Transgenesis enables mapping of segmental ganglia in the leech Helobdella austinensis.

The analysis of how neural circuits function in individuals and change during evolution is simplified by the existence of neurons identified as homologous within and across species. Invertebrates, including leeches, have been used for these purposes in part because their nervous systems comprise a high proportion of identified neurons, but technical limitations make it challenging to assess the full extent to which assumptions of stereotypy hold true. Here, we introduce Minos plasmid-mediated transgenesis as a tool for introducing transgenes into the embryos of the leech Helobdella austinensis (Spiralia; Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae). We identified an enhancer driving pan-neuronal expression of markers, including histone2B:mCherry, which allowed us to enumerate neurons in segmental ganglia. Unexpectedly, we find that the segmental ganglia of adult transgenic Helobdella contain fewer and more variable numbers of neurons than in previously examined leech species.

Acceleration of genome rearrangement in clitellate annelids.

Comparisons of multiple metazoan genomes have revealed the existence of ancestral linkage groups (ALGs), genomic scaffolds sharing sets of orthologous genes that have been inherited from ancestral animals for hundreds of millions of years (Simakov et al. 2022; Schultz et al. 2023) These ALGs have persisted across major animal taxa including Cnidaria, Deuterostomia, Ecdysozoa and Spiralia. Notwithstanding this general trend of chromosome-scale conservation, ALGs have been obliterated by extensive genome rearrangements in certain groups, most notably including Clitellata (oligochaetes and leeches), a group of easily overlooked invertebrates that is of tremendous ecological, agricultural and economic importance (Charles 2019; Barrett 2016). To further investigate these rearrangements, we have undertaken a comparison of 12 clitellate genomes (including four newly sequenced species) and 11 outgroup representatives. We show that these rearrangements began at the base of the Clitellata (rather than progressing gradually throughout polychaete annelids), that the inter-chromosomal rearrangements continue in several clitellate lineages and that these events have substantially shaped the evolution of the otherwise highly conserved Hox cluster.

Slit-Robo expression in the leech nervous system: insights into eyespot evolution.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Head transcriptome profiling of glossiphoniid leech (Helobdella austinensis) reveals clues about proboscis development.

Cephalization refers to the evolutionary trend towards the concentration of neural tissues, sensory organs, mouth and associated structures at the front end of bilaterian animals. Comprehensive studies on gene expression related to the anterior formation in invertebrate models are currently lacking. In this study, we performed de novo transcriptional profiling on a proboscis-bearing leech (Helobdella austinensis) to identify differentially expressed genes (DEGs) in the anterior versus other parts of the body, in particular to find clues as to the development of the proboscis. Between the head and the body, 132 head-specific DEGs were identified, of which we chose 11 to investigate their developmental function during embryogenesis. Analysis of the spatial expression of these genes using in situ hybridization showed that they were characteristically expressed in the anterior region of the developing embryo, including the proboscis. Our results provide information on the genes related to head formation and insights into the function of proboscis-related genes during organogenesis with the potential roles of genes not yet characterized.

Glossiphoniid leeches as a touchstone for studies of development in clitellate annelids.

My goals in this chapter are to share my enthusiasm for studying the biology of leeches, to place this work in context by presenting my rationale for studying non-traditional biological models in general, and to sample just three of the questions that intrigue me in leech biology, namely segmentation, genome evolution and neuronal fate specification. I first became excited about the idea of using leeches as a subject of investigation as an undergraduate in 1970 and have been engaged in this work since I arrived at Berkeley as a postdoc in 1976, intending to study leech neurobiology. Both my research interests and the rationale for the work have expanded greatly since then. What follows is a fragmentary personal and historical account-the interested reader may find more comprehensive treatments elsewhere (Kuo et al., 2020; Shankland & Savage, 1997; Shain, 2009; Weisblat & Huang, 2001; Weisblat & Kuo, 2009, 2014; Weisblat & Winchell, 2020).

A tale of two leeches: Toward the understanding of the evolution and development of behavioral neural circuits.

