Dynamic relationship among extracellular matrix and body wall cells in Hirudo verbana morphogenesis.

A great bulk of recent experimental evidence suggests the key role of the complex crosstalk between the extracellular matrix (ECM) and the cellular component of tissues during morphogenesis and embryogenesis. In particular, remodeling of the ECM and of its physical interactions pattern with surrounding cells represent two crucial processes that might be involved in muscle development. However, little information is available on this topic, especially on invertebrate species. To obtain new insights on how tuning the ECM microenvironment might drive cellular fate during embryonic development, we used the invertebrate medicinal leech Hirudo verbana as a valuable experimental model, due to its simple anatomy and the recapitulation of many aspects of the basic biological processes of vertebrates. Our previous studies on leech post-embryonic development have already shown the pivotal role of ECM changes during the growth of the body wall and the role of Yes-associated protein 1 (YAP1) in mechanotransduction. Here, we suggest that the interactions between stromal cell telocytes and ECM might be crucial in driving the organization of muscle layers during embryogenesis. Furthermore, we propose a possible role of the pleiotropic enzyme HvRNASET2 as a possible modulator of collagen deposition and ECM remodeling not only during regenerative processes (as previously demonstrated) but also in embryogenesis.

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.

Reflections on Model Organisms in Evolutionary Developmental Biology.

This chapter reflects on and makes explicit the distinctiveness of reasoning practices associated with model organisms in the context of evolutionary developmental research. Model organisms in evo-devo instantiate a unique synthesis of model systems strategies from developmental biology and comparative strategies from evolutionary biology that negotiate a tension between developmental conservation and evolutionary change to address scientific questions about the evolution of development and the developmental basis of evolutionary change. We review different categories of model systems that have been advanced to understand practices found in the life sciences in order to comprehend how evo-devo model organisms instantiate this synthesis in the context of three examples: the starlet sea anemone and the evolution of bilateral symmetry, leeches and the origins of segmentation in bilaterians, and the corn snake to understand major evolutionary change in axial and appendicular morphology.

Spatiotemporal Expression of Anticoagulation Factor Antistasin in Freshwater Leeches.

Antistasin, which was originally discovered in the salivary glands of the Mexican leech Haementeria officinalis, was newly isolated from Helobdella austinensis. To confirm the temporal expression of antistasin during embryogenesis, we carried out semi-quantitative RT-PCR. Hau-antistasin1 was uniquely expressed at stage 4 of the cleavage and was strongly expressed in the late stages of organogenesis, as were other antistasin members. In order to confirm the spatial expression of antistasin, we performed fluorescence in situ hybridization in the late stages of organogenesis. The expression of each antistasin in the proboscis showed a similar pattern and varied in expression in the body. In addition, the spatial expression of antistasin orthologs in different leeches showed the possibility of different function across leech species. Hau-antistasin1 was expressed in the same region as hedgehog, which is a known mediator of signal transduction pathway. Hau-antistasin1 is probably a downstream target of Hedgehog signaling, involved in segment polarity signal pathway.

On the origin of leeches by evolution of development.

Leeches are a unique group of annelids arising from an ancestor that would be characterized as a freshwater oligochaete worm. Comparative biology of the oligochaetes and the leeches reveals that body plan changes in the oligochaete-to-leech transition probably occurred by addition or modification of the terminal steps in embryonic development and that they were likely driven by a change in the feeding behavior in the ancestor of leeches. In this review article, developmental changes that are associated with the evolution of several leech-specific traits are discussed. These include (1) the evolution of suckers, (2) the loss of chaetae, (3) the loss of septa, and (4) a fixed number of segments. An altered developmental fate of the teloblast is further proposed to be a key factor contributing to the fixation of the segment number, and the evolutionary change in teloblast development may also account for the loss of the ability to regenerate the lost body segments in the leech.

Temporal and spatial expression of the Fox gene family in the Leech Helobdella austinensis.

The Forkhead box (Fox) gene family is an evolutionarily ancient gene family named after the Drosophila melanogaster forkhead gene (fkh). Fox genes are highly conserved transcription factors critical for embryogenesis and carcinogenesis. In the current study, we report a whole-genome survey of Fox genes and their expression patterns in the leech Helobdella austienesis. Phylogenetic analysis suggests that some Fox genes of leeches are correlated with other Lophotrochozoa and vertebrate Fox genes. Here we have performed semiquantitative reverse transcription polymerase chain reaction and whole-mount in situ hybridization of Fox genes throughout the embryonic development of H. austinensis. We found that each one of the leech Fox genes (FoxA1, FoxA3, FoxC, FoxL2, FoxO1, and FoxO2) is expressed in a specific set of cells or tissue type. From Stages 9-11, Hau-FoxA1 was expressed in the foregut of the anterior region, and Hau-FoxL2 was expressed in mesodermal muscle fiber. Hau-FoxA3 was temporally expressed in the ventral neuroectoderm. At Stage 11, Hau-FoxC was expressed in the foregut. Hau-FoxO genes have a ubiquitous expression. Our results provide more insight on the evolutionary linkage and role of the Fox gene function in Bilateria.

Duplicated FoxA genes in the leech Helobdella: Insights into the evolution of direct development in clitellate annelids.

BACKGROUND: As an adaptation to the land, the clitellate annelid had reorganized its embryogenesis to develop "directly" without the ancestral planktonic larval stage. To study the evolution of gut development in the directly developing clitellates, we characterized the expression pattern of the conserved gut gene, FoxA, in the embryonic development of the leech. RESULTS: The leech has three FoxA paralogs. Hau-FoxA1 is first expressed in a subset of endoderm cells and then in the foregut and the midgut. Hau-FoxA2 is expressed in the stomodeum, which is secondarily derived from the anterior ectoderm in the clitellates rather than the tissue around the blastopore, the ancestral site of mouth formation in Phylum Annelida. Hau-FoxA3 is expressed during the morphogenesis of segmental ganglia from the ectodermal teloblast lineages, a clitellate-specific trait. Hau-FoxA1 and Hau-FoxA2 are also expressed during the morphogenesis of the leech-specific front sucker. CONCLUSIONS: The expression patterns suggested that Hau-FoxA1 carries out most of the conserved function in the endoderm and gut development, while the other two duplicates appear to have evolved unique novel functions in the directly developing clitellate embryos. Therefore, neofunctionalization and co-option of FoxA might have made a significant contribution to the evolution of direct development in Clitellata. Developmental Dynamics 247:763-778, 2018. © 2018 Wiley Periodicals, Inc.

Regional and segmental differences in the embryonic expression of a putative leech Hox gene, Lox2, by central neurons immunoreactive to FMRFamide-like neuropeptides.

We performed immunofluorescence experiments using a rat polyclonal antibody on formaldehyde-fixed whole-mount embryos to characterize the expression of a putative leech Hox gene, Lox2, during embryonic development. The main goal was to determine whether the differentiation of subsets of FMRFamide-like immunoreactive (FLI) neurons coincide with the expression domain of Lox2. The earliest expression of Lox2 was detected in relatively large, prominent nuclei in the posterior region at embryonic day 4, a very early stage. Lox2 expression was also detected in subsets of central neurons (neurons located in the CNS) located in midbody ganglia 6 (M6)-M21. In addition, Lox2 was expressed by a number of segment-specific and segmentally repeated central FLI neurons. Lox2-positive FLI neurons of interest included some of those previously identified: the rostral most ventral (RMV) neurons, the circular ventral (CV) neurons, and cell 261. The paired RMVs, which are located in all midbody ganglia, expressed Lox2 only in M7-M19. The CV neurons, specialized motor neurons that innervate the circular ventral muscles of the body wall, expressed Lox2 in M7-M19. The putative cell 261 expressed Lox2 in M7-M12, where Lox1 is also expressed. FMRFamide staining in putative segmental homologs of cell 261 was not detected in other segmental ganglia. Our results suggest a role for Lox2 in very early embryonic development (before the formation of the CNS), and in the differentiation of segmentally repeated and region-specific FLI neurons.

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.

Developmental transition to bilaterally symmetric cell divisions is regulated by Pax-mediated transcription in embryos of the leech Helobdella austinensis.

The leech embryo develops by spiral cleavage, and establishes the symmetry properties of its adult body plan through the bilaterally symmetric divisions of mesodermal proteloblast DM″ and ectodermal proteloblast DNOPQ‴. We here show that transcriptional inhibitors α-amanitin and actinomycin D specifically disrupt the symmetry and orientation of these two proteloblast cell divisions while having no apparent effect on the timing or geometry of other divisions. Transcriptional inhibition had a similar effect on both proteloblasts, i.e. cytokinesis was highly asymmetric and the cleavage plane roughly orthogonal to that seen during normal development. These findings suggest that zygotic gene product(s) are required, either directly or indirectly, for the correct placement of the proteloblast cleavage furrow. The same phenotypes were also observed following in vivo expression of dominant-negative Pax gene constructs. These dominant-negative phenotypes depended on protein/DNA interaction, and could be rescued by coexpression of full length Pax proteins. However, symmetric cleavage of the mesodermal proteloblast was rescued by full length constructs of either Hau-Paxß1 or Hau-Pax2/5/8, while only Hau-Paxß1 rescued the symmetry of ectodermal cleavage. We conclude that both proteloblasts need Pax-mediated transcription to adopt their normally symmetric cleavage patterns, but differ in terms of the specific Pax proteins required. The implication of these findings for the evolution of spiral cleavage is discussed.

Fixation/permeabilization: new alternative procedure for immunofluorescence and mRNA in situ hybridization of vertebrate and invertebrate embryos.

A new procedure is described to visualize the spatial pattern of expression of proteins and mRNAs in cryosections or whole-mounted leech, Drosophila, zebrafish, and chick embryos. Our principal contribution is in the use of a nonconventional fixation/permeabilization procedure based on the use of formaldehyde or paraformaldehyde combined with a short C-chain carboxylic acid. Detergents, methanol, and proteinases were omitted. Hybridization procedures were modified from those of routinely used protocols developed for the same embryos. Results showed that cytoskeletal and other cytoplasmic proteins, as well as different mRNAs, were clearly visualized in the expected regions of the embryos. Our procedure has several advantages over currently used protocols: is simpler, produces better general preservation of cells, yields reliable results, and can be used for embryos of different taxa at different developmental stages. It is hypothesized that short C-chain aliphatic carboxylic acids modulate the cross-linking effect of aldehyde fixatives on cell proteins.

Identification of leech embryonic neurons that express a Hox gene required for the differentiation of a paired, segment-specific motor neuron.

This study investigated the embryonic expression and function of the Hox gene Lox1 in the simple, well-characterized central nervous system (CNS) of the medicinal leech Hirudo medicinalis. Lox1 was expressed in an anterior-posterior domain, extending from the posterior aspect of the fourth segment (rostral neuromere 4, R4) to the seventeenth segment (midbody ganglion 13, M13). Lox1 expression was also found in both sex organ primordia (male and female). Lox1 expression was not detected in every cell of the ganglia included in its domain. It was detected in a specific subset that included several segmentally iterated neurons and segment-specific neurons. Several central neurons (neurons located in the central nervous system - CNS) that coexpressed both Lox1 and FMRFamide-like peptides were identified using antibody staining of leech embryos and epifluorescence and confocal microscopy. RNA interference was used to block the expression of Lox1. The expression pattern and the effect of RNA interference indicate that Lox1 is required for the differentiation of a segment-specific pair of motor neurons, the RPE (rostral penile evertor) neurons, which appear only in midbody ganglion 6 (M6) and innervate the male sex organ.

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.

Identification of 3'UTR sequence elements and a teloplasm localization motif sufficient for the localization of Hro-twist mRNA to the zygotic animal and vegetal poles.

The early localization of mRNA transcripts is critical in sorting cell fate determinants in the developing embryo. In the glossiphoniid leech, Helobdella robusta, maternal mRNAs, such as Hro-twist, localize to the zygotic teloplasm. Ten seven nucleotide repeat elements (AAUAAUA) called ARE2 and a predicted secondary structural motif, called teloplasm localization motif (TLM), are present in the 3'UTR of Hro-twist mRNA. We used site-directed mutagenesis, deletions, and microinjection of labeled, exogenous transcripts to determine if ARE2 elements, and the TLM, play a role in Hro-twist mRNA localization. Deleting the poly-A tail and the cytoplasmic polyadenylation element (CPE) had no effect on Hro-twist mRNA localization. Site-directed mutagenesis of nucleotides that altered ARE2 element sequences or the TLM suggest that the ARE2 elements and the TLM are important for Hro-twist mRNA localization to the teloplasm of pre-cleavage zygotes. Hro-Twist protein expression data suggest that the localization of Hro-twist transcripts in zygotes and stage two embryos is not involved in ensuring mesoderm specification, as Hro-Twist protein is expressed uniformly in most cells before gastrulation. Our data may support a shared molecular mechanism for leech transcripts that localize to the teloplasm.

The medicinal leech genome encodes 21 innexin genes: different combinations are expressed by identified central neurons.

Gap junctional proteins are important components of signaling pathways required for the development and ongoing functions of all animal tissues, particularly the nervous system, where they function in the intracellular and extracellular exchange of small signaling factors and ions. In animals whose genomes have been sufficiently sequenced, large families of these proteins, connexins, pannexins, and innexins, have been found, with 25 innexins in the nematode Caenorhabditis elegans Starich et al. (Cell Commun Adhes 8: 311-314, 2001) and at least 37 connexins in the zebrafish Danio rerio Cruciani and Mikalsen (Biol Chem 388:253-264, 2009). Having recently sequenced the medicinal leech Hirudo verbana genome, we now report the presence of 21 innexin genes in this species, nine more than we had previously reported from the analysis of an EST-derived transcriptomic database Dykes and Macagno (Dev Genes Evol 216: 185-97, 2006); Macagno et al. (BMC Genomics 25:407, 2010). Gene structure analyses show that, depending on the leech innexin gene, they can contain from 0 to 6 introns, with closely related paralogs showing the same number of introns. Phylogenetic trees comparing Hirudo to another distantly related leech species, Helobdella robusta, shows a high degree of orthology, whereas comparison to other annelids shows a relatively low level. Comparisons with other Lophotrochozoans, Ecdyzozoans and with vertebrate pannexins suggest a low number (one to two) of ancestral innexin/pannexins at the protostome/deuterostome split. Whole-mount in situ hybridization for individual genes in early embryos shows that ∼50% of the expressed innexins are detectable in multiple tissues. Expression analyses using quantitative PCR show that ∼70% of the Hirudo innexins are expressed in the nervous system, with most of these detected in early development. Finally, quantitative PCR analysis of several identified adult neurons detects the presence of different combinations of innexin genes, a property that may underlie the participation of these neurons in different adult coupling circuits.

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.

Evolution of development: diversified dorsoventral patterning.

Patterning of the dorsoventral axis by graded BMP signaling is conserved in the evolution of animals. However, this system has also proven to be highly adaptable, as is now highlighted by its short-range function in the leech Helobdella.

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.