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Over the past 14 years, the Neuroscience Research Opportunities to Increase Diversity (NeuroID) program, funded by the National Institute of Neurological Diseases and Stroke (NINDS), has played a transformative role in training numerous undergraduate Hispanic students within The University of Puerto Rico-Rio Piedras (UPR-RP). This innovative Neuroscience-based research training initiative has successfully guided dozens of Hispanic students toward graduate programs in Neuroscience, significantly contributing to the enhancement of diversity within the academic and scientific fields. The achievements of the NeuroID program can be attributed to three key objectives. Firstly, the establishment of a comprehensive and innovative program has provided Hispanic undergraduate students with invaluable insights into various facets of a research career in neuroscience. Secondly, the program has fostered a robust mentorship network that supports selected students throughout their journey to become neuroscientists. Thirdly, it has strengthened the neuroscience network in Puerto Rico by bridging the gap between undergraduate teaching universities and research laboratories in top-tier institutions across the mainland United States., (Copyright © 2024 Faculty for Undergraduate Neuroscience.)

In holothurians, the regenerative process following evisceration involves the development of a "rudiment" or "anlage" at the injured end of the mesentery. This regenerating anlage plays a pivotal role in the formation of a new intestine. Despite its significance, our understanding of the molecular characteristics inherent to the constituent cells of this structure has remained limited. To address this gap, we employed state-of-the-art scRNA-seq and HCR-FISH analyses to discern the distinct cellular populations associated with the regeneration anlage. Through this approach, we successfully identified thirteen distinct cell clusters. Among these, two clusters exhibit characteristics consistent with putative mesenchymal cells, while another four show features akin to coelomocyte cell populations. The remaining seven cell clusters collectively form a large group encompassing the coelomic epithelium of the regenerating anlage and mesentery. Within this large group of clusters, we recognized previously documented cell populations such as muscle precursors, neuroepithelial cells and actively proliferating cells. Strikingly, our analysis provides data for identifying at least four other cellular populations that we define as the precursor cells of the growing anlage. Consequently, our findings strengthen the hypothesis that the coelomic epithelium of the anlage is a pluripotent tissue that gives rise to diverse cell types of the regenerating intestinal organ. Moreover, our results provide the initial view into the transcriptomic analysis of cell populations responsible for the amazing regenerative capabilities of echinoderms.

Wnt signaling has been shown to play multiple roles in regenerative processes, one of the most widely studied of which is the regeneration of the intestinal luminal epithelia. Most studies in this area have focused on self-renewal of the luminal stem cells; however, Wnt signaling may also have more dynamic functions, such as facilitating intestinal organogenesis. To explore this possibility, we employed the sea cucumber Holothuria glaberrima that can regenerate a full intestine over the course of 21 days after evisceration. We collected RNA-seq data from various intestinal tissues and regeneration stages and used these data to define the Wnt genes present in H. glaberrima and the differential gene expression (DGE) patterns during the regenerative process. Twelve Wnt genes were found, and their presence was confirmed in the draft genome of H. glaberrima. The expressions of additional Wnt-associated genes, such as Frizzled and Disheveled , as well as genes from the Wnt/β-catenin and Wnt/Planar Cell Polarity (PCP) pathways, were also analyzed. DGE showed unique distributions of Wnt in early- and late-stage intestinal regenerates, consistent with the Wnt/β-catenin pathway being upregulated during early-stages and the Wnt/PCP pathway being upregulated during late-stages. Our results demonstrate the diversity of Wnt signaling during intestinal regeneration, highlighting possible roles in adult organogenesis.