In the animal kingdom, behavioral traits encompass a broad spectrum of biological phenotypes that have critical roles in adaptive evolution, but an EvoDevo approach has not been broadly used to study behavior evolution. Here, we propose that, by integrating two leech model systems, each of which has already attained some success in its respective field, it is possible to take on behavioral traits with an EvoDevo approach. We first identify the developmental changes that may theoretically lead to behavioral evolution and explain why an EvoDevo study of behavior is challenging. Next, we discuss the pros and cons of the two leech model species, Hirudo, a classic model for invertebrate neurobiology, and Helobdella, an emerging model for clitellate developmental biology, as models for behavioral EvoDevo research. Given the limitations of each leech system, neither is particularly strong for behavioral EvoDevo. However, the two leech systems are complementary in their technical accessibilities, and they do exhibit some behavioral similarities and differences. By studying them in parallel and together with additional leech species such as Haementeria, it is possible to explore the different levels of behavioral development and evolution.

Reproductive differences among species, and between individuals and cohorts, in the leech genus Helobdella (Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae), with implications for reproductive resource allocation in hermaphrodites.

Leeches and oligochaetes comprise a monophyletic group of annelids, the Clitellata, whose reproduction is characterized by simultaneous hermaphroditism. While most clitellate species reproduce by cross-fertilization, self-fertilization has been described within the speciose genus Helobdella. Here we document the reproductive life histories and reproductive capacities for three other Helobdella species. Under laboratory conditions, both H. robusta and H. octatestisaca exhibit uniparental reproduction, apparently reflecting self-fertility, and suggesting that this trait is ancestral for the genus. However, the third species, H. austinensis, seems incapable of reproduction by self-fertilization, so we inferred its reproductive life history by analyzing reproduction in breeding cohorts. Comparing the reproductive parameters for H. robusta reproducing in isolation and in cohorts revealed that reproduction in cohorts is dramatically delayed with respect to that of isolated individuals, and that cohorts of leeches coordinate their cocoon deposition in a manner that is not predicted from the reproductive parameters of individuals reproducing in isolation. Finally, our comparisons of reproductive capacity for individuals versus cohorts for H. robusta, and between different sizes of cohorts for H. austinensis, reveal differences in resource allocation between male and female reproductive roles that are consistent with evolutionary theory.

Behavioral analysis of substrate texture preference in a leech, Helobdella austinensis.

Leeches in the wild are often found on smooth surfaces, such as vegetation, smooth rocks or human artifacts such as bottles and cans, thus exhibiting what appears to be a "substrate texture preference". Here, we have reproduced this behavior under controlled circumstances, by allowing leeches to step about freely on a range of silicon carbide substrates (sandpaper). To begin to understand the neural mechanisms underlying this texture preference behavior, we have determined relevant parameters of leech behavior both on uniform substrates of varying textures, and in a behavior choice paradigm in which the leech is confronted with a choice between rougher and smoother substrate textures at each step. We tested two non-exclusive mechanisms which could produce substrate texture preference: (1) a Differential Diffusion mechanism, in which a leech is more likely to stop moving on a smooth surface than on a rough one, and (2) a Smoothness Selection mechanism, in which a leech is more likely to attach its front sucker (prerequisite for taking a step) to a smooth surface than to a rough one. We propose that both mechanisms contribute to the texture preference exhibited by leeches.

Expression patterns of duplicated snail genes in the leech Helobdella.

snail gene family members are zinc-finger transcription factors with key roles in morphogenesis. Involvement of snail family genes in mesoderm formation has been observed in insects and mammals. The snail genes are also involved in cell motility, neural differentiation, cell fate, survival decision, and left-right identity. The functions of snail genes have been studied primarily among ecdysozoans and deuterostomes, with relatively little work carried out in lophotrochozoans. In this study, we isolated two snail homologs (Hau-snail1 and Hau-snail2) from the leech Helobdella austinensis. We characterized the temporal and spatial expression patterns of these two genes by semi-quantitative RT-PCR and in situ hybridization. The expression of Hau-snail1 and Hau-snail2 correlates with ventral nerve cord (VNC) development, segmental mesoderm, and with a ring of cells that comes to lie at the base of the leech proboscis, respectively, showing similarity to the divergent expression of duplicated snail genes in polychaetes. Our results do not support the function of lophotrochozoan snail genes in mesoderm specification.

Spatiotemporal expression of a twist homolog in the leech Helobdella austinensis.

Genes of the twist family encode bHLH transcription factors known to be involved in the regulation and differentiation of early mesoderm. Here, we report our characterization of Hau-twist, a twist homolog from the leech Helobdella austinensis, a tractable lophotrochozoan representative. Hau-twist was expressed in segmental founder cells of the mesodermal lineage, in subsets of cells within the mesodermal lineage of the germinal plate, in circumferential muscle fibers of a provisional integument during segmentation and organogenesis stages and on the ventral side of the developing proboscis. Thus, consistent with other systems, our results suggest that twist gene of the leech Helobdella might function in mesoderm differentiation.

