Regeneration of Hydra from Reaggregated Cells

Separated cells of hydra reaggregate and develop into normal animals. The regeneration of serially grafted aggregates derived from different parts of hydra tissue demonstrates that the polarity of morphogenesis in hydra is the result of the cellular composition of the tissue, not cellular orientation.     

水螅营养积累对基盘组织更新进程的影响

目的观察大乳头水螅（Hydra magnipapillata ）基盘组织更新进程,探讨水螅营养积累对基盘组织更新进程的影响。方法设定水螅喂食频率梯度（代表不同的营养积累水平）,记录和观察喂食频率对水螅更新基盘组织进程的影响。通过ABTS细胞化学染色法检测水螅基盘分子标志物过氧化物酶的表达,观察水螅老基盘组织脱落后水螅体主体新生基盘组织的再生过程。结果喂食频率对水螅更新基盘组织进程有明显的影响。水螅基盘组织更新的标准过程如下;在一定的喂食频率下培养水螅,水螅体出芽区逐渐有芽体产生,随后在出芽区和基盘之间靠近芽体的位置出现缢痕,最后水螅体在缢痕处断裂为水螅体主体和老基盘组织两部分。缢痕断裂后对水螅体主体保持既定的喂食频率,其伤口能愈合但不能再生出新的基盘组织;对其降低喂食频率直至其伤口上方的芽体全部脱落后伤口处重新启动新生基盘组织的再生进程。另外,脱落的老基盘组织有两种不同的命运,即大部分老基盘组织不能发育成正常水螅体、最终解体;而小部分的老基盘组织能发育成正常的水螅体。结论水螅营养积累可能促进基盘组织更新进程,靠近断裂伤口处的芽体能抑制水螅体主体新生基盘的再生进程。     

水螅异常极性体轴的形成及矫正进程的观察

目的:如何建立和维持体轴是一个基本的发育生物学问题,而淡水水螅是适合进行形态发生和个体发育调控机制研究的重要模式生物。本文观察了大乳头水螅异常极性体轴的形成及矫正进程,初步探讨水螅极性体轴的维持和调控机制。方法:先切取水螅的整个头部,再获得带二根触手的口区组织。通过ABTS细胞化学染色法检测水螅基盘分子标志物过氧化物酶的表达,判别水螅基盘组织(水螅足区的末端)是否形成。结果:从40块口区组织再生得到的水螅个体中有1例极性体轴发育异常的个体,其身体两端均发育成头区,且两端的头区均具有捕食能力。随后水螅其中一端头区的触手逐渐萎缩、退化,最终该端头区转化成具有吸附能力的基盘组织。结论:水螅组织的再生涉及极性体轴的重建,而一些特殊因素可能造成临时性的水螅极性体轴调控紊乱。本研究表明水螅具备自我矫正异常极性体轴的能力。另外,本研究结果显示水螅触手可以萎缩直至退化,该现象涉及的细胞学过程可能是非常复杂的,有可能涉及到触手细胞的凋亡转化过程,也可能是触手的高度分化细胞仍然具备去分化能力、去分化后再转移到身体其他地方,其具体机制值得进一步探究。     

Hydra, a niche for cell and developmental plasticity

The silencing of genes whose expression is restricted to specific cell types and/or specific regeneration stages opens avenues to decipher the molecular control of the cellular plasticity underlying head regeneration in hydra. In this review, we highlight recent studies that identified genes involved in the immediate cytoprotective function played by gland cells after amputation; the early dedifferentiation of digestive cells into blastema-like cells during head regeneration, and the early late proliferation of neuronal progenitors required for head patterning. Hence, developmental plasticity in hydra relies on spatially restricted and timely orchestrated cellular modifications, where the functions played by stem cells remain to be characterized.     

The septate junction limits mobility of lipophilic markers in plasma membranes ofHydra vulgaris (attenuata)

Summary Fluorescent lipophilic probes were used to study the role of septate junctions in maintaining distinct apical and basolateral domains of plasma membranes in epithelial cells of hydra. In short-term experiments, a 16-carbon chain aminofluorescein probe (AFC₁₆) was localized to the apical plasma membranes of ectodermal and endodermal epithelial cells when presented in the culture medium or injected into the gastric lumen, but did not demarcate basolateral membranes. In longer term experiments, basolateral membranes were stained and the staining was independent of temperature conditions. A dual 18-carbon chain indocarbocyanine probe (DiIC₁₈) gradually diffused across the septate junction to label basolateral membranes at room temperature, but not at 4°C. DiIC₁₈ also filled and stained certain mounted nematocytes. The results indicate that in hydra, lipophilic probes may be limited in mobility within the membrane plane by the septate junctions in a manner similar to vertebrate tight junctions, and that apical membranes of mature nematocytes are differentially permeable.     

