-3), Drosophila has three (robo1, robo2 and robo3) , and vertebrates have four (Dutt1/Robo1, Robo2, Robo3/Rig-1, Robo4/Magic Roundabout) [7, 12].
Slit binding has been demonstrated biochemically for most Robos, except Robo4. Functionally, Slit ligands are known to be crucial for the formation of projections from the eye to brain [12,13,14,15], and for being involved in defining the position and boundaries of optic chiasm . Furthermore, Slit ligands regulate axon pathfinding along the entire tract , repel migrating neurons—including cerebral cortical neurons, GABAergic interneurons and olfactory interneurons [17,18,19]—and stimulate the elongation and branching of sensory axons . Robo receptors in Drosophila transduce a repellent signal at the midline, and the combinatorial expression of the three Robos specifies the lateral positioning of longitudinal axon tracts . Additionally, Slits and Robos are dynamically expressed in complementary patterns in the migratory pathways of neural crest cells in chicks . These studies seem to suggest that this ligand-receptor complex is involved in everything that concerns the relation between neurons and midline, and that this system might have acquired additional functions compared to the urbilaterian nervous system .
The present study aims to identify and characterize the expression of Slit/Robo orthologs in leeches as representatives of lophotrochozoans. We found one slit (Hau-slit) and two robo genes (Hau-robo1 and Hau-robo2) in the Helobdella genome, and that their transcripts are expressed dynamically in late stages of development in the leech Helobdella austinensis, varying both temporally and spatially along the ventral nerve cord and eyespots. Our results are consistent with a conserved role for the Slit-Robo interaction in neurogenesis, midline formation, and eyespot development in the Lophotrochozoa. Furthermore, based on comparison of the expression of slit and robo orthologs in another glossiphoniid leech, Alboglossiphonia lata, we provide a new insight into the molecular basis of evolutionary variation of eyespot development among leeches.
Sequence retrieval and phylogenetic analyses
Phylogenetic analyses for slit and duplicated robo orthologs were conducted using a dataset representing diverse metazoan species. Results from these analyses clearly indicate that the leech slit orthologs are members of the lophotrochozoan slit family supported by high bootstrap values (Fig. 1A). A molecular phylogeny of Slit protein amino acid sequences reflects the known phylogenetic relationships among vertebrates and protostomes, except Schmidtea (Lophotrochozoa; Platyhelminthes) and Caenorhabditis (Ecdysozoa; Nematoda), which appear as outliers due to long-branch attraction, as is often the case. Within protostomes, the Ecdysozoa and Lophotrochozoa are captured with modest supports, but the annelid clade (represented here by Alboglossiphonia, Capitella, Helobdella, and Platynereis) is not recovered as a monophyletic clade. A molecular phylogeny based on the Robo amino acid sequences produced similar results (except Lottia joining Schmidtea as an outlier), and indicates that the duplication of Robo occurred in the ancestor of glossiphoniid leeches after their divergence from molluscan ancestor (Fig. 1B).
Temporal expression pattern using semi-quantitative RT-PCR
Semi-quantitative RT-PCR (sqRT-PCR) was performed to obtain a developmental expression profile for Hau-slit, Hau-robo1 and Hau-robo2 (Fig. 1C). 18S ribosomal RNA was used as an internal standard to control for variations in the efficiency of RNA extraction and reverse transcription reaction . Hau-slit and Hau-robo1 transcripts were not detected in zygotic stages prior to stage 8, indicating that these genes are zygotic, and then decreased in stage 11. Hau-robo2 was expressed at lower levels than Hau-slit and Hau-robo1; its transcript was first detected at stage 9. Similar to Hau-slit and Hau-robo1, transcript levels for Hau-robo2 peaked during stage 10.
slit and robo expression patterns during development in the Leech Helobdella
Following the RT-PCR results described above, we used double fluorescence whole mount in situ hybridization (FWMISH) to characterize slit and robo expression patterns in Helobdella embryos during stage 8 to early stage 9 of development. During stage 8, the four ipsilateral ectodermal bandlets and the underlying mesoderm bandlet converge at the posterior end of the embryo to form the germinal band. The left and right germinal bands are stretched by the posterior addition of new blast cells and the oriented division of blast cell clones along the A-P axis. In late stage 8 to early stage 9, it moves circumferentially from the dorsal region and meets at the expected ventral midline of the embryo to form the germinal plate .