Leeches of the genus Helobdella as model organisms for Evo-Devo studies.

Model organisms are important tools in modern biology and have been used elucidate mechanism underlying processes, such as development, heredity, neuronal signaling, and phototropism, to name but a few. In this context, the use of model organisms is predicated on uncovering evolutionarily conserved features of biological processes in the expectation that the findings will be applicable to organisms that are either inaccessible or intractable for direct experimentation. For the most part, particular species have been adapted as model organisms because they can be easily reared and manipulated in the laboratory. In contrast, a major goal in the field of evolutionary developmental biology (Evo-Devo) is to identify and elucidate the differences in developmental processes among species associated with the dramatic range of body plans among organisms, and how these differences have emerged over time in various branches of phylogeny. At first glance then, it would appear that the concept of model organisms for Evo-Devo is oxymoronic. In fact, however, laboratory-compatible, experimentally tractable species are of great use for Evo-Devo, subject to the condition that the ensemble of models investigated should reflect the range of taxonomic diversity, and for this purpose glossiphoniid leeches are useful. Four decades ago (1975), leeches of the species-rich genus Helobdella (Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae) were collected in Stow Lake, Golden Gate Park, San Francisco, CA (USA). These and other Helobdella species may be taken as Evo-Devo models of leeches, clitellate annelids, and the super-phylum Lophotrochozoa. Here we depict/discuss the biology/taxonomy of these Evo-Devo systems, and the challenges of identifying species within Helobdella. In addition, we document that H. austinensis has been established as a new model organism that can easily be cultivated in the laboratory. Finally, we provide an updated scheme illustrating the unique germ line/soma-differentiation during early development and speculate on the mechanisms of sympatric speciation in this group of aquatic annelids.

Differential expression of conserved germ line markers and delayed segregation of male and female primordial germ cells in a hermaphrodite, the leech helobdella.

In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy that minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa, and nanos) and microinjected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for self-fertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to other animals.

Developmental biology of the leech Helobdella.

Glossiphoniid leeches of the genus Helobdella provide experimentally tractable models for studies in evolutionary developmental biology (Evo-Devo). Here, after a brief rationale, we will summarize our current understanding of Helobdella development and highlight the near term prospects for future investigations, with respect to the issues of: D quadrant specification; the transition from spiral to bilaterally symmetric cleavage; segmentation, and the connections between segmental and non-segmental tissues; modifications of BMP signaling in dorsoventral patterning and the O-P equivalence group; germ line specification and genome rearrangements. The goal of this contribution is to serve as a summary of, and guide to, published work.

Insights into bilaterian evolution from three spiralian genomes.

Current genomic perspectives on animal diversity neglect two prominent phyla, the molluscs and annelids, that together account for nearly one-third of known marine species and are important both ecologically and as experimental systems in classical embryology. Here we describe the draft genomes of the owl limpet (Lottia gigantea), a marine polychaete (Capitella teleta) and a freshwater leech (Helobdella robusta), and compare them with other animal genomes to investigate the origin and diversification of bilaterians from a genomic perspective. We find that the genome organization, gene structure and functional content of these species are more similar to those of some invertebrate deuterostome genomes (for example, amphioxus and sea urchin) than those of other protostomes that have been sequenced to date (flies, nematodes and flatworms). The conservation of these genomic features enables us to expand the inventory of genes present in the last common bilaterian ancestor, establish the tripartite diversification of bilaterians using multiple genomic characteristics and identify ancient conserved long- and short-range genetic linkages across metazoans. Superimposed on this broadly conserved pan-bilaterian background we find examples of lineage-specific genome evolution, including varying rates of rearrangement, intron gain and loss, expansions and contractions of gene families, and the evolution of clade-specific genes that produce the unique content of each genome.

Regional differences in BMP-dependence of dorsoventral patterning in the leech Helobdella.

