An interview with Celina Juliano.

Celina Juliano is an Assistant Professor at UC Davis, where she uses Hydra as a model system to understand development and regeneration. She is co-founder of the Cnidarian Model Systems Meeting (Cnidofest) biennial conference and the OpenHydra Hydra resource platform. This year, she was awarded the Elizabeth D. Hay New Investigator award for outstanding developmental biology research during the early stages of her independent career by the Society for Developmental Biology (SDB). Following the virtual SDB 2020 meeting, we met with Celina over Zoom to hear more about her life and career.

Differential expression of BMP inhibitors gremlin and noggin in Hydra suggests distinct roles during budding and patterning of tentacles.

BACKGROUND: Mechanisms regulating BMP and Wnt pathways and their interactions are not well studied in Hydra. RESULTS: We report identification of BMP inhibitor gremlin, comparison of its expression with that of noggin and possible antagonism between Wnt and BMP signaling in Hydra. Gremlin is expressed in body column with high levels in budding region and in early buds. Noggin, on the other hand, is expressed in the hypostome, base of tentacles, lower body column, and basal disc. During budding, noggin is expressed at the sites of tentacle emergence. This was confirmed in ectopic tentacles in polyps treated with alsterpaullone (ALP), a GSK-3ß inhibitor that leads to upregulation of Wnt pathway. RT-PCR data show that upregulation of Wnt is accompanied by downregulation of bmp 5-8b though noggin and gremlin remain unaltered till 24 hours. CONCLUSIONS: Different expression patterns of gremlin and noggin suggest their roles in budding and patterning of tentacles, respectively. Further, bmp 5-8b inhibition by activated Wnt signaling does not directly involve noggin and gremlin in Hydra. Our data suggest that Wnt/BMP antagonism may have evolved early for defining the oral-aboral axis, while the involvement of BMP antagonists during axial patterning is a recent evolutionary acquisition within the Bilateria lineage.

Nodal signalling determines biradial asymmetry in Hydra.

In bilaterians, three orthogonal body axes define the animal form, with distinct anterior-posterior, dorsal-ventral and left-right asymmetries. The key signalling factors are Wnt family proteins for the anterior-posterior axis, Bmp family proteins for the dorsal-ventral axis and Nodal for the left-right axis. Cnidarians, the sister group to bilaterians, are characterized by one oral-aboral body axis, which exhibits a distinct biradiality of unknown molecular nature. Here we analysed the biradial growth pattern in the radially symmetrical cnidarian polyp Hydra, and we report evidence of Nodal in a pre-bilaterian clade. We identified a Nodal-related gene (Ndr) in Hydra magnipapillata, and this gene is essential for setting up an axial asymmetry along the main body axis. This asymmetry defines a lateral signalling centre, inducing a new body axis of a budding polyp orthogonal to the mother polyp's axis. Ndr is expressed exclusively in the lateral bud anlage and induces Pitx, which encodes an evolutionarily conserved transcription factor that functions downstream of Nodal. Reminiscent of its function in vertebrates, Nodal acts downstream of ß-Catenin signalling. Our data support an evolutionary scenario in which a 'core-signalling cassette' consisting of ß-Catenin, Nodal and Pitx pre-dated the cnidarian-bilaterian split. We presume that this cassette was co-opted for various modes of axial patterning: for example, for lateral branching in cnidarians and left-right patterning in bilaterians.

A small molecule screen identifies a novel compound that induces a homeotic transformation in Hydra.

Developmental processes such as morphogenesis, patterning and differentiation are continuously active in the adult Hydra polyp. We carried out a small molecule screen to identify compounds that affect patterning in Hydra. We identified a novel molecule, DAC-2-25, that causes a homeotic transformation of body column into tentacle zone. This transformation occurs in a progressive and polar fashion, beginning at the oral end of the animal. We have identified several strains that respond to DAC-2-25 and one that does not, and we used chimeras from these strains to identify the ectoderm as the target tissue for DAC-2-25. Using transgenic Hydra that express green fluorescent protein under the control of relevant promoters, we examined how DAC-2-25 affects tentacle patterning. Genes whose expression is associated with the tentacle zone are ectopically expressed upon exposure to DAC-2-25, whereas those associated with body column tissue are turned off as the tentacle zone expands. The expression patterns of the organizer-associated gene HyWnt3 and the hypostome-specific gene HyBra2 are unchanged. Structure-activity relationship studies have identified features of DAC-2-25 that are required for activity and potency. This study shows that small molecule screens in Hydra can be used to dissect patterning processes.

FGFR-ERK signaling is an essential component of tissue separation.

Formation of a constriction and tissue separation between parent and young polyp is a hallmark of the Hydra budding process and controlled by fibroblast growth factor receptor (FGFR) signaling. Appearance of a cluster of cells positive for double phosphorylated ERK (dpERK) at the late separation site indicated that the RAS/MEK/ERK pathway might be a downstream target of the Hydra Kringelchen FGFR. In fact, inhibition of ERK phosphorylation by the MEK inhibitor U0126 reversibly delayed bud detachment and prevented formation of the dpERK-positive cell cluster indicating de novo-phosphorylation of ERK at the late bud base. In functional studies, a dominant-negative Kringelchen FGFR prevented bud detachment as well as appearance of the dpERK-positive cell cluster. Ectopic expression of full length Kringelchen, on the other hand, induced a localized rearrangement of the actin cytoskeleton at sites of constriction, localized ERK-phosphorylation and autotomy of the body column. Our data suggest a model in which (i) the Hydra FGFR targets, via an unknown pathway, the actin cytoskeleton to induce a constriction and (ii) FGFR activates MEK/ERK signaling at the late separation site to allow tissue separation.

Organizer formation in Hydra is disrupted by thalidomide treatment.

Thalidomide is a drug that is well known for its teratogenic properties in humans. Surprisingly, thalidomide does not have teratogenic effects on mouse development. We investigated the effect of thalidomide on patterning in hydra, an early metazoan with a very simple axial symmetry. Hydra develops asexually via Wnt-dependent organizer formation, leading to the budding of a new organism. We observe both induction and inhibition of organizer formation depending on cellular context. Interestingly, thalidomide treatment altered budding and the developing organizer, but had little effect on the adult. Expression of Hybra1, a marker of the organizer increased upon thalidomide treatment. However when the organizer is induced by ectopic activation of Wnt signaling via GSK3 inhibition, thalidomide suppresses induction. We show that inhibition of Wnt signaling is not mediated by induction of the BMP pathway. We show that thalidomide activity on organizer formation in hydra depends on the activity of casein kinase1 and the abundance of ß-catenin. Finally, we find that interstitial cells, multipotent cells which give rise to nemoatocytes, neural, digestive and germline cells, are partially responsible for the inhibitory effect of thalidomide.

In an early branching metazoan, bacterial colonization of the embryo is controlled by maternal antimicrobial peptides.

Early embryos of many organisms develop outside the mother and are immediately confronted with myriads of potential colonizers. How these naive developmental stages control and shape the bacterial colonization is largely unknown. Here we show that early embryonic stages of the basal metazoan Hydra are able to control bacterial colonization by using maternal antimicrobial peptides. Antimicrobial peptides of the periculin family selecting for a specific bacterial colonization during embryogenesis are produced in the oocyte and in early embryos. If overexpressed in hydra ectodermal epithelial cells, periculin1a drastically reduces the bacterial load, indicating potent antimicrobial activity. Unexpectedly, transgenic polyps also revealed that periculin, in addition to bactericidal activity, changes the structure of the bacterial community. These findings delineate a role for antimicrobial peptides both in selecting particular bacterial partners during development and as important components of a "be prepared" strategy providing transgenerational protection.

Critical behavior and axis defining symmetry breaking in Hydra embryonic development.

The formation of a hollow cellular sphere is often one of the first steps of multicellular embryonic development. In the case of Hydra, the sphere breaks its initial symmetry to form a foot-head axis. During this process a gene, ks1, is increasingly expressed in localized cell domains whose size distribution becomes scale-free at the axis-locking moment. We show that a physical model based solely on the production and exchange of ks1-promoting factors among neighboring cells robustly reproduces the scaling behavior as well as the experimentally observed spontaneous and temperature-directed symmetry breaking.

beta-catenin plays a central role in setting up the head organizer in hydra.

In an adult hydra the head organizer, located in the hypostome, is constantly active in maintaining the structure of the animal in the context of its steady state tissue dynamics. Several Wnt genes, TCF, and elevated levels of beta-catenin are expressed in the hypostome as well as during the formation of a new organizer region in developing buds suggesting they play a role in the organizer. Transgenic hydra were generated in which a modified hydra beta-catenin gene driven by an actin promoter is continuously expressed at a high level throughout the animal. These animals formed heads and secondary axes in multiple locations along the body column. Transplantation experiments indicate they have a high and stable level of head organizer activity throughout the body columns. However, none of the Wnt genes are expressed in the body columns of these transgenic animals. Further, in alsterpaullone-treated animals, which results in a transient rise in head organizer activity throughout the body column, the time of expression of the Wnt genes is much shorter than the time of the elevated level of head inducing activity. These results for the first time provide direct functional evidence that beta-catenin plays a crucial role in the maintenance and activity of the head organizer and suggest that Wnt ligands may be required only for the initiation but not in maintenance of the organizer in Hydra.

Notch signalling defines critical boundary during budding in Hydra.

Boundary formation is an important mechanism of development and has been studied in a number of bilaterian model organisms where it is often controlled by Notch, FGF and Wnt signalling. Tissue boundaries are also formed in simple pre-bilaterian animals. The boundary between parent and bud during asexual reproduction in the fresh water polyp Hydra vulgaris is an example. The Hydra homolog of the FGF-receptor FGFR (kringelchen) and some components of the Wnt signalling pathway are expressed at this boundary, but their precise functions are unknown. In this work we have discovered an important role for Notch signalling at this boundary. Notch signalling is needed to sharpen the kringelchen expression zone during the final budding stages from an initially broad band into a clear line demarcating the boundary between bud and parent. Expression of the Notch target gene HyHes and the putative matrix metalloprotease MMP-A3 was observed at the boundary shortly before the bud began to constrict and differentiate foot cells. When Notch signalling was inhibited with the presenilin inhibitor DAPT the expression pattern for kringelchen changed dramatically into a diffused pattern. The expression of both HyHes and MMP-A3 was abolished. Moreover, morphogenesis of the bud was not completed and buds did not constrict, failed to form a foot and never detached from the parent. This resulted in the formation of two-headed animals. We suggest that the function of Notch signalling during budding in Hydra is in promoting the formation of two stripes of differing gene expression, which are needed to differentiate the foot of the bud and a progressing narrowing of the mesoglea on the side of the parent.

Early evolution of the LIM homeobox gene family.

BACKGROUND: LIM homeobox (Lhx) transcription factors are unique to the animal lineage and have patterning roles during embryonic development in flies, nematodes and vertebrates, with a conserved role in specifying neuronal identity. Though genes of this family have been reported in a sponge and a cnidarian, the expression patterns and functions of the Lhx family during development in non-bilaterian phyla are not known. RESULTS: We identified Lhx genes in two cnidarians and a placozoan and report the expression of Lhx genes during embryonic development in Nematostella and the demosponge Amphimedon. Members of the six major LIM homeobox subfamilies are represented in the genomes of the starlet sea anemone, Nematostella vectensis, and the placozoan Trichoplax adhaerens. The hydrozoan cnidarian, Hydra magnipapillata, has retained four of the six Lhx subfamilies, but apparently lost two others. Only three subfamilies are represented in the haplosclerid demosponge Amphimedon queenslandica. A tandem cluster of three Lhx genes of different subfamilies and a gene containing two LIM domains in the genome of T. adhaerens (an animal without any neurons) indicates that Lhx subfamilies were generated by tandem duplication. This tandem cluster in Trichoplax is likely a remnant of the original chromosomal context in which Lhx subfamilies first appeared. Three of the six Trichoplax Lhx genes are expressed in animals in laboratory culture, as are all Lhx genes in Hydra. Expression patterns of Nematostella Lhx genes correlate with neural territories in larval and juvenile polyp stages. In the aneural demosponge, A. queenslandica, the three Lhx genes are expressed widely during development, including in cells that are associated with the larval photosensory ring. CONCLUSIONS: The Lhx family expanded and diversified early in animal evolution, with all six subfamilies already diverged prior to the cnidarian-placozoan-bilaterian last common ancestor. In Nematostella, Lhx gene expression is correlated with neural territories in larval and juvenile polyp stages. This pattern is consistent with a possible role in patterning the Nematostella nervous system. We propose a scenario in which Lhx genes play a homologous role in neural patterning across eumetazoans.

Multiple Wnts are involved in Hydra organizer formation and regeneration.

Wnt genes and beta-catenin signaling are involved in axial patterning processes in vertebrate embryogenesis in setting up the Spemann-Mangold organizer in amphibian embryos. An organizer with a similar function is present in the hypostome of an adult Hydra polyp. Previously, a Hydra ortholog of Wnt3 (HyWnt3), which is expressed in the hypostome, has been described. Here, ten additional Hydra Wnt genes have been identified. Of these, six (HyWnt1, -7, -9/10a, -9/10c, -11, and -16) are expressed in the adult hypostome. And, as is HyWnt3, these six Wnt genes are also expressed when a new head organizer is formed during head regeneration and bud formation. The kinetics of Wnt gene expressions during head regeneration suggests that a cascade of consecutive Wnt activation accompanies regeneration, and HyWnt3 begins this cascade. Recombinant HyWnt3 protein induced body column tissue to undergo head formation. It also increased the head formation capacity in the head regeneration-deficient mutant strain reg-16 to that of wild-type strains. In addition our data reveal striking similarities in the molecular basis of the organizer in Hydra and axis polarization in chordates (e.g. Spemann's organizer) as well as it's role in regeneration suggesting a conserved function of Wnt signaling in setting up this ancient metazoan signaling center.

Cell signaling molecules in hydra: insights into evolutionarily ancient functions of signaling pathways.

Hydra, a Cnidarian believed to have been evolved about 60 million years ago, has been a favorite model for developmental biologists since Abraham Trembley introduced it in 1744. However, the modern renaissance in research on hydra was initiated by Alfred Gierer when he established a hydra laboratory at the Max Plank Institute in Göttingen in the late 1960s. Several signaling mechanisms that regulate development and pattern formation in vertebrates, including humans, have been found in hydra. These include Wnt, BMP, VEGF, FGF, Notch, and RTK signaling pathways. We have been using hydra to understand the evolution of cell signaling for the past several years. In this article, I will summarize the work on cell signaling pathways in hydra with emphasis on our own work. We have identified and characterized, for the first time, the hydra homologs of the BMP inhibitors Noggin and Gremlin, the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and several receptor tyrosine kinases (RTKs). Our work, along with that of others, clearly demonstrates that these pathways arose early in evolution to carry out functions that were often quite different from their functions in more complex animals. Apart from providing insights into morphogenesis and pattern formation in adult, budding and regenerating hydra, these findings bring out the utility of hydra as a model system to study evolutionarily ancient, in contrast to recently acquired, functions of various biological molecules.

Transgenesis in Hydra to characterize gene function and visualize cell behavior.

The freshwater polyp Hydra is a cnidarian used as a model organism in a number of fields, including the study of the origin and evolution of developmental mechanisms, aging, symbiosis and host-microbe interactions. Here, we describe a procedure for the establishment of stable transgenic Hydra lines by embryo microinjection. The three-stage protocol comprises (i) the design and preparation of a transgenic construct, (ii) the microinjection of the vector into early embryos of Hydra vulgaris, and (iii) the selection and enrichment of mosaic animals in order to develop uniformly transgenic clonal lines. The preparation of a transgenic construct requires ~2 weeks, and transgenic lines can be obtained within 3 months. The method allows constitutive or inducible gain- and loss-of-function approaches, as well as in vivo tracing of individual cells. Hydra polyps carrying transgenic cells reveal functional properties of the ancestral circuitry controlling animal development.

Molecular signature of an ancient organizer regulated by Wnt/ß-catenin signalling during primary body axis patterning in Hydra.

Wnt/ß-catenin signalling has been shown to play a critical role during head organizer formation in Hydra. Here, we characterized the Wnt signalling regulatory network involved in formation of the head organizer. We found that Wnt signalling regulates genes that are important in tissue morphogenesis. We identified that majority of transcription factors (TFs) regulated by Wnt/ß-catenin signalling belong to the homeodomain and forkhead families. Silencing of Margin, one of the Wnt regulated homeodomain TFs, results in loss of the ectopic tentacle phenotype typically seen upon activation of Wnt signalling. Furthermore, we show that the Margin promoter is directly bound and regulated by ß-catenin. Ectopic expression of Margin in zebrafish embryos results in body axis abnormalities suggesting that Margin plays a role in axis patterning. Our findings suggest that homeobox TFs came under the regulatory umbrella of Wnt/ß-catenin signalling presumably resulting in the evolution of primary body axis in animal phyla.

Models for the generation of the embryonic body axes: ontogenetic and evolutionary aspects.

Coelenterates including hydra are assumed to be close to the last common ancestor before bilaterality evolved. Models based on local self-enhancement and long-range inhibition account for pattern formation and regeneration along this ancestral axis. The body of a hydra-like ancestor evolved into the brain and heart of higher organisms, accounting for the close relationship of both patterning processes. Bilateria require a long-extended organizing region to pattern their dorsoventral axis. Models reveal the difficulties in the generation of such a stripe-like organizer and account for different mechanisms realized in vertebrates and insects. Common pathways involved in hydra budding and in the formation of appendages in higher organisms suggest a possible link.

Emergence of form in embryogenesis.

The development of form in an embryo is the result of a series of topological and informational symmetry breakings. We introduce the vector-reaction-diffusion-drift (VRDD) system where the limit cycle of spatial dynamics is morphogen concentrations with Dirac delta-type distributions. This is fundamentally different from the Turing reaction-diffusion system, as VRDD generates system-wide broken symmetry. We developed 'fundamental forms' from spherical blastula with a single organizing axis (rotational symmetry), double axis (mirror symmetry) and triple axis (no symmetry operator in three dimensions). We then introduced dynamics for cell differentiation, where genetic regulatory states are modelled as a finite-state machine (FSM). The state switching of an FSM is based on local morphogen concentrations as epigenetic information that changes dynamically. We grow complicated forms hierarchically in spatial subdomains using the FSM model coupled with the VRDD system. Using our integrated simulation model with four layers (topological, physical, chemical and regulatory), we generated life-like forms such as hydra. Genotype-phenotype mapping was investigated with continuous and jump mutations. Our study can have applications in morphogenetic engineering, soft robotics and biomimetic design.

Real time dynamics of ß-catenin expression during Hydra development, regeneration and Wnt signalling activation.

Understanding the dynamic cellular behaviours driving morphogenesis and regeneration is a long-standing challenge in biology. Live imaging, together with genetically encoded reporters, may provide the necessary tool to address this issue, permitting the in vivo monitoring of the spatial and temporal expression dynamics of a gene of interest during a variety of developmental processes. Canonical Wnt/ß-catenin signalling controls a plethora of cellular activities during development, regeneration and adulthood throughout the animal kingdom. Several reporters have been produced in animal models to reveal sites of active Wnt signalling. In order to monitor in vivo Wnt/ß-catenin signalling activity in the freshwater polyp Hydra vulgaris, we generated a ß-cat-eGFP transgenic Hydra, in which eGFP is driven by the Hydra ß-catenin promoter. We characterized the expression dynamics during budding, regeneration and chemical activation of the Wnt/ß-cat signalling pathway using light sheet fluorescence microscopy. Live imaging of the ß-cat-eGFP lines recapitulated the previously reported endogenous expression pattern of ß-catenin and revealed the dynamic appearance of novel sites of Wnt/ß-catenin signalling, that earlier evaded detection by mean of in situ hybridization. By combining the Wnt activity read-out efficiency of the ß-catenin promoter with advanced imaging, we have created a novel model system to monitor in real time the activity of Hydra ß-cat regulatory sequences in vivo, and open the path to reveal ß-catenin modulation in many other physiological contexts.

The publications of embryologist Lev V. Beloussov.

A list of papers and books of the late Lev V. Beloussov was compiled and is available in Word and EndNote Supplements. The breadth of his work is briefly described.

Structure and expression of STK, a src-related gene in the simple metazoan Hydra attenuata.

Both cDNA clones and a genomic DNA clone encoding a 509-amino-acid protein that is 64% similar to chicken pp60c-src were isolated from the simple metazoan Hydra attenuata. We have designated this gene STK, for src-type kinase. Features of the amino acid sequence of the protein encoded by the STK gene suggest that it is likely to be myristoylated and regulated by phosphorylation in a manner similar to that found for pp60c-src. The genomic sequence encoding the protein was found to be interrupted by at least two introns, one of which was located in a position identical to that of one of the introns in the chicken src gene. The STK gene was expressed during early development of H. attenuata and at high levels in the epithelial cells of adult polyps. Probing of Hydra proteins with an antibody to phosphotyrosine indicated that the major phosphotyrosine-containing protein in H. attenuata may be the STK protein itself. H. attenuata is the simplest organism from which a protein-tyrosine kinase gene has been isolated. The presence of such a gene in the evolutionarily ancient phylum Cnidaria suggests that protein-tyrosine kinase genes arose concomitantly with or shortly after the appearance of multicellular organisms.