Throughout stage 8, Hau-slit and its presumptive receptor Hau-robo1 were expressed in longitudinally iterated dots, which were located at the leading edges (the prospective ventral region) of the left and right germinal bands. As the germinal bands coalesce in a zipper-like fashion in anteroposterior (A-P) progression along the future ventral midline to form a germinal plate, the dots came into direct apposition along the midline (Fig. 2A).
The cross sections of double FWMISH-stained early stage 9 embryos revealed further details of the Hau-slit and Hau-robo1 expression patterns (Fig. 2A, bottom row). Hau-slit expression at the midline occurs primarily in three discrete levels. First, a deep layer of Hau-slit expressing cells appear to be in or immediately adjacent to the presumptive visceral mesoderm; lateral domains of Hau-slit occur at this same level, near the lateral edges of the germinal plate. Midline expression of Hau-slit occurs also in two other layers, which appear to mark the ventral and dorsal surfaces of the segmental ganglia, respectively. The deep, middle and superficial layers of Hau-slit expression are centered at the midline, but each layer is several cell diameters in width. In addition, the middle and superficial layers are connected by a narrow, dorsoventrally oriented strand of expression. Hau-robo1 expression occurs in two broader domains that appear to complement the domains of Hau-slit expression. One domain of Hau-robo1 expression consists of most or all cells in the segmental ganglia. The other domain is a thin layer of cells, apparently in visceral mesoderm, extending between the deep and lateral domains of Hau-slit expression.
Germ layer localization of slit and robo expressing cells in Helobdella germinal plate
To examine the embryonic origin of the cells expressing Hau-slit and Hau-robo1, we carried out FWMISH on embryos, in which the M (mesoderm), N (primary, ventral neuro-ectoderm), and OPQ (dorsal and lateral ectoderm) lineages had been labeled by the injection of fluorescently labeled dextran amines (Fig. 2Ba, b). Both genes were expressed in subsets of cells in both the M and N lineages (Fig. 2Bc, h). Hau-robo1 was more broadly expressed in neural lineage than did Hau-slit (Fig. 2Be, f), and unlike the latter, it was expressed in a few cells within the OPQ lineage (Fig. 2Bg, h).
To determine whether the expression of Hau-slit is required for germinal plate formation, we performed a knockdown of the Hau-slit by injecting dsRNA into DNOPQ cells of stage 4C embryos (see Materials and Methods for details). In knockdown embryos, the spatial expression of Hau-slit was reduced in the anterior germinal plate, and coalescence of the bilateral germinal bands into the germinal plates was disturbed compared to control embryos at late stage 9 (Fig. 2Ca, b). This observation was further supported by sqRT-PCR, which showed a decreased expression of Hau-slit mRNA in knockdown embryos (Fig. 2Cc). However, the disturbed coalescence of germinal bands resulted in the laceration of knockdown embryos beyond stage 9, precluding any further observation.
slit and robo are expressed in various tissues during late development
Throughout stage 9, Hau-slit is expressed in dorsal ganglia of the head and developing ventral segmental ganglia (Fig. 3Aa). Meanwhile, Hau-robo1 is expressed in dorsal ganglia of the head and in developing ventral segmental ganglia, but the transcript levels are higher in the anterior to middle ventral segmental ganglia than in the posterior region from mid to late stage 9 (Fig. 3Ad).
At stage 10, the expression of Hau-slit persisted along the entire ventral segmental ganglia and the anteriormost part of the developing everted proboscis, as well as in the dorsal midline (Fig. 3Ab). At late stage 10, Hau-slit is expressed along the entire ventral segmental ganglia, dorsal midline and rectum (Fig. 3Ac). At early-to-mid stage 10, Hau-robo1 is expressed strongly along all the proboscis and segmental ventral ganglia (Fig. 3Ae). At late stage 10, Hau-robo1 is expressed in a pattern of sequential dots along the entire ventral segmental ganglia, and as dots scattered all over the foregut, which surrounds the invaginated proboscis and midgut. Furthermore, Hau-robo1 is expressed strongly in the caudal region (Fig. 3Af).
Consistent with the low overall levels of Hau-robo2 detected in our RT-PCR analysis, Hau-robo2 is expressed in a highly restricted pattern, which corresponds to a bilateral pair of anterodorsal spot gradually increasing in intensity from stage 9 to 10 (Fig. 3Ah-j).
For a more detailed view of the Hau-slit and Hau-robo1 expression, we examined stained late stage 10 embryos in section (Fig. 3B). The examination of serially sectioned material revealed that Hau-slit is expressed in a sparsely distributed set of cells in the radial muscle of the proboscis. In addition, Hau-slit transcripts are detected in visceral mesoderm and/or endoderm of the intestine (Fig. 3Ba-c). On the other hand, Hau-robo1 is expressed in the outer ring of the proboscis sheath—i.e., longitudinal muscle of the proboscis, radial muscle precursor, and ventral segmental ganglia, as well as in a precursor of presumptive reproductive organs  (Fig. 3Bd-e).
The pattern of Hau-slit expression observed within segmental tissues at the end of stage 8 has been transformed by late stage 10 as follows: Within the deep layer, Hau-slit expression in the midline appears to have disappeared (compare Fig. 2AI bottom row to Fig. 3Bb). Furthermore, the transcript domains near the lateral edges of the germinal plate meet at the dorsal midline, reflecting the spreading of the germinal plate around the circumference of the yolk to generate the body tube from stage 9 to 10 . The superficial layer of expression is reduced to a narrow domain of midline cells in the body wall. Moreover, the middle layer of Hau-slit expression is now comprised of a few bilateral pairs of cells within the ganglia and at its dorsal surface (Fig. 3Bc).
Ganglionic expression of slit and robo
To examine the ganglionic expression of Hau-slit and Hau-robo1 in detail, we combined FWMISH for Hau-slit and Hau-robo1 with immunostaining of acetylated-α-tubulin to mark developing axon tracts in stage 10 embryos. As previously described in another glossiphoniid leech species , immunostaining revealed prominent bilaterally paired longitudinal axon tracts of connective nerves, three main pairs of segmental nerves (previously named AA, MA and PP) exiting each ganglion, and three main commissures in the neuropil of each ganglion (Fig. 4a, d). The longitudinal connectives and neuropil are dorsally situated, with cell bodies on the ventral and lateral aspects of the ganglia (Fig. 4a', d'). The patterns revealed by a combined immunostaining of acetylated-α-tubulin and FWISH for Hau-slit and Hau-robo1 are consistent with the conclusion that these genes are expressed in specific subsets of ganglionic cells. For Hau-slit, we observe a pair of prominent cells flanking the ventral midline between the MP and PP nerves (Fig. 4b, c). At the midline itself, Hau-slit is expressed in two bilateral pairs of cells on the dorsal side of the neuropil and one pair of anteroposteriorly arrayed cells on the ventral side of the neuropil (Fig. 4a’-c’ and data not shown). Based on our lineage tracing analysis (Fig. 2Bb and Bd), we found the previously described M lineage-derived connective muscle cells and N lineage-derived neuropil glia as likely candidates for the source of this expression . Hau-robo1 is expressed in pairs of relatively large cells in the interstices separating the three segmental nerves on each side of the ganglia, and smaller cells positioned along the segmental nerves in the periphery (Fig. 4e, f).
slit and robo expressed in the eyespots
By late stage 11, Hau-slit is expressed at the margin of the anterior sucker, in eyespots, and as a narrow longitudinal patch between the eyespots, which corresponds to cerebral ganglia (Fig. 5Aa-a’ and 5Ba). It is also expressed in the developing ventral segmental ganglia, in visceral mesoderm and/or endoderm of the muscle associated with the rectum, and strongly in the body region (Fig. 5Aa). Hau-robo1 is expressed in a single bilaterally paired set of cells in the anterior (head) region, corresponding to the pigmented eyespots (Fig. 5Ab, b', and Bb, b'). It is also expressed in the nerve cord, visceral mesoderm, presumptive primordial germline, and in the posterior sucker (Fig. 5Ab). Consistent with the results obtained with single probes, the expression of Hau-slit and Hau-robo1 partially overlaps in the eyespots in these late stage 11 specimens (Fig. 5Ca”, b).