In the leech Helobdella, the ectoderm exhibits a high degree of morphological homonomy between body segments, but pattern elements in lateral ectoderm arise via distinct cell lineages in the segments of the rostral and midbody regions. In each of the four rostral segments, a complete set of ventrolateral (O fate) and dorsolateral (P fate) ectodermal pattern elements arises from a single founder cell, op. In the 28 midbody and caudal segments, however, there are two initially indeterminate o/p founder cells; the more dorsal of these is induced to adopt the P fate by BMP5-8 emanating from the dorsalmost ectoderm, while the more ventral cell assumes the O fate. Previous work has suggested that the dorsoventral patterning of O and P fates differs in the rostral region, but the role of BMP signaling in those segments has not been investigated. We show here that suppression of dorsal BMP5-8 signaling (which effects a P-to-O fate change in the midbody) has no effect on the patterning of O and P fates in the rostral region. Furthermore, ectopic expression of BMP5-8 in the ventral ectoderm (which induces an O-to-P fate change in the midbody) has no effect in the rostral region. Finally, expression of a dominant-negative BMP receptor (which induces a P-to-O fate change in the midbody) fails to affect O/P patterning in the rostral region. Thus, the rostral segments appear to use some mechanism other than BMP signaling to pattern O and P cell fates along the dorsoventral axis. From a mechanistic standpoint, the OP lineage of the rostral segments and the O-P equivalence group of the midbody and caudal segments constitute distinct developmental modules that rely to differing degrees on positional cues from surrounding ectoderm in order to specify homonomous cell fates.

Intermediate filament genes as differentiation markers in the leech Helobdella.

The intermediate filament (IF) cytoskeleton is a general feature of differentiated cells. Its molecular components, IF proteins, constitute a large family including the evolutionarily conserved nuclear lamins and the more diverse collection of cytoplasmic intermediate filament (CIF) proteins. In vertebrates, genes encoding CIFs exhibit cell/tissue type-specific expression profiles and are thus useful as differentiation markers. The expression of invertebrate CIFs, however, is not well documented. Here, we report a whole-genome survey of IF genes and their developmental expression patterns in the leech Helobdella, a lophotrochozoan model for developmental biology research. We found that, as in vertebrates, each of the leech CIF genes is expressed in a specific set of cell/tissue types. This allows us to detect earliest points of differentiation for multiple cell types in leech development and to use CIFs as molecular markers for studying cell fate specification in leech embryos. In addition, to determine the feasibility of using CIFs as universal metazoan differentiation markers, we examined phylogenetic relationships of IF genes from various species. Our results suggest that CIFs, and thus their cell/tissue-specific expression patterns, have expanded several times independently during metazoan evolution. Moreover, comparing the expression patterns of CIF orthologs between two leech species suggests that rapid evolutionary changes in the cell or tissue specificity of CIFs have occurred among leeches. Hence, CIFs are not suitable for identifying cell or tissue homology except among very closely related species, but they are nevertheless useful species-specific differentiation markers.

A new molecular logic for BMP-mediated dorsoventral patterning in the leech Helobdella.

Bone morphogenetic protein (BMP) signaling is broadly implicated in dorsoventral (DV) patterning of bilaterally symmetric animals [1-3], and its role in axial patterning apparently predates the birth of Bilateria [4-7]. In fly and vertebrate embryos, BMPs and their antagonists (primarily Sog/chordin) diffuse and interact to generate signaling gradients that pattern fields of cells [8-10]. Work in other species reveals diversity in essential facets of this ancient patterning process, however. Here, we report that BMP signaling patterns the DV axis of segmental ectoderm in the leech Helobdella, a clitellate annelid (superphylum Lophotrochozoa) featuring stereotyped developmental cell lineages, but the detailed mechanisms of DV patterning in Helobdella differ markedly from fly and vertebrates. In Helobdella, BMP2/4s are expressed broadly, rather than in dorsal territory, whereas a dorsally expressed BMP5-8 specifies dorsal fate by short-range signaling. A BMP antagonist, gremlin, is upregulated by BMP5-8 in dorsolateral, rather than ventral territory, and yet the BMP-antagonizing activity of gremlin is required for normal ventral cell fates. Gremlin promotes ventral fates without disrupting dorsal fates by selectively inhibiting BMP2/4s, not BMP5-8. Thus, DV patterning in the development of the leech revealed unexpected evolutionary plasticity of the conserved BMP patterning system, presumably reflecting its adaptation to different modes of embryogenesis.

Lineage analysis of micromere 4d, a super-phylotypic cell for Lophotrochozoa, in the leech Helobdella and the sludgeworm Tubifex.