Nerve commitment during head regeneration in hydra.

Most important event in head regeneration in hydra is a wave of conversion of many interstitial cells into nerve cells. Experimental evidence lends support to the idea that the commitment of interstitial cells into nerve cells is the first morphogenetic prerequisite for emergence of head structures, when the number of nerve cells increases. This increase in nerve cells is delayed when regeneration occurs at a site lower in the body column.     

A novel hydra matrix metalloproteinase (HMMP) functions in extracellular matrix degradation, morphogenesis and the maintenance of differentiated cells in the foot process

As a member of Cnidaria, the body wall of hydra is structurally reduced to an epithelial bilayer with an intervening extracellular matrix (ECM). Biochemical and cloning studies have shown that the molecular composition of hydra ECM is similar to that seen in vertebrates and functional studies have demonstrated that cell-ECM interactions are important to developmental processes in hydra. Because vertebrate matrix metalloproteinases (MMPs) have been shown to have an important role in cell-ECM interactions, the current study was designed to determine whether hydra has homologues of these proteinases and, if so, what function these enzymes have in morphogenesis and cell differentiation in this simple metazoan. Utilizing a PCR approach, a single hydra matrix metalloproteinase, named HMMP was identified and cloned. The structure of HMMP was similar to that of vertebrate MMPs with an overall identity of about 35%. Detailed structural analysis indicated some unique features in (1) the cysteine-switch region of the prodomain, (2) the hinge region preceding the hemopexin domain, and (3) the hemopexin domain. Using a bacterial system, HMMP protein was expressed and folded to obtain an active enzyme. Substrate analysis studies indicated that recombinant HMMP could digest a number of hydra ECM components such as hydra laminin. Using a fluorogenic MMP substrate assay, it was determined that HMMP was inhibited by peptidyl hydroxamate MMP inhibitors, GM6001 and matlistatin, and by human recombinant TIMP-1. Whole-mount in situ studies indicated that HMMP mRNA was expressed in the endoderm along the entire longitudinal axis of hydra, but at relatively high levels at regions where cell-transdifferentiation occurred (apical and basal poles). Functional studies using GM6001 and TIMP-1 indicated that these MMP inhibitors could reversibly block foot regeneration. Blockage of foot regeneration was also observed using antisense thio-oligo nucleotides to HMMP introduced into the endoderm of the basal pole using a localized electroporation technique. Studies with adult intact hydra found that GM6001 could also cause the reversible de-differentiation or inhibition of transdifferentiation of basal disk cells of the foot process. Basal disk cells are adjacent to those endoderm cells of the foot process that express high levels of HMMP mRNA. In summary, these studies indicate that hydra has at least one MMP that is functionally tied to morphogenesis and cell transdifferentiation in this simple metazoan.     

A new biochemical marker for foot-specific cell differentiation in hydra

Summary Mucous cells in the basal disk of hydra contain a peroxidase-like enzyme allowing specific staining of these cells with substrates for peroxidases. The peroxidase activity provides an excellent marker for foot mucous cell, differentiation and was used to follow the reappearance of footspecific cells during foot regeneration after amputation. By choosing the appropriate either soluble or precipitable substrate the peroxidase reaction was used both for a qualitative and for a quantitative evaluation of foot-specific differentiation in hydra. For histological studies diaminobenzidien was found to be a suitable substrate which forms a dark brown precipitate within the cells containing the peroxidase activity. For a quantitative evaluation of foot regeneration the soluble substrate 2,2-azino-di(3-ethyl-benzthiazoline-sulfonic acid-6) ammonium salt was used which after reaction with the enzyme gives rise to a diffusible green reaction product the concentration of which can be measured by its specific absorption at 415 nm. Based on the diffusible enzyme product a new quantitative assay for foot regenration was developed and applied to confirm the effect and specificity of morphogenetic substances which either inhibit or activate foot or head regeneration in hydra.     

Regeneration of hydra damaged by zolone PM insecticide

The zolone PM (phosalone) insecticide in the suspensions used destroys parts of the hypostome, ruins many buds and budless hydras. In the insecticide suspension a hydra shrinks and assumes the smallest surface. It is due to this that the surface mucous layer thickens and epidermal and gastrodermal cells become highly squeezed together. The insecticide penetrates the hypostomal opening and destroys hypostomal cells but does not act specifically upon a certain type of cells. Gradually, other cells replace the destroyed ones. They arrive from the gastrodermal region. Among them, zymogen cells, capable of differentiation und dedifferentiation into other types of cells, play the most significant part.     

Epithelial morphogenesis in hydra requires de novo expression of extracellular matrix components and matrix metalloproteinases

As a member of the phylum Cnidaria, the body wall of hydra is organized as an epithelium bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM). Previous studies have established the general molecular structure of hydra ECM and indicate that it is organized as two subepithelial zones that contain basement membrane components such as laminin and a central fibrous zone that contains interstitial matrix components such as a unique type I fibrillar collagen. Because of its simple structure and high regenerative capacity, hydra has been used as a developmental model to study cell-ECM interaction during epithelial morphogenesis. The current study extends previous studies by focusing on the relationship of ECM biogenesis to epithelial morphogenesis in hydra, as monitored during head regeneration or after simple incision of the epithelium. Histological studies indicated that decapitation or incision of the body column resulted in an immediate retraction of the ECM at the wound site followed by a re-fusion of the bilayer within 1 hour. After changes in the morphology of epithelial cells at the regenerating pole, initiation of de novo biogenesis of an ECM began within hours while full reformation of the mature matrix required approximately 2 days. These processes were monitored using probes to three matrix or matrix-associated components: basement membrane-associated hydra laminin beta1 chain (HLM-beta1), interstitial matrix-associated hydra fibrillar collagen (Hcol-I) and hydra matrix metalloproteinase (HMMP). While upregulation of mRNA for both HLM-beta1 and Hcol-I occurred by 3 hours, expression of the former was restricted to the endoderm and expression of the latter was restricted to the ectoderm. Upregulation of HMMP mRNA was also associated with the endoderm and its expression paralleled that for HLM-beta1. As monitored by immunofluorescence, HLM-beta1 protein first appeared in each of the two subepithelial zones (basal lamina) at about 7 hours, while Hcol-I protein was first observed in the central fibrous zone (interstitial matrix) between 15 and 24 hours. The same temporal and spatial expression pattern for these matrix and matrix-associated components was observed during incision of the body column, thus indicating that these processes are a common feature of the epithelium in hydra. The correlation of loss of the ECM, cell shape changes and subsequent de novo biogenesis of matrix and matrix-associated components were all functionally coupled by antisense experiments in which translation of HLM-beta1 and HMMP was blocked and head regeneration was reversibly inhibited. In addition, inhibition of translation of HLM-beta1 caused an inhibition in the appearance of Hcol-I into the ECM, thus suggesting that binding of HLM-beta1 to the basal plasma membrane of ectodermal cells signaled the subsequent discharge of Hcol-I from this cell layer into the newly forming matrix. Given the early divergence of hydra, these studies point to the fundamental importance of cell-ECM interactions during epithelial morphogenesis.     

Cnidarian and bilaterian promoters can direct GFP expression in transfected hydra

Complete sexual development is not easily amenable to experimentation in hydra. Therefore, the analysis of gene function and gene regulation requires the introduction of exogenous DNA in a large number of cells of the hydra polyps and the significant expression of reporter constructs in these cells. We present here the procedure whereby we coupled DNA injection into the gastric cavity to electroporation of the whole animal in order to efficiently transfect hydra polyps. We could detect GFP fluorescence in both endodermal and ectodermal cell layers of live animals and in epithelial as well as interstitial cell types of dissociated hydra. In addition, we could confirm GFP protein expression by showing colocalisation between GFP fluorescence and anti-GFP immunofluorescence. Finally, when a FLAG epitope was inserted in-frame with the GFP coding sequence, GFP fluorescence also colocalised with anti-FLAG immunofluorescence. This GFP expression in hydra cells was directed by various promoters, either homologous, like the hydra homeobox cnox-2 gene promoter, or heterologous, like the two nematode ribosomal protein S5 and L28 gene promoters, and the chicken beta-actin gene promoter. This strategy provides new tools for dissecting developmental molecular mechanisms in hydra; more specifically, the genetic regulations that take place in endodermal cells at the time budding or regeneration is initiated.     

Mechanisms of nutritive endocytosis. III. A freeze-fracture study of phagocytosis by digestive cells of Chlorohydra

Freeze-fracture was used to compare the ultrastructure of plasma with phagosomal membranes of digestive cells of green hydra. Changes in both the pattern and density of intramembrane particles (IMP) were evident during the transition from plasma to phagosomal membrane. Small particle-free regions and associated aggregates of three to eight IMP were observed in presumptive adherent and enveloping plasma membranes, as well as in fully formed phagosomal membranes. Larger particle-free regions were observed as rims around the tips of enveloping membranes which had nearly completed enclosure of particles. The density of IMP in newly formed phagosomal membranes was 1.5-fold greater than that in the parent plasma membrane from which they derived, and was greater also than in older phagosomal membranes.     

Identification and characterization of hydra metalloproteinase 2 (HMP2): a meprin-like astacin metalloproteinase that functions in foot morphogenesis

Several members of the newly emerging astacin metalloproteinase family have been shown to function in a variety of biological events, including cell differentiation and morphogenesis during both embryonic development and adult tissue differentiation. We have characterized a new astacin proteinase, hydra metalloproteinase 2 (HMP2) from the Cnidarian, Hydra vulgaris. HMP2 is translated from a single mRNA of 1.7 kb that contains a 1488 bp open reading frame encoding a putative protein product of 496 amino acids. The overall structure of HMP2 most closely resembles that of meprins, a subgroup of astacin metalloproteinases. The presence of a transient signal peptide and a putative prosequence indicates that HMP2 is a secreted protein that requires post-translational processing. The mature HMP2 starts with an astacin proteinase domain that contains a zinc binding motif characteristic of the astacin family. Its COOH terminus is composed of two potential protein-protein interaction domains: an "MAM" domain (named after meprins, A-5 protein and receptor protein tyrosine phosphatase mu) that is only present in meprin-like astacin proteinases; and a unique C-terminal domain (TH domain) that is also present in another hydra metalloproteinase, HMP1, in Podocoryne metalloproteinase 1 (PMP1) of jellyfish and in toxins of sea anemone. The spatial expression pattern of HMP2 was determined by both mRNA whole-mount in situ hybridization and immunofluorescence studies. Both morphological techniques indicated that HMP2 is expressed only by the cells in the endodermal layer of the body column of hydra. While the highest level of HMP2 mRNA expression was observed at the junction between the body column and the foot process, immunofluorescence studies indicated that HMP2 protein was present as far apically as the base of the tentacles. In situ analysis also indicated expression of HMP2 during regeneration of the foot process. To test whether the higher levels of HMP2 mRNA expression at the basal pole related to processes underlying foot morphogenesis, antisense studies were conducted. Using a specialized technique named localized electroporation (LEP), antisense constructs to HMP2 were locally introduced into the endodermal layer of cells at the basal pole of polyps and foot regeneration was initiated and monitored. Treatment with antisense to HMP2 inhibited foot regeneration as compared to mismatch and sense controls. These functional studies in combination with the fact that HMP2 protein was expressed not only at the junction between the body column and the foot process, but also as far apically as the base of the tentacles, suggest that this meprin-class metalloproteinase may be multifunctional in hydra.     

RNAi gene silencing affects cell and developmental plasticity in hydra

The recent establishment of gene silencing through RNA interference upon feeding opens avenues to decipher the genetic control of regeneration in hydra. Following that approach, we identified three main stages for head regeneration. Immediately post-amputation, the serine protease inhibitor Kazal1 gene produced by the gland cells prevents from an excessive autophagy in regenerating tips. This cytoprotective function, or self-preservation, is similar to that played by Kazal-type proteins in the mammalian exocrine pancreas, in homeostatic or post-injury conditions, likely reflecting an evolutionarily conserved mechanism linking cell survival to tissue repair. Indeed, in wild-type hydra, within the first hours following mid-gastric section, an extensive cellular remodelling is taking place, including phenotypic cellular transitions and cell proliferation. The activation of the MAPK pathway, which leads to the RSK-dependent CREB phosphorylation, is required for these early cellular events. Later, at the early-late stage, the expression of the Gsx/cnox-2 ParaHox gene in proliferating apical neuronal progenitors is required for the de novo neurogenesis that precedes the emergence of the tentacle rudiments. Hence, head regeneration in wild-type hydra relies on spatially restricted and timely orchestrated cellular modifications, which display similarities with those reported during vertebrate epimorphic regeneration. These results suggest some conservation across evolution of the mechanisms driving the post-amputation reactivation of developmental programs.     

Head regeneration in wild-type hydra requires de novo neurogenesis

Because head regeneration occurs in nerve-free hydra mutants, neurogenesis was regarded as dispensable for this process. Here, in wild-type hydra, we tested the function of the ParaHox gsx homolog gene, cnox-2, which is a specific marker for bipotent neuronal progenitors, expressed in cycling interstitial cells that give rise to apical neurons and gastric nematoblasts (i.e. sensory mechanoreceptor precursors). cnox-2 RNAi silencing leads to a dramatic downregulation of hyZic, prdl-a, gsc and cnASH, whereas hyCOUP-TF is upregulated. cnox-2 indeed acts as an upstream regulator of the neuronal and nematocyte differentiation pathways, as cnox-2(-) hydra display a drastic reduction in apical neurons and gastric nematoblasts, a disorganized apical nervous system and a decreased body size. During head regeneration, the locally restricted de novo neurogenesis that precedes head formation is cnox-2 dependent: cnox-2 expression is induced in neuronal precursors and differentiating neurons that appear in the regenerating tip; cnox-2 RNAi silencing reduces this de novo neurogenesis and delays head formation. Similarly, the disappearance of cnox-2(+) cells in sf-1 mutants also correlates with head regeneration blockade. Hence in wild-type hydra, head regeneration requires the cnox-2 neurogenic function. When neurogenesis is missing, an alternative, slower and less efficient, head developmental program is possibly activated.     

Autophagy and self-preservation: a step ahead from cell plasticity?

Silencing the SPINK-related gene Kazal1 in hydra gland cells induces an excessive autophagy of both gland and digestive cells, leading to animal death. Moreover, during regeneration, autophagosomes are immediately detected in regenerating tips, where Kazal1 expression is lowered. When Kazal1 is completely silenced, hydra no longer survive the amputation stress (Chera S, de Rosa R, Miljkovic-Licina M, Dobretz K, Ghila L, Kaloulis K, Galliot B. Silencing of the hydra serine protease inhibitor Kazal1 gene mimics the human Spink1 pancreatic phenotype. J Cell Sci 2006; 119:846-57). These results highlight the essential digestive and cytoprotective functions played by Kazal1 in hydra. In mammals, autophagy of exocrine pancreatic cells is also induced upon SPINK1/Spink3 inactivation, whereas Spink3 is activated in injured pancreatic cells. Hence SPINKs, by preventing an excessive autophagy, appear to act as key players of the stress-induced self-preservation program. In hydra, this program is a prerequisite to the early cellular transition, whereby digestive cells of the regenerating tips transform into a head-organizer center. Enhancing the self-preservation program in injured tissues might therefore be the condition for unmasking their potential cell and/or developmental plasticity.     

Structure and function of an early divergent form of laminin in hydra: a structurally conserved ECM component that is essential for epithelial morphogenesis

As a major component of the extracellular matrix (ECM), laminin has been found in many vertebrate and invertebrate organisms. Its molecular structure is very similar across species lines and its biological function in the ECM has been extensively studied. In an effort to study ECM structure and function in hydra, we have cloned a partial hydra laminin alpha chain and the full-length hydra laminin beta chain using ECM-enriched cDNA libraries. Analysis of deduced amino acid sequences indicated that both polypeptides have high sequence similarity to a number of invertebrate and vertebrate laminin alpha and beta subunits. Rotary shadow analysis of isolated hydra laminin indicates it has a heterotrimeric organization that is characteristic of vertebrate laminins. A putative integrin-class protein was also identified using a cell-binding peptide sequence from the laminin beta chain as an affinity probe, indicating that integrins are possible cell surface receptors in hydra. In agreement with previous results for the hydra laminin beta chain, in situ hybridization experiments revealed that hydra laminin alpha chain mRNA is restricted to endodermal cells. As with a number of other hydra ECM components, higher levels of laminin alpha chain mRNA are localized to regions where cell migration and differentiation are actively undertaken such as the base of tentacles, the peduncle region, buds, regenerating tentacles, and at the head end during regeneration. The role of laminin in morphogenesis was studied using an antisense approach and the results indicated that translation of the laminin alpha chain is required for head regeneration.