In contrast, Hau-robo2 is expressed in four bilaterally paired sets of cells in the region that corresponds to a developing pair of pigmented eyespots and three pairs of extra ocular photoreceptor cells (PRCs) (Fig. 5Ac, c’ and Bc). All four pairs of eyespots express r-opsin transcripts during late stage 11 (Fig. 5Bd, d'), as reported by .
Eyespots by robo2 expression in leeches
Unlike Helobdella showing a single pair of pigmented eyespots (Fig. 6Ae), another glossiphoniid leech species, Alboglossiphonia lata (Ala), is characterized by having three pairs of pigmented eyespots (Fig. 6Aa). This interspecific difference makes the comparison of slit and robo orthologs in the two species particularly interesting. In fact, we found some notable similarities and differences in the expression of Ala-slit relative to its ortholog in Helobdella. First, we observed slit expression in a pair of longitudinally orientated midline patches of tissue between the multiple eyespots in Alboglossiphonia (Fig. 6Ab), compared to just one in Helobdella (Fig. 5Ba). Second, we were unable to detect any Ala-slit expression within the eyespots themselves in Alboglossiphonia, although this failure could simply result from higher levels of autofluorescence in Alboglossiphonia embryos. Unfortunately, the increased autofluorescence also made it impossible to carry out double-FWMISH in this species. Intriguingly, both Ala-robo1 and Ala-robo2 are expressed in all three pairs of pigmented eyespots, consistent with the co-expression of both robo paralogs in only the pigmented eyespots in Helobdella (Fig. 5Bb, c). In addition, we also observed the internal structure of pigmented eyespots whether they are composed of photoreceptor cells and pigmented supportive cells (PSCs) as previously described . In the given section view of both species, it was corroborated that the PRCs and pigmented supportive cells were clearly visible is 3 pairs of eyespots in A. lata (Fig. 6Ba’-a’’’), which were structurally identical with H. austinensis (Fig. 6Bb, b’).
Here, we have identified and characterized the expression of homologs of slit and robo genes in two glossiphonid leech species, Helobdella austinensis and Alboglossiphonia lata. We found that these species contain a single slit gene, as do all other protostomes that have been examined (Fig. 1A) [7, 26]. In the case of robo, however, we found two copies in each species, which is also consistent with other protostomes where two or more robo paralogs are found. Intriguingly, our phylogenetic analysis indicates that robo duplications have occurred independently at least three times within the mollusk, flatworm and leech lineages (Fig. 1B) . Together, these findings suggest that the Slit ligand is subject to more severe evolutionary constraints than the Robo receptor.
Our semi-quantitative RT-PCR results indicate the expression of Hau-slit and Haurobo1 throughout the segmentation and organogenesis in Helobdella austinensis (Fig. 1C). This result was corroborated by FWMISH. Hau-slit is expressed in the leading (prospective ventral) edge of the coalescing germinal bands, and in the lateral (prospective dorsal) edge of the expanding germinal plate, suggesting its potential role in the formation of both ventral and dorsal midlines (Fig. 2AI). Both mesodermal and ectodermal lineages express Hau-slit and Hau-robo1 (Fig. 2Bc, d–g, h), and knockdown of Hau-slit in the DNOPQ lineage demonstrates its tight association with the coalescence of germinal band during stage 8 (Fig. 2Ca, b). This result suggests a potential role of Hau-slit in the epibolic migration of germinal bands, similar to the previously reported role of Hau-netrin-1 .
From their multiple sites of expression, Hau-slit and Hau-robo1 appear to be involved in patterning both non-segmental (proboscis and gut) and segmental tissues (Fig. 3B). A rearward progressing wave of slit and robo gene expression, especially clear for Hau-robo1, correlates with a segmental differentiation (Fig. 3Aa–f). In contrast, the expression of Hau-robo2 is confined to developing eyespots and associated phaosomes, indicating a derived and specialized function of this gene (Fig. 3Ah–j and Fig. 5Ac, Bc and 6Ae’).
Hau-slit expression in particular occurs in a narrow longitudinal pattern along both the ventral and dorsal midlines (Fig. 3Ba, b), indicating its conserved function in midline signaling, and is consistent with the role this gene plays in repelling migrating neurons that form ganglia and connectives from the midline (Fig. 4a–c). The presence of Hau-slit transcripts between the developing eyespots might also indicate the repelling role of this gene during eyespot formation, in which it is involved in defining the position and boundaries of these organs (Fig. 5Ba). Slit ligands are known to be involved in the formation of projections from the eye to brain, and in axon pathfinding regulation along the tract in vertebrates [13,14,15,16, 19]. It is interesting to note that in Drosophila, Robo receptors are associated with repelling signals at the midline . Thus, studies of vertebrates and insects imply a conserved involvement of the Slit/Robo system in everything that concerns the relation between neurons and midline . Not surprisingly, our observations on the expression of slit and robo genes along the developing segmental ganglia also suggest a similar role of Slit/Robo in neural patterning in the Lophotrochozoa. We speculate that the expression of slit and robo during later organogenesis, in association with proboscis, eyespots, crop and reproductive organs, may indicate that the Slit/Robo system is involved in the formation of peripheral nerves that interconnect major internal organs.
In the past, light sensitive photoreceptor cells were considered to exist in just two types—ciliary and rhabdomeric photoreceptors [29,30,31]. In the Annelida, however, a third cell type, the phaosomal photoreceptor, has been described, which is characterized by a dramatically infolded apical surface so that the sensory microvilli appear to occupy an internal cavity of the cell [25, 31,32,33]. Phaosomal photoreceptor cells are rare in the Polychaeta, but may be the only photoreceptor cell type present in the Clitellata. It has been suggested that phaosomal receptors are originally derived from rhabdomeric receptors—for example, they express rhabdomeric opsins—and that clitellate eyes have re-evolved after being lost during the evolution of clitellates from their polychaete ancestors. The loss of ancestral annelid eyes in that transition could have occurred in association with the emergence to direct development and/or the occupation of endobenthic habitats in the clitellate stem lineage [25, 31].
In the work reported here, we observed that the combinatorial gene expression of the robo1 and robo2 genes correlates with the distinction between pigmented and unpigmented (cryptic) eyespots of Helobdella austinensis. At late stage 10, Hau-robo2 is consistently expressed in the developing pigmented eyespots (Fig. 3Aj), and subsequently in the three pairs of unpigmented, thus cryptic, eyespots at late stage 11 (Fig. 5Ab–c’, Bc). Cryptic eyespots have been previously observed in Helobdella robusta based on the expression patterns of opsin and gq-protein genes . These results suggest that the expression of robo2 might be critical for the development of eyespots, including the phaosomal photoreceptors. In contrast to Hau-robo2, Hau-robo1 is expressed only in the pigmented eyespots of Helobodella, suggesting that robo1 act as a decision maker for the development of pigmented epithelium and/or other features that are unique to pigmented eyespots (Fig. 5Ab, Bb).
The idea that robo1 and robo2 operate concertedly in the specification of pigmented (requiring both robo1 and robo2) and unpigmented (robo2 only) eyespots is further supported by their expression patterns in Alboglossiphonia lata, the second glossiphoniid leech species we examined. In contrast to Helobdella, which exhibits one pair of pigmented and three pairs of unpigmented eyespots, Alboglossiphonia presents three pairs of eyespots that are all pigmented with combination of PRCs and PSCs, in which the expression of both robo1 and robo2 is confirmed (Fig. 6A, B). Thus, we speculate that robo2 is a key factor in specifying the baseline state of cryptic eyespot differentiation, and that the addition of robo1 expression is required to specify fully differentiated pigmented eyespots (Fig. 6C).
Leeches as a group vary widely in the number and distribution of their eyespots . Such a diversity in eyespot among leeches is not surprising given their long evolutionary history over ~ 360 million years , as well as pervasive ecological shifts in habitats and lifestyle . In fact, the variation in pigmented and cryptic eyespots observed in the present study is particularly fascinating since the two species under investigation both belong to the family Glossiphoniidae, representing only one of the earliest branching leech lineages . Our finding elucidates the undoubted importance of robo expression in the differentiation of eyespots in leeches, and set the framework for studying the genetic and developmental basis underlying the eyespot evolution across all leeches, and even annelids as a whole.
Our results of the slit and robo gene characterization in Helobdella support their conserved role in the development of nervous system, including the midline formation and eyespot development in the Lophotrochozoa. A comparison with another glossiphonid leech, Alboglossiphonia, allows us to highlight the importance of differential robo expression in the loss of pigmented eyespot pairs across the family. Based on the results presented here, we intend to expand our investigation to other early-diverging and paraphyletic species of leeches showing intermediate characteristics to shed light on the eyespot evolution. Finally, our results provide relevant information for evo-devo studies related to nervous system evolution across Bilateria.
Embryos were obtained from a laboratory colony of the species Helobdella austinensis and Alboglossiphonia lata as described elsewhere [38,39,40,41]. The specimens were cared for once daily by changing solution and the bowl was scrubbed manually to get rid of any residual waste. They were stored in a BOD incubator at 22 ℃.
Alignment and phylogenetic analyses
Two separate Slit and Robo data set were built with respective Slit and Robo homologs from representatives of the major metazoan clades with a total of 21 amino acid sequences for Slit and 30 for Robo. Slit and Robo protein sequences were retrieved from the UniProt database and GenBank. Alignments were carried out using MUSCLE (Multiple Sequence Comparison by Log- Expectation) in implemented in MEGA X software. The aligned sequences were analyzed with MEGA X software  using the model selected by finding best fit models . The Maximum Likelihood analyses were conducted by generating 1,000 bootstrap replicates.
Developmental semi-quantitative RT-PCR
Total RNA samples were prepared from H. austinensis embryos at selected stages  with RNA Wiz (Ambion, Austin, TX, USA) according to the manufacturer's instructions, using 40 embryos for each stage. The total RNA obtained (3 µg) was reverse transcribed using a first-strand cDNA synthesis kit (BD Biosciences, Palo Alto, CA, USA). The PCR mixture (50 μl) contained 10 × Taq buffer, 0.3 U Taq polymerase (PerkinElmer, Wellesley, MA, USA), 2.5 mM of dNTPs, 5 pmol of each set of primers (for the details of primer information, see Additional file 1: Table S1), and 50 ng of cDNA from the selected stages as template. The PCR reactions were performed under the following cycling conditions: an initial denaturation at 94 °C for 5 min, followed by 25–35 cycles of denaturation at 94 °C for 30 s, and elongation at 72 °C for 10 min. A 6 μl aliquot of each PCR reaction was removed after 25 cycles, while the remaining material underwent five additional cycles of amplification. The 18 rRNA sequence was used as an internal standard (QuantumRNA, Ambion). The extent of amplification was chosen empirically to avoid saturation of the amplified bands. To quantify PCR products, each sample was run in a 1.5% agarose gel, and then stained with ethidium bromide. Band intensity was measured with an Alphalmager (Alpha Innotech Corp., San Leandro, CA, USA) using the Alphaease (v3.3b) program.
Probe synthesis for in situ hybridization
To isolate the H. austinesis slit and robo gene, previously published sequences, we used BLAST implemented in the whole draft-genome reference (http://genome.jgi.doe.gov/Helro1/Helro1.home.html). Three candidate genes (protein id: Hau-slit 103882; Hau-robo1 189275; Hau-robo2 188972) were screened. In the A. lata transcriptome data, one slit and two robo transcripts were found. The slit and robo specific primers were designed to amplify the fragments (For detailed primer sequences, see Additional file 1: Table S1). These amplified fragments were cloned into pGEM T vector (Promega, Madison, WI, USA). RNAprobes labeled with digoxigenin or fluorescein were made using the MEGAscript kit (Ambion, Austin, TX, USA) and DIG or Fluorescein RNA Labeling Mix (Roche, Basel, Switzerland), according to the manufacturer's instructions. The synthesized RNA probes were applied to each sample at a final concentration of 2 ng/μl.
Fluorescence in situ hybridization
We carried out double fluorescence whole mount in situ hybridization (FWMISH) using the NEN tyramide signal amplification (TSA) Plus kit (PerkinElmer, Wellesley, MA, USA). The protocol was identical to that in Cho et al. 2010, where stage 8 to 11 embryos were rinsed once in 1 × PBTw and blocked for 2 h at room temperature in a solution of 0.1 M Tris–HCl pH 7.5, 0.15 M NaCl, and 0.1% Tween 20 (TNT) containing 1% NEN TSA blocking reagent (collectively designated TNB). Embryos were incubated overnight at 4 °C with peroxidase-conjugated anti-digoxygenin or anti-fluorescein (Roche, Basel, Switzerland) at a 1:1000 dilution in TNB. Subsequent washes (3 × 20 min) in TNT at room temperature were followed by a single 1 × 30 min rinse in NEN TSA Plus amplification solution. The color reaction was initiated by adding a 1:50 dilution of reconstituted cyanine-3 tyramide in NEN amplification solution. For fluorescence microscopy, embryos were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Sigma) to visualize cell nuclei. FWMISH was visualized using a Leica compound microscope and a spinning disk confocal microscope (Leica, Wetzlar, HE, Germany).
Lineage tracing and cross sectioning
To determine the embryonic origin of the cells expressing specific Hau-slit and Hau-robo1, teloblasts or proteloblasts of embryos in stages 5 and 6 were injected with a fluorescently labeled, fixable dextran lineage tracer, fluorescein-conjugated dextran amine (FDA), as previously described . Injected embryos were cultured in Helobdella triserialis saline (HTR) at 23 °C to the desired embryonic stage, then fixed, and processed by FWMISH as described above. After FWMISH, the embryos were dehydrated in ethanol and propylene oxide, followed by infiltration with plastic embedding medium (PolyBed, Polysciences, Inc.). Then embryos were sectioned by using a glass knife on a microtome (MT-2B; Sorvall, Newtown, CT, USA) or hand cut by razor blade into 0.1 mm sections. Sections were imaged on a Leica compound microscope to obtain combined fluorescence images (Leica, Wetzlar, HE, Germany).
Hau-slit Double-Stranded RNA (dsRNA) Preparation
Single-stranded RNAs were produced from opposing strands of a full-length cDNA clone in pBluescript II (Stratagene), by in vitro transcription with the T3 and T7 polymerases (Ambion) (For the details, see Additional file 1: Table S1). The plasmid DNA was then removed by using DNaseI from the RNase-free kit (Ambion). The two single-stranded RNAs were allowed to anneal by mixing equal amounts of each strand, heating to 85 °C, and cooling gradually to 40 °C. The quality of the annealed dsRNA was checked by electrophoresis on an agarose gel. NOPQ proteloblasts injected with Hau-slit dsRNA at 100 ng/μl. Injected embryos were cultured in a modified HL medium (9.6 mM NaCl, 1.2 mM KCl, 2 mM MgCl2, 8 mM CaCl2, 1 mM maleic acid, pH6.6) supplied with antibiotics (0.05 mg/ml tetracycline, 100 units/ml penicillin, 100 units/ml Streptomycin).
After rehydrating stage 10 embryos, they were pre-incubated in 5% mercaptoethanol and 1% Triton in 0.1 M Tris–HCl (pH 7.5) at 37 °C on shaking incubator (rpm 60) for an hour. Following three washes with PBT, the embryos were incubated in Block solution (1:9 10X Roche Western Blocking Reagent in PBT) for two hours. Then, embryos were incubated with a monoclonal anti-acetylated-α-Tubulin antibody (Sigma, T-7451) in Blocking Solution (1:500) at 4 °C for 72 h. After three consecutive washes with PBT, embryos were incubated with a secondary antibody (Abcam, ab150113) in Blocking Solution (1:1000) at 4 °C for 48 h. Consequently, embryos were washed with PBT overnight. Then embryos were stained with DAPI in PBT (1:1000) at room temperature in the dark overnight. After washing with PBT 3 more times, embryos were embedded in 30%, 50% 20 min and 87% glycerol and 2.5 mg/ml of DABCO in 1xPBS. Embryos were imaged by fluorescence microscopy on a Nikon SMZ18 Stereomicroscope and LEICA DM6 B.
Availability of data and materials
Related sequencing data have been uploaded to NCBI’s GeneBank. Other data supporting the findings of the present study are available from the corresponding author upon reasonable request.
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We would like to thank the participants in this study.
This work was conducted during the research year of Chungbuk National University in 2019. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1A6A1A06046235, 2023R1A2C1007868). This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR202305101, NIBR202333201).
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Kwak, HJ., Medina-Jiménez, B.I., Park, S.C. et al. Slit-Robo expression in the leech nervous system: insights into eyespot evolution. Cell Biosci 13, 70 (2023). https://doi.org/10.1186/s13578-023-01019-1
- Gene duplication
- Axon guidance