Radial glia is a cell type traditionally associated with the developing nervous system, particularly with the formation of cortical layers in the mammalian brain. Nonetheless, some of these cells, or closely related types, called radial glia-like cells are found in adult central nervous system structures, functioning as neurogenic progenitors in normal homeostatic maintenance and in response to injury. The heterogeneity of radial glia-like cells is nowadays being probed with molecular tools, primarily by the expression of specific genes that define cell types. Similar markers have identified radial glia-like cells in the nervous system of non-vertebrate organisms. In this review, we focus on adult radial glia-like cells in neurogenic processes during homeostasis and in response to injury. We highlight our results using a non-vertebrate model system, the echinoderm Holothuria glaberrima where we have described a radial glia-like cell that plays a prominent role in the regeneration of the holothurian central nervous system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Miranda-Negrón and García-Arrarás.)

Injury to the central nervous system (CNS) results in permanent damage and lack of function in most vertebrate animals, due to their limited regenerative capacities. In contrast, echinoderms can fully regenerate their radial nerve cord (RNC) following transection, with little to no scarring. Investigators have associated the regenerative capacity of some organisms to the stress response and inflammation produced by the injury. Here, we explore the gene activation profile of the stressed holothurian CNS. To do this, we performed RNA sequencing on isolated RNC explants submitted to the stress of transection and enzyme dissection and compared them with explants kept in culture for 3 days following dissection. We describe stress-associated genes, including members of heat-shock families, ubiquitin-related pathways, transposons, and apoptosis that were differentially expressed. Surprisingly, the stress response does not induce apoptosis in this system. Other genes associated with stress in other animal models, such as hero proteins and those associated with the integrated stress response, were not found to be differentially expressed either. Our results provide a new viewpoint on the stress response in the nervous system of an organism with amazing regenerative capacities. This is the first step in deciphering the molecular processes that allow echinoderms to undergo fully functional CNS regeneration, and also provides a comparative view of the stress response in other organisms.

The cellular events underlying intestine regrowth in the sea cucumber Holothuria glaberrima have been described by our group. Currently, the molecular and signaling mechanisms involved in this process are being explored. One of the limitations to our investigations has been the absence of suitable cell culture methodologies, required to advance the regeneration studies. An in vitro system, where regenerating intestine explants can be studied in organ culture, was established previously by our group. However, a detailed description of the histological properties of the cultured gut explants was lacking. Here, we used immunocytochemical techniques to study the potential effects of the culture conditions on the histological characteristics of explants, comparing them to the features observed during gut regeneration in our model in vivo . Additionally, the explant outgrowths were morphologically described by phase-contrast microscopy and SEM. Remarkably, intestine explants retain most of their original histoarchitecture for up to 10 days, with few changes as culture time increases. The most evident effects of the culture conditions on explants over culture time were the reduction in the proliferative rate, the loss of the polarity in the localization of proliferating cells, and the appearance of a subpopulation of putative spherulocytes. Finally, cells that migrated from the gut explants could form net-like monolayers, firmly attached to the culture substrate. Overall, regenerating explants in organ culture represent a powerful tool to perform short-term studies of processes associated with gut regeneration in H. glaberrima under controlled conditions., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

The microbiota, the set of microorganisms associated with a particular environment or host, has acquired a prominent role in the study of many physiological and developmental processes. Among these, is the relationship between the microbiota and regenerative processes in various organisms. Here we introduce the concept of the microbiota and its involvement in regeneration-related cellular events. We then review the role of the microbiota in regenerative models that extend from the repair of tissue layers to the regeneration of complete organs or animals. We highlight the role of the microbiota in the digestive tract, since it accounts for a significant percentage of an animal microbiota, and at the same time provides an outstanding system to study microbiota effects on regeneration. Lastly, while this review serves to highlight echinoderms, primarily holothuroids, as models for regeneration studies, it also provides multiple examples of microbiota-related interactions in other processes in different organisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Díaz-Díaz, Rodríguez-Villafañe and García-Arrarás.)