The super-phylum Lophotrochozoa contains the plurality of extant animal phyla and exhibits a corresponding diversity of adult body plans. Moreover, in contrast to Ecdysozoa and Deuterostomia, most lophotrochozoans exhibit a conserved pattern of stereotyped early divisions called spiral cleavage. In particular, bilateral mesoderm in most lophotrochozoan species arises from the progeny of micromere 4d, which is assumed to be homologous with a similar cell in the embryo of the ancestral lophotrochozoan, more than 650 million years ago. Thus, distinguishing the conserved and diversified features of cell fates in the 4d lineage among modern spiralians is required to understand how lophotrochozoan diversity has evolved by changes in developmental processes. Here we analyze cell fates for the early progeny of the bilateral daughters (M teloblasts) of micromere 4d in the leech Helobdella sp. Austin, a clitellate annelid. We show that the first six progeny of the M teloblasts (em1-em6) contribute five different sets of progeny to non-segmental mesoderm, mainly in the head and in the lining of the digestive tract. The latter feature, associated with cells em1 and em2 in Helobdella, is seen with the M teloblast lineage in a second clitellate species, the sludgeworm Tubifex tubifex and, on the basis of previously published work, in the initial progeny of the M teloblast homologs in molluscan species, suggesting that it may be an ancestral feature of lophotrochozoan development.

Evolutionary dynamics of the wnt gene family: a lophotrochozoan perspective.

The wnt gene family encodes a set of secreted glycoproteins involved in key developmental processes, including cell fate specification and regulation of posterior growth (Cadigan KM, Nusse R. 1997. Wnt signaling: a common theme in animal development. Genes Dev. 11:3286-3305.; Martin BL, Kimelman D. 2009. Wnt signaling and the evolution of embryonic posterior development. Curr Biol. 19:R215-R219.). As for many other gene families, evidence for expansion and/or contraction of the wnt family is available from deuterostomes (e.g., echinoderms and vertebrates [Nusse R, Varmus HE. 1992. Wnt genes. Cell. 69:1073-1087.; Schubert M, Holland LZ, Holland ND, Jacobs DK. 2000. A phylogenetic tree of the Wnt genes based on all available full-length sequences, including five from the cephalochordate amphioxus. Mol Biol Evol. 17:1896-1903.; Croce JC, Wu SY, Byrum C, Xu R, Duloquin L, Wikramanayake AH, Gache C, McClay DR. 2006. A genome-wide survey of the evolutionarily conserved Wnt pathways in the sea urchin Strongylocentrotus purpuratus. Dev Biol. 300:121-131.]) and ecdysozoans (e.g., arthropods and nematodes [Eisenmann DM. 2005. Wnt signaling. WormBook. 1-17.; Bolognesi R, Farzana L, Fischer TD, Brown SJ. 2008. Multiple Wnt genes are required for segmentation in the short-germ embryo of Tribolium castaneum. Curr Biol. 18:1624-1629.]), but little is known from the third major bilaterian group, the lophotrochozoans (e.g., mollusks and annelids [Prud'homme B, Lartillot N, Balavoine G, Adoutte A, Vervoort M. 2002. Phylogenetic analysis of the Wnt gene family. Insights from lophotrochozoan members. Curr Biol. 12:1395.]). To obtain a more comprehensive scenario of the evolutionary dynamics of this gene family, we exhaustively mined wnt gene sequences from the whole genome assemblies of a mollusk (Lottia gigantea) and two annelids (Capitella teleta and Helobdella robusta) and examined them by phylogenetic, genetic linkage, intron-exon structure, and embryonic expression analyses. The 36 wnt genes obtained represent 11, 12, and 9 distinct wnt subfamilies in Lottia, Capitella, and Helobdella, respectively. Thus, two of the three analyzed lophotrochozoan genomes retained an almost complete ancestral complement of wnt genes emphasizing the importance and complexity of this gene family across metazoans. The genome of the leech Helobdella reflects significantly more dynamism than those of Lottia and Capitella, as judged by gene duplications and losses, branch length, and changes in genetic linkage. Finally, we performed a detailed expression analysis for all the Helobdella wnt genes during embryonic development. We find that, although the patterns show substantial overlap during early cleavage stages, each wnt gene has a unique expression pattern in the germinal plate and during tissue morphogenesis. Comparisons of the embryonic expression patterns of the duplicated wnt genes in Helobdella with their orthologs in Capitella reveal extensive regulatory diversification of the duplicated leech wnt genes.