Gut regeneration in holothurians: a snapshot of recent developments.

Visceral regeneration in sea cucumbers has been studied since early last century; however, it is only within the last 15 years that real progress has been made in understanding the cellular and molecular events involved. In the present review, we bring together these recent studies, providing readers with basic information on the anatomy and histology of the normal gut and detailing the changes in tissue organization and gene expression that occur during the regenerative process. We discuss the nature and possible sources of cells involved in the formation of the intestinal regenerate as well as the role of cell death and proliferation in this process. In addition, we compare gut formation during regeneration and during embryogenesis. Finally, we describe the molecular studies that have helped advance regenerative studies in holothurians and integrate the gene expression information with data on cellular events. Studies on visceral regeneration in these echinoderms provide a unique view that complements regeneration studies in other animal phyla, which are mainly focused on whole-animal regeneration or appendage regeneration.

The enteric nervous system of echinoderms: unexpected complexity revealed by neurochemical analysis.

Echinoderms are one of the most important groups of metazoans from the point of view of evolution, ecology and abundance. Nevertheless, their nervous system has been little studied. Particularly unexplored have been the components of the nervous system that lie outside the ectoneural and hyponeural divisions of the main nerve ring and radial nerve cords. We have gathered information on the nervous components of the digestive tract of echinoderms and demonstrate an unexpected level of complexity in terms of neurons, nerve plexi, their location and neurochemistry. The nervous elements within the digestive system consist of a distinct component of the echinoderm nervous system, termed the enteric nervous system. However, the association between the enteric nervous system and the ectoneural and hyponeural components of the nervous system is not well established. Our findings also emphasize the importance of the large lacunae in the neurobiology of echinoderms, a feature that should be addressed in future studies.

Visceral regeneration in holothurians.

Holothurians, or sea cucumbers, exhibit two processes that have intrigued biologists for decades: autotomy and regeneration. Autotomy includes the loss of body parts by evisceration or fission, and regeneration is the extraordinary process by which the lost organs are replaced. In this article, we review the literature on evisceration, transection, and visceral regeneration in holothurians and compare these processes in different orders and lower taxa. Focusing mainly on the digestive tube, we analyze regeneration from a cellular perspective, considering especially the origin, migration, and proliferation of the cellular components of the regenerated organ. The data highlight the most interesting aspects of holothurian regeneration and indicate those critical problems requiring new information and new approaches.

Identification of Hox Gene Sequences in the Sea Cucumber Holothuria glaberrima Selenka (Holothuroidea: Echinodermata).

The Echinodermata is a unique animal group forming an early branch in the deuterostomes phylogenetic tree. In echinoids and asteroids a single Hox cluster with nine cognates of the vertebrate Hox paralogous groups has been reported, but no data are available from other echinoderm classes. We report here nine Hox-type sequences from the sea cucumber Holothuria glaberrima, a member of the class Holothuroidea. Partial homeodomain sequences were amplified by polymerase chain reaction from genomic DNA and from a regenerating gastrointestinal tract complementary DNA library. Sequence analyses suggest that the holothuroid cluster has at least three genes of the anterior, one of the medial, and five of the posterior groups. This is the first evidence of five posterior sequences in echinoderms.

Differential regulation of NPY mRNA expression in embryonic sympathetic and chromaffin cultures by NGF.

RT-PCR analysis of NPY mRNA expression in chick embryonic sympathoadrenal cells in culture showed that NGF increases sympathetic but not adrenal NPY mRNA content. These results show that the previously reported differential effect at the protein level can also be detected at the mRNA level, suggesting a pre-translational point of regulation. The differential NGF effect in such closely related phenotypes is particularly relevant to studies of plasticity and differentiation.

Serum amyloid A protein in an echinoderm: its primary structure and expression during intestinal regeneration in the sea cucumber Holothuria glaberrima.

Serum amyloid A (SAA) proteins comprise a family of highly conserved apolipoproteins found in all mammals thus far investigated, and also in ducks and salmonid fishes. However, no invertebrate SAA homologues have been detected to date. Here we report the characterization of the first SAA homologue in a nonvertebrate deuterostome, the echinoderm Holothuria glaberrima. A 971-base-pair cDNA was obtained from a regenerating intestine cDNA library. The clone contains a 369-nucleotide open reading frame corresponding to a 122-amino-acid protein exhibiting a high degree of homology to members of the SAA superfamily. Sequence alignments of the holothuroid and vertebrate SAA proteins make evident a remarkable degree of conservation, even between phylogenetically disparate groups. Northern blots and immunohistochemistry show that SAA expression increases during regeneration of the holothuroid digestive tract as compared with normal nonregenerating tissue, and that the SAA protein is expressed by cells of the coelomic epithelium of the regenerating intestine. While SAA expression during the initial wound healing stage of regeneration is minimal, it increases during subsequent stages, peaking at day 15 of regeneration, concomitantly with lumen formation and the organization of the muscular layers of the regenerating digestive tract. Although in vertebrates SAA proteins may be part of a well-conserved anti-inflammatory mechanism, their exact biological function remains obscure. Our results suggest the possibility that SAA proteins, although structurally conserved, may possess enough functional diversity to participate in processes other than anti-inflammatory responses.

Analysis of mice carrying targeted mutations of the glucocorticoid receptor gene argues against an essential role of glucocorticoid signalling for generating adrenal chromaffin cells.

Molecular mechanisms underlying the generation of distinct cell phenotypes is a key issue in developmental biology. A major paradigm of determination of neural cell fate concerns the development of sympathetic neurones and neuroendocrine chromaffin cells from a common sympathoadrenal (SA) progenitor cell. Two decades of in vitro experiments have suggested an essential role of glucocorticoid receptor (GR)-mediated signalling in generating chromaffin cells. Targeted mutation of the GR should consequently abolish chromaffin cells. The present analysis of mice lacking GR gene product demonstrates that animals have normal numbers of adrenal chromaffin cells. Moreover, there are no differences in terms of apoptosis and proliferation or in expression of several markers (e.g. GAP43, acetylcholinesterase, adhesion molecule L1) of chromaffin cells in GR-deficient and wild-type mice. However, GR mutant mice lack the adrenaline-synthesizing enzyme PNMT and secretogranin II. Chromaffin cells of GR-deficient mice exhibit the typical ultrastructural features of this cell phenotype, including the large chromaffin granules that distinguish them from sympathetic neurones. Peripherin, an intermediate filament of sympathetic neurones, is undetectable in chromaffin cells of GR mutants. Finally, when stimulated with nerve growth factor in vitro, identical proportions of chromaffin cells from GR-deficient and wild-type mice extend neuritic processes. We conclude that important phenotypic features of chromaffin cells that distinguish them from sympathetic neurones develop normally in the absence of GR-mediated signalling. Most importantly, chromaffin cells in GR-deficient mice do not convert to a neuronal phenotype. These data strongly suggest that the dogma of an essential role of glucocorticoid signalling for the development of chromaffin cells must be abandoned.

Regeneration of the enteric nervous system in the sea cucumber Holothuria glaberrima.

Among higher metazoans, echinoderms exhibit the most impressive capacity for regeneration. Holothurians, or sea cucumbers, respond to adverse stimuli by autotomizing and ejecting their visceral organs, which are then regenerated. Neuronal fibers and cell bodies are present within the viscera, but previous regeneration studies have not accounted for the nervous component. We used light microscopic immunocytochemistry and ultrastructural studies to describe the regeneration of the enteric nervous system in the sea cucumber Holothuria glaberrima. This study provides evidence that the enteric nervous system of this echinoderm regenerates after evisceration and that in 3-5 weeks the regenerated system is virtually identical to that of noneviscerated animals. The regeneration of the enteric nervous system occurs parallel to the regeneration of other organ components. Nerve fibers and cells are observed within the mesenterial thickenings that give rise to the new intestine and within the internal connective tissue prior to lumen formation. We also used bromodeoxyuridine incorporation to show that proliferation of the neuronal population occurs in the regenerating intestine. The regeneration of the nervous system commands high interest because members of the closely related phylum Chordata either lack or have a very limited capacity to regenerate their nervous system. Thus, holothurians provide a model system to study enteric nervous system regeneration in deuterostomes.

Growth factors effects on the expression of morphological and biochemical properties of avian embryonic sympathetic cells. Emphasis on NGF.

Growth factors are known to be important agents in the differentiation and modulation of neuronal phenotypes. We have analyzed the effect of several growth factors on the modulation of morphological and biochemical properties of avian embryonic sympathetic neurons. The growth factors studied include: nerve growth factor (NGF), neurotrophin-3 (NT-3), brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGF-beta1). Morphological properties were analyzed by immunocytochemistry to neurofilament proteins and visualization of fibers after glyoxylic acid-induced fluorescence. Biochemical modulation was determined by radioimmunoanalysis for the peptides enkephalin (ENK), somatostatin (SS) and neuropeptide Y (NPY) and by HPLC-electrochemistry quantification of catecholamines. Similar to previous results using chromaffin cell cultures [R. Ramírez-Ordóñez, J.E. García-Arrarás, Peptidergic, catecholaminergic and morphological properties of avian chromaffin cells are modulated distinctively by growth factors, Dev. Brain Res., 87 (1995) 160-171], we found a dissociation in the modulation of biochemical and morphological properties, however, the effect of specific factors differed between the chromaffin and sympathetic cultures. We have focused on NGF to analyze its effect on the sympathetic peptide phenotypes and its lack of an effect on the chromaffin cell peptide phenotypes. The results presented here, establish interesting differences between chromaffin cells and sympathetic neurons that are of importance to studies of cell lineage and differentiation.

Developmental expression of galanin-like immunoreactivity by members of the avian sympathoadrenal cell lineage.

The developmental coexpression of galanin-like immunoreactivity with the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) was studied in the avian embryo sympathoadrenal system using double-labeling immunocytochemistry. Galanin-like immunoreactivity is expressed by various catecholaminergic cell populations, namely sympathoblasts, chromaffin and small intensely fluorescent (SIF) cells, but not by principal neurons of the paravertebral sympathetic ganglia. Both galanin and somatostatin immunoreactivities are coexpressed in the adrenal and sympathetic ganglion primordia by the neural precursors, but the subsequent expression pattern of both peptides differs. Our results support the hypothesis that early sympathoblasts express a large repertoire of neuroactive substances and that the expression of these becomes restricted during further development as the sympathoblasts become principal neurons.

Cellular mechanisms of intestine regeneration in the sea cucumber, Holothuria glaberrima Selenka (Holothuroidea:Echinodermata).

Echinoderms are the deuterostome group with the most striking capacity to regenerate lost body parts. In particular, members of the class Holothuroidea are able to regenerate most of their internal organs following a typical evisceration process. Such formation of new viscera in an adult organism provides a unique model to study the process of organogenesis. We have studied this process in the sea cucumber Holothuria glabberrima by describing the spatial and temporal pattern of cellular events that occur during intestine regeneration following chemically induced evisceration. Regeneration begins as a thickening of the mesenteries that supported the autotomized organs to the body wall. The mesenterial thickening consists of tissues where most of the cellular populations found in the normal intestine are already present. However, the cell numbers differ, particularly those of hemocytes and amoebocytes, suggesting that some of these cells play an important role in the formation of the solid rod of hypertrophic mesentery that characterizes the intestinal primordia. The appearance of the luminal epithelium, together with the formation of the lumen, occurs during the second week of regeneration by proliferation and extensive migration of cells from the esophagus and cloacal ends into the thickenings. At this stage all tissue layers are present, but it takes an additional week for them to exhibit the proportions typical of the normal organ. Cell division, as determined by BrdU labeling, mainly occurs in the coelomic epithelia of the hypertrophic mesentery and in the regenerating luminal epithelium. Our study provides evidence that the process of new organ formation in holothurians can be described as an intermediate process showing characteristics of both epimorphic and morphallactic phenomena.

Reduced acetylcholinesterase (AChE) activity in adrenal medulla and loss of sympathetic preganglionic neurons in TrkA-deficient, but not TrkB-deficient, mice.

TrkA high-affinity receptors are essential for the normal development of sympathetic paravertebral neurons and subpopulations of sensory neurons. Paravertebral sympathetic neurons and chromaffin cells of the adrenal medulla share an ontogenetic origin, responsiveness to NGF, and expression of TrkA. Which aspects of development of the adrenal medulla might be regulated via TrkA are unknown. In the present study we demonstrate that mice deficient for TrkA, but not the neurotrophin receptor TrkB, show an early postnatal progressive reduction of acetylcholinesterase (AChE) enzymatic activity in the adrenal medulla and in preganglionic sympathetic neurons within the thoracic spinal cord, which are also significantly reduced in number. Quantitative determinations of specific AChE activity revealed a massive decrease (-62%) in the adrenal gland and a lesser, but still pronounced, reduction in the thoracic spinal cord (-40%). Other markers of the adrenal medulla and its innervation, including various neuropeptides, chromogranin B, secretogranin II, amine transporters, the catecholamine-synthesizing enzymes tyrosine hydroxylase and PNMT, synaptophysin, and L1, essentially were unchanged. Interestingly, AChE immunoreactivity appeared unaltered, too. Preganglionic sympathetic neurons, in contrast to adrenal medullary cells, do not express TrkA. They must, therefore, be affected indirectly by the TrkA knock-out, possibly via a retrograde signal from chromaffin cells. Our results suggest that signaling via TrkA, but not TrkB, may be involved in the postnatal regulation of AChE activity in the adrenal medulla and its preganglionic nerves.

Development of galanin- and enkephalin-like immunoreactivities in the sympathoadrenal lineage of the avian embryo. In vivo and in vitro studies.

The neurochemical differentiation of the sympathoadrenal nervous system has been analyzed by focusing on the developmental expression of two neuropeptides, galanin and enkephalin. Both peptides are expressed early in the formation of the sympathetic ganglia and adrenal gland. Expression in the adrenal persists during embryogenesis to hatching while expression in the sympathetic is lost as sympathoblasts differentiate into neurons. Galanin expression and its modulation by nerve growth factor (NGF) and dexamethasone (Dex) was also studied in vitro. Differential effects of these factors were found on adrenal versus sympathetic cultures. However, the results coincided with proposed role of the factors in inducing either neuronal properties (NGF) or chromaffin characteristics (Dex).

Galanin-like immunoreactivity in the sea cucumber Holothuria glaberrima.

Galanin, a neuropeptide of 29 amino acids originally purified from porcine small intestine, has been found in most vertebrate groups, where it is present in both the central and peripheral nervous systems. In this study, galanin-like immunoreactivity was detected in an invertebrate, the sea cucumber Holothuria glaberrima. In this organism, fibers and cells expressing galanin-like immunoreactivity were found in the enteric nerve plexuses of the esophagus, and large and small intestine, particularly in the serosal layer. Immunoreactivity was also found in the ectoneural portion of the radial nerve and in nervous elements within the body wall. Preabsorbtion tests indicated that the observed immunoreactivity was not due to cross-reactivity of the antibody with other peptides also found in the intestinal plexuses. The immunoreactivity to galanin was quantified in various tissues using radioimmunoassay and partially characterized with high performance liquid chromatography. These results show that although the holothurian peptide shows common immunological determinants with porcine galanin, it differs with regard to certain biochemical properties.

Peptidergic, catecholaminergic and morphological properties of avian chromaffin cells are modulated distinctively by growth factors.

Most neurons and endocrine cells are known to co-express a 'classical neurotransmitter' with one or more neuropeptides. Although their expression has been shown to be modulated by differentiation factors, it is not known if particular combinations of neurotransmitter/neuropeptide(s) are co-regulated. We have analyzed the effect of nerve growth factor (NGF), neurotrophin-3 (NT-3), brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-beta 1) on the modulation of neuroactive substances co-expressed by avian chromaffin cells. The content of the neuropeptides neuropeptide Y (NPY), enkephalin (ENK) and somatostatin (SS) was measured by radioimmunoanalysis, and the content of the catecholamines norepinephrine (NE) and epinephrine (E) by high pressure liquid chromatography-electrochemistry (HPLC-EC). In addition, the morphological differentiation of chromaffin cells in response to the growth factors was assessed. All of the studied factors had distinct effects on the chromaffin content of neuropeptides and catecholamines. Our results show that the modulation of CAs and neuropeptides, and among the neuropeptides themselves is completely dissociated. Moreover, the cellular responses to the different growth factors show that neurochemical properties are modulated independently of morphological ones.

Depolarization effects on the peptidergic phenotypes of chick sympathetic and adrenal cells.

Depolarizing stimuli are among the factors known to influence the phenotypic plasticity of nerve cells. In order to determine the prevalence of the depolarization effects in terms of cell and neuropeptide phenotypes, we have analyzed the effect of potassium (K+)-induced depolarization on the avian sympathoadrenal system. The expression of three peptidergic phenotypes, somatostatin (SS), neuropeptide Y (NPY) and enkephalin (Enk) by two cell types, adrenal and sympathetic, was studied under different depolarizing regimens. Cells from the sympathetic paravertebral ganglion and adrenal gland of 10-11-day chick embryos were cultured and the peptide levels were measured by radioimmunoassays. Chronic depolarization causes differential effects on the peptidergic phenotypes increasing NPY and Enk but decreasing SS in both adrenal and sympathetic cultures. However, shorter exposures to depolarizing stimuli revealed diverse effects on NPY and Enk phenotypes and even between adrenal and sympathetic cells. Moreover, the maintenance of the effects after removal of the depolarizing stimuli showed additional differences among the phenotypes. Our results are not compatible with a previously established hypothesis stating that depolarization increases the synthesis of whichever neurotransmitters a neuron is already producing. They provide evidence indicating that the depolarization effect is much more complex than originally thought, and serve to initiate an in depth probe into the effect of depolarization of cellular plasticity.

Localization of the heptapeptide GFSKLYFamide in the sea cucumber Holothuria glaberrima (Echinodermata): a light and electron microscopic study.

Two peptides, Gly-Phe-Ser-Lys-Leu-Tyr-Phe-NH2 (GFSKLYFamide) and Ser-Gly-Tyr-Ser-Val-Leu-Tyr-Phe-NH2 (SGYSVLYFamide), recently isolated from the sea cucumber Holothuria glaberrima [Díaz-Miranda et al. (1992) Biol. Bull. 182:241-247] represent the first neuropeptides isolated from holothurians. Using an antibody against GFSKLYFa, we describe here the localization and distribution pattern of GFSKLYFa-like immunoreactivity in H. glaberrima, where immunoreactive fibers form a prominent and extensive peptidergic nervous system component. Neuron-like cells and nerve fibers expressing GFSKLYFa-like immunoreactivity are found in the ectoneural and hyponeural divisions of the radial nerve cords as well as in the digestive, haemal, respiratory, and reproductive systems; in the tentacles; and in tube feet. Neuroendocrine-like cells are found in the mucosal layer of the intestine. Ultrastructure immunocytochemical analysis revealed that, in nerve cells and fibers in the serosal layer of the intestine, the immunoreactivity is concentrated in vesicles. The immunoreactive nerve fibers are found mainly within a dense nerve plexus overlying and in close contact with smooth muscle cells of the intestine. The exclusive expression of GFSKLYFa-like immunoreactivity in neuronal or neuroendocrine tissue together with the close apposition of some fibers to muscle cells suggests that GFSKLYFa acts as a neuromuscular transmitter or neuromodulator in H. glaberrima. The wide occurrence of GFSKLYFa-like immunoreactivity throughout the nervous system of the sea cucumber suggests that GFSKLYFa plays an important role in the control of multiple action systems, including digestion, respiration, circulation, reproduction, and locomotion.

Pharmacological action of the heptapeptide GFSKLYFamide in the muscle of the sea cucumber Holothuria glaberrima (Echinodermata).

The holothurian neuropeptide GFSKLYFamide (Gly-Phe-Ser-Lys-Leu-Tyr-Phe-NH2), GFSKLYFa, was characterized recently and shown to be present in nerve fibers that apparently innervate various muscle systems. We have studied the potential neurotransmitter role of this peptide by assaying its effects on the contractility of visceral and somatic muscles. GFSKLYFa in nanomolar concentrations induces a relaxation of the muscle tension in the intestine. A similar effect is observed on the longitudinal muscle bands of the body wall of the sea cucumber. The relaxing action of GFSKLYFa is dose dependent suggesting that its action is mediated by receptors present in the muscle cells. In addition, GFSKLYFa induces the relaxation of the acetylcholine contracted intestine. Our investigation provides additional evidence indicating that GFSKLYFa might be a neurotransmitter acting at the neuromuscular junctions of the sea cucumber Holothuria glaberrima.

The syntaxin family of vesicular transport receptors.

Syntaxins A and B are nervous system-specific proteins implicated in the docking of synaptic vesicles with the presynaptic plasma membrane. A family of syntaxin-related proteins from rat has been identified that shares 23%-84% amino acid identity. Each of the six syntaxins terminate with a carboxy-terminal hydrophobic domain that anchors the protein on the cytoplasmic surface of cellular membranes. The syntaxins display a broad tissue distribution and, when expressed in COS cells, are targeted to different subcellular compartments. Microinjection studies suggest that the nervous system-specific syntaxin 1A is important for calcium-regulated secretion from neuro-endocrine PC12 cells. These results indicate that the syntaxins are a family of receptors for intracellular transport vesicles and that each target membrane may be identified by a specific member of the syntaxin family.