Which genes and gene signaling pathways mediate regenerative processes? In recent years, multiple studies, using a variety of animal models, have aimed to answer this question. Some answers have been obtained from transcriptomic and genomic studies where possible gene and gene pathway candidates thought to be involved in tissue and organ regeneration have been identified. Several of these studies have been done in echinoderms, an animal group that forms part of the deuterostomes along with vertebrates. Echinoderms, with their outstanding regenerative abilities, can provide important insights into the molecular basis of regeneration. Here we review the available data to determine the genes and signaling pathways that have been proposed to be involved in regenerative processes. Our analyses provide a curated list of genes and gene signaling pathways and match them with the different cellular processes of the regenerative response. In this way, the molecular basis of echinoderm regenerative potential is revealed, and is available for comparisons with other animal taxa., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Medina-Feliciano and García-Arrarás.)

Regeneration is a key developmental process by which organisms recover vital tissue and organ components following injury or disease. A growing interest is focused on the elucidation and characterization of the molecular mechanisms involved in these regenerative processes. We have now analyzed the possible role of the Wnt/β-catenin pathway on the regeneration of the intestine in the sea cucumber Holothuria glaberrima. For this we have studied the expression in vivo of Wnt-associated genes and have implemented the use of Dicer-substrate interference RNA (DsiRNA) to knockdown the expression of β-catenin transcript on gut rudiment explants. Neither cell dedifferentiation nor apoptosis were affected by the reduction of β-catenin transcripts in the gut rudiment explants. Yet, the number of proliferating cells decreased significantly following the interference, suggesting that the Wnt/β-catenin signaling pathway plays a significant role in cell proliferation, but not in cell dedifferentiation nor apoptosis during the regeneration of the intestine. The development of the in vitro RNAi protocol is a significant step in analyzing specific gene functions involved in echinoderm regeneration., (Copyright © 2021. Published by Elsevier Inc.)

The increased antibiotics usage in biomedical and agricultural settings has been well documented. Antibiotics have now been shown to exert effects outside their purposive use, including effects on physiological and developmental processes. We explored the effect of various antibiotics on intestinal regeneration in the sea cucumber Holothuria glaberrima . For this, holothurians were eviscerated and left to regenerate for 10 days in seawater with different penicillin/streptomycin-based cocktails (100 µg/mL PS) including: 100 µg/mL kanamycin (KPS), 5 µg/mL vancomycin (VPS), and 4 µg/mL (E4PS) or 20 µg/mL (E20PS) erythromycin. Immunohistological and histochemical analyses were performed to analyze regenerative processes, including rudiment size, extracellular matrix (ECM) remodeling, cell proliferation, and muscle dedifferentiation. A reduction in muscle dedifferentiation was observed in all antibiotic-treated animals. ECM remodeling was decreased by VPS, E4PS, and E20PS treatments. In addition, organisms subjected to E20PS displayed a significant reduction in the size of their regenerating rudiments while VPS exposure altered cell proliferation. MTT assays were used to discard the possibility that the antibiotics directly affect holothurian metabolic activity while bacterial cultures were used to test antibiotic effects on holothurian enteric microbiota. Our results demonstrate a negative effect on intestinal regeneration and strongly suggest that these effects are due to alterations in the microbial community.

Echinoderms comprise a group of animals with impressive regenerative capabilities. They can replace complex internal organs following injury or autotomy. In holothurians or sea cucumbers, cellular processes of intestinal regeneration have been extensively studied. The molecular machinery behind this faculty, however, remains to be understood. Here we assembled and annotated a de novo transcriptome using RNA-seq data consisting of regenerating and non-regenerating intestinal tissues from the sea cucumber Holothuria glaberrima. Comparisons of differential expression were made using the mesentery as a reference against 24 h and 3 days regenerating intestine, revealing a large number of differentially expressed transcripts. Gene ontology and pathway enrichment analysis showed evidence of increasing transcriptional activity. Further analysis of transcripts associated with transcription factors revealed diverse expression patterns with mechanisms involving developmental and cancer-related activity that could be related to the regenerative process. Our study demonstrates the broad and diversified gene expression profile during the early stages of the process using the mesentery as the focal point of intestinal regeneration. It also establishes the genes that are the most important candidates in the cellular processes that underlie regenerative responses.

Regeneration is one of the most fascinating and yet least understood biological processes. Echinoderms, one of the closest related invertebrate groups to humans, can contribute to our understanding of the genetic basis of regenerative processes. Among echinoderms, sea cucumbers have the ability to grow back most of their body parts following injury, including the intestine and nervous tissue. The cellular and molecular events underlying these abilities in sea cucumbers have been most extensively studied in the species Holothuria glaberrima . However, research into the regenerative abilities of this species has been impeded due to the lack of adequate genomic resources. Here, we report the first draft genome assembly of H. glaberrima and demonstrate its value for future genetic studies. Using only short sequencing reads, we assembled the genome into 89,105 scaffolds totaling 1.1 gigabases with an N50 of 25 kilobases. Our BUSCO assessment of the genome resulted in 894 (91.4%) complete and partial genes from 978 genes queried. We incorporated transcriptomic data from several different life history stages to annotate 51,415 genes in our final assembly. To demonstrate the usefulness of the genome, we fully annotated the melanotransferrin ( Mtf) gene family, which have a potential role in the regeneration of the sea cucumber intestine. Using these same data, we extracted the mitochondrial genome, which showed high conservation to that of other holothuroids. Thus, these data will be a critical resource for ongoing studies of regeneration and other studies in sea cucumbers., Competing Interests: Conflict of Interest: SP was employed by Iridian Genomes, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The cellular mechanisms underlying the amazing ability of sea cucumbers to regenerate their autotomized intestines have been widely described by us and others. However, the signaling pathways that control these mechanisms are unknown. Previous studies have shown that Wnt homologs are upregulated during early intestinal regenerative stages, suggesting that the Wnt/β-catenin pathway is active during this process. Here, we used small molecules, putative disruptors of the Wnt pathway, to determine the potential role of the canonical Wnt pathway on intestine regeneration in the sea cucumber Holothuria glaberrima. We evaluated their effects in vivo by using histological analyses for cell dedifferentiation, cell proliferation and apoptosis. We found that iCRT14, an alleged Wnt pathway inhibitor, decreased the size of the regenerating intestine, while LiCl, a presumed Wnt pathway activator, increased its size. The possible cellular mechanisms by which signaling pathway disruptors affect the gut rudiment size were further studied in vitro, using cultures of tissue explants and additional pharmacological agents. Among the tested signaling activators, those that act through GSK-3 inhibition, LiCl, 1-Azakenpaullone, and CHIR99021 were found to increase muscle cell dedifferentiation, while the inhibitor iCRT14 blocked cell dedifferentiation. Differently, cell proliferation was reduced by all GSK-3 inhibitors, as well as by iCRT14 and C59, which interferes with Wnt ligand secretion. The in vivo temporal and spatial pattern of β-catenin activity was determined using an antibody against phosphorylated β-catenin and shown to correlate with cell proliferative activity. In vitro treatment using C59 decreased the number of cells immunostained for nuclear phosphorylated β-catenin. Our results showed that the cell dedifferentiation observed during intestinal regeneration can be decoupled from the cell proliferation event and that these cellular processes can be modulated by particular signaling pathway inhibitors and activators. These results open the door for future studies where the cellular signaling pathways involved at each regeneration stage can be determined., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Almost every organism has the ability of repairing damaged tissues or replacing lost and worn out body parts, nevertheless the degree of the response substantially differs between each species. Adult sea cucumbers from the Holothuria glaberrima species can eviscerate various organs and the intestinal system is the first one to regenerate. This process involves the formation of a blastema-like structure that derives from the torn mesentery edges by the intervention of specific cellular processes (e.g., cell dedifferentiation and division). Still, the genetic networks controlling the regenerative response in this model system are just starting to be unraveled. In this work we examined if and how the retinoic acid (RA) signaling pathway is involved in the regenerative response of this deuterostome. We first identified and characterized the holothurian orthologs for short chain dehydrogenase/reductase 7 (SDR7) and aldehyde dehydrogenase family 8A1 (ALDH8A1), two enzymes respectively associated with retinaldehyde and RA anabolism. We then showed that the SDR7 transcript was differentially expressed during specific stages of intestinal regeneration while ALDH8A1 did not show significant differences in regenerating tissues when compared to those of normal (non-eviscerated) organisms. Finally, we investigated the consequences of modulating RA signaling during intestinal regeneration using pharmacological tools. We showed that application of an inhibitor (citral) of the enzyme synthesizing RA or a retinoic acid receptor (RAR) antagonist (LE135) resulted in organisms with a significantly smaller intestinal rudiment when compared to those treated with DMSO (vehicle). The two inhibitors caused a reduction in cell division and cell dedifferentiation in the new regenerate when compared to organisms treated with DMSO. Results of treatment with tazarotene (an RAR agonist) were not significantly different from the control. Taken together, these results suggest that the RA signaling pathway is regulating the cellular processes that are crucial for intestinal regeneration to occur. Thus, RA might be playing a role in echinoderm regeneration that is similar to what has been described in other animal systems.

High-throughput 16S rRNA gene sequencing has been used to identify the intestinal microbiota of many animal species, but that of marine invertebrate organisms remains largely unknown. There are only a few high-throughput sequencing studies on the intestinal microbiota of echinoderms (non-vertebrate Deuterostomes). Here we describe the intestinal microbiota of the sea cucumber Holothuria glaberrima, an echinoderm, well-known for its remarkable power of regeneration. We characterized the microbiota from the anterior descending intestine, the medial intestine (these two comprise the small intestine) and the posterior descending intestine (or large intestine), using pyrosequencing to sequence the V4 region of the 16S rRNA gene. We compared animals in their natural marine environment and in sea-water aquaria. A total of 8,172 OTU's were grouped in 10 bacterial phyla, 23 classes, 44 orders, 83 families, 127 genera and 1 group of unknown bacteria, present across the digestive tract of 10 specimens. The results showed that the anterior intestine is dominated by Proteobacteria (61%) and Bacteroidetes (22%), the medium intestine is similar but with lower Bacteroidetes (4%), and the posterior intestine was remarkably different, dominated by Firmicutes (48%) and Bacteroidetes (35%). The structure of the community changed in animals kept in aquaria, which had a general dominance of Firmicutes and Bacteroidetes, regardless the intestinal segment. Our results evidence that in the natural sea environment, there is intestinal segment differentiation in the microbiota of H. glaberrima, which is lost in artificial conditions. This is relevant for physiological studies, such as mechanisms of digestive regeneration, which might be affected by the microbiota., Competing Interests: The authors have declared that no competing interests exist.

Retinoic acid receptors (RAR) and retinoid X receptors (RXR) are ligand-mediated transcription factors that synchronize intricate signaling networks in metazoans. Dimer formation between these two nuclear receptors mediates the recruitment of co-regulatory complexes coordinating the progression of signaling cascades during developmental and regenerative events. In the present study we identified and characterized the receptors for retinoic acid in the sea cucumber Holothuria glaberrima; a model system capable of regenerative organogenesis during adulthood. Molecular characterizations revealed the presence of three isoforms of RAR and two of RXR as a consequence of alternative splicing events. Various analyses including: primary structure sequencing, phylogenetic analysis, protein domain prediction, and multiple sequence alignment further confirmed their identity. Semiquantitative reverse transcription PCR analysis of each receptor isoform herein identified showed that the retinoid receptors are expressed in all tissues sampled: the mesenteries, respiratory trees, muscles, gonads, and the digestive tract. During regenerative organogenesis two of the receptors (RAR-L and RXR-T) showed differential expression in the posterior segment while RAR-S is differentially expressed in the anterior segment of the intestine. This work presents the first description of the components relaying the signaling for retinoic acid within this model system., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Regeneration of the damaged central nervous system is one of the most interesting post-embryonic developmental phenomena. Two distinct cellular events have been implicated in supplying regenerative neurogenesis with cellular material - generation of new cells through cell proliferation and recruitment of already existing cells through cell migration. The relative contribution and importance of these two mechanisms is often unknown., Methods: Here, we use the regenerating radial nerve cord (RNC) of the echinoderm Holothuria glaberrima as a model of extensive post-traumatic neurogenesis in the deuterostome central nervous system. To uncouple the effects of cell proliferation from those of cell migration, we treated regenerating animals with aphidicolin, a specific inhibitor of S-phase DNA replication. To monitor the effect of aphidicolin on DNA synthesis, we used BrdU immunocytochemistry. The specific radial glial marker ERG1 was used to label the regenerating RNC. Cell migration was tracked with vital staining with the lipophilic dye DiI., Results: Aphidicolin treatment resulted in a significant 2.1-fold decrease in cell proliferation. In spite of this, the regenerating RNC in the treated animals did not differ in histological architecture, size and cell number from its counterpart in the control vehicle-treated animals. DiI labeling showed extensive cell migration in the RNC. Some cells migrated from as far as 2 mm away from the injury plane to contribute to the neural outgrowth., Conclusions: We suggest that inhibition of cell division in the regenerating RNC of H. glaberrima is compensated for by recruitment of cells, which migrate into the RNC outgrowth from deeper regions of the neuroepithelium. Neural regeneration in echinoderms is thus a highly regulative developmental phenomenon, in which the size of the cell pool can be controlled either by cell proliferation or cell migration, and the latter can neutralize perturbations in the former.

Extracellular matrix remodeling is an essential component of regenerative processes in metazoans. Among these animals, holothurians (sea cucumbers) are distinguished by their great regenerative capacities. We have previously shown that fibrous collagen as well as other fibrous components disappear from the connective tissue (CT) early during intestinal regeneration, and later return as the organ primordia form. We now report on changes of the nonfibrous component of the CT. We have used Alcian Blue staining and an antibody, Proteoglycan Like-1 (PGL-1), that recognizes a proteoglycan-like antigen to identify the presence of proteoglycans in normal and regenerating intestines. Our results show that early in regeneration, the ground substance resembles that of the mesentery, the structure from where the new intestine originates. As regeneration proceeds, Alcian Blue staining and PGL-1 labeling reorganize, so that by 4 weeks the normal intestinal CT pattern is achieved. Together with our previous findings, the data suggest that CT components that might be detrimental to regeneration disappear early on, while those that might be beneficial to regeneration, such as proteoglycans, are present throughout the regenerative process., (© 2016 The Histochemical Society.)

The Echinodermata comprise an interesting branch in the phylogenetic tree of deuterostomes. Their radial symmetry which is reflected in their nervous system anatomy makes them a target of interest in the study of nervous system evolution. Until recently, the study of the echinoderm nervous system has been hindered by a shortage of neuronal markers. However, in recent years several markers of neuronal and fiber subpopulations have been described. These have been used to identify subpopulations of neurons and fibers, but an integrative study of the anatomical relationship of these subpopulations is wanting. We have now used eight commercial antibodies, together with three antibodies produced by our group to provide a comprehensive and integrated description and new details of the echinoderm neuroanatomy using the holothurian Holothuria glaberrima (Selenka, 1867) as our model system. Immunoreactivity of the markers used showed: (1) specific labeling patterns by markers in the radial nerve cords, which suggest the presence of specific nerve tracts in holothurians. (2) Nerves directly innervate most muscle fibers in the longitudinal muscles. (3) Similar to other deuterostomes (mainly vertebrates), their enteric nervous system is composed of a large and diverse repertoire of neurons and fiber phenotypes. Our results provide a first blueprint of the anatomical organization of cells and fibers that form the holothurian neural circuitry, and highlight the fact that the echinoderm nervous system shows unexpected diversity in cell and fiber types and their distribution in both central and peripheral nervous components.

Adult neurogenesis, generation of new functional cells in the mature central nervous system (CNS), has been documented in a number of diverse organisms, ranging from humans to invertebrates. However, the origin and evolution of this phenomenon is still poorly understood for many of the key phylogenetic groups. Echinoderms are one such phylum, positioned as a sister group to chordates within the monophyletic clade Deuterostomia. They are well known for the ability of their adult organs, including the CNS, to completely regenerate after injury. Nothing is known, however, about production of new cells in the nervous tissue under normal physiological conditions in these animals. In this study, we show that new cells are continuously generated in the mature radial nerve cord (RNC) of the sea cucumber Holothuria glaberrima. Importantly, this neurogenic activity is not evenly distributed, but is significantly more extensive in the lateral regions of the RNC than along the midline. Some of the new cells generated in the apical region of the ectoneural neuroepithelium leave their place of origin and migrate basally to populate the neural parenchyma. Gene expression analysis showed that generation of new cells in the adult sea cucumber CNS is associated with transcriptional activity of genes known to be involved in regulation of various aspects of neurogenesis in other animals. Further analysis of one of those genes, the transcription factor Myc, showed that it is expressed, in some, but not all radial glial cells, suggesting heterogeneity of this CNS progenitor cell population in echinoderms.

Background: Adult echinoderms can completely regenerate major parts of their central nervous system even after severe injuries. Even though this capacity has long been known, the molecular mechanisms that drive fast and complete regeneration in these animals have remained uninvestigated. The major obstacle for understanding these molecular pathways has been the lack of functional genomic studies on regenerating adult echinoderms., Results: Here, we employ RNA interference-mediated gene knockdown to characterize the role of Myc during the early (first 48 hours) post-injury response in the radial nerve cord of the sea cucumber Holothuria glaberrima. Our previous experiments identified Myc as the only pluripotency-associated factor, whose expression significantly increased in the wounded CNS. The specific function(s) of this gene, however, remained unknown. Here we demonstrate that knockdown of Myc inhibits dedifferentiation of radial glia and programmed cell death, the two most prominent cellular events that take place in the regenerating sea cucumber nervous system shortly after injury., Conclusions: In this study, we show that Myc overexpression is required for proper dedifferentiation of radial glial cells and for triggering the programmed cell death in the vicinity of the injury. Myc is thus the first transcription factor, whose functional role has been experimentally established in echinoderm regeneration.

There is limited information on the regeneration of the enteric nervous system (ENS) following major reconstruction of the digestive tract. We have studied ENS regeneration in the sea cucumber Holothuria glaberrima which undergoes an organogenic process forming a new digestive tract at the tip of the mesentery. Our results show that (1) a degeneration of nerve fibers occurs early in the regeneration process, prior to eventual regeneration; (2) nerve fibers that innervate the regenerating intestine are of extrinsic and intrinsic origin; (3) innervation by extrinsic fibers occurs in a gradient that begins in the proximal area of the regenerate; (4) late events include the appearance of nerve fibers that project from the serosa into the connective tissue and of nerve bundles in the mesothelial layer; (5) neurons and neuroendocrine cells appear early following the formation of the epithelial layers. Our results provide not only a comparative biological approach to study ENS regeneration but also an alternative point of view for the study of enteric neuropathologies and for the innervation of organs made in vitro.

Background: Echinoderms are emerging as important models in regenerative biology. Significant amount of data are available on cellular mechanisms of post-traumatic repair in these animals, whereas studies of gene expression are rare. In this study, we employ high-throughput sequencing to analyze the transcriptome of the normal and regenerating radial nerve cord (a homolog of the chordate neural tube), in the sea cucumber Holothuria glaberrima., Results: Our de novo assembly yielded 70,173 contigs, of which 24,324 showed significant similarity to known protein-coding sequences. Expression profiling revealed large-scale changes in gene expression (4,023 and 3,257 up-regulated and down-regulated transcripts, respectively) associated with regeneration. Functional analysis of sets of differentially expressed genes suggested that among the most extensively over-represented pathways were those involved in the extracellular matrix (ECM) remodeling and ECM-cell interactions, indicating a key role of the ECM in regeneration. We also searched the sea cucumber transcriptome for homologs of factors known to be involved in acquisition and/or control of pluripotency. We identified eleven genes that were expressed both in the normal and regenerating tissues. Of these, only Myc was present at significantly higher levels in regeneration, whereas the expression of Bmi-1 was significantly reduced. We also sought to get insight into which transcription factors may operate at the top of the regulatory hierarchy to control gene expression in regeneration. Our analysis yielded eleven putative transcription factors, which constitute good candidates for further functional studies. The identified candidate transcription factors included not only known regeneration-related genes, but also factors not previously implicated as regulators of post-traumatic tissue regrowth. Functional annotation also suggested that one of the possible adaptations contributing to fast and efficient neural regeneration in echinoderms may be related to suppression of excitotoxicity., Conclusions: Our transcriptomic analysis corroborates existing data on cellular mechanisms implicated in regeneration in sea cucumbers. More importantly, however, it also illuminates new aspects of echinoderm regeneration, which have been scarcely studied or overlooked altogether. The most significant outcome of the present work is that it lays out a roadmap for future studies of regulatory mechanisms by providing a list of key candidate genes for functional analysis.

Background: Unlike the mammalian central nervous system (CNS), the CNS of echinoderms is capable of fast and efficient regeneration following injury and constitutes one of the most promising model systems that can provide important insights into evolution of the cellular and molecular events involved in neural repair in deuterostomes. So far, the cellular mechanisms of neural regeneration in echinoderm remained obscure. In this study we show that radial glial cells are the main source of new cells in the regenerating radial nerve cord in these animals., Results: We demonstrate that radial glial cells of the sea cucumber Holothuria glaberrima react to injury by dedifferentiation. Both glia and neurons undergo programmed cell death in the lesioned CNS, but it is the dedifferentiated glial subpopulation in the vicinity of the injury that accounts for the vast majority of cell divisions. Glial outgrowth leads to formation of a tubular scaffold at the growing tip, which is later populated by neural elements. Most importantly, radial glial cells themselves give rise to new neurons. At least some of the newly produced neurons survive for more than 4 months and express neuronal markers typical of the mature echinoderm CNS., Conclusions: A hypothesis is formulated that CNS regeneration via activation of radial glial cells may represent a common capacity of the Deuterostomia, which is not invoked spontaneously in higher vertebrates, whose adult CNS does not retain radial glial cells. Potential implications for biomedical research aimed at finding the cure for human CNS injuries are discussed.

Research on the involvement of retroelements in developmental processes has been gaining momentum recently; however, most of the studies published so far have been focused on embryonic development. This commentary presents two recent papers, which document significant changes in transcriptional activity of retroelements in two different model systems, salamander limb regeneration and regeneration of radial organs in the sea cucumber Holothuria glaberrima . We hypothesize that transcriptional activity of the retrotransposons can be specifically controlled by the host and may play some hitherto unrecognized role in regeneration.

Members of the calbindin subfamily serve as markers of subpopulations of neurons within the vertebrate nervous system. Although markers of these proteins are widely available and used, their application to invertebrate nervous systems has been very limited. In this study we investigated the presence and distribution of members of the calbindin subfamily in the sea cucumber Holothuria glaberrima (Selenka, 1867). Immunohistological experiments with antibodies made against rat calbindin 1, parvalbumin, and calbindin 2, showed that these antibodies labeled cells and fibers within the nervous system of H. glaberrima. Most of the cells and fibers were co-labeled with the neural-specific marker RN1, showing their neural specificity. These were distributed throughout all of the nervous structures, including the connective tissue plexi of the body wall and podia. Bioinformatics analyses of the possible antigen recognized by these markers showed that a calbindin 2-like protein present in the sea urchin Strongylocentrotus purpuratus, corresponded to the calbindin-D32k previously identified in other invertebrates. Western blots with anti-calbindin 1 and anti-parvalbumin showed that these markers recognized an antigen of approximately 32 kDa in homogenates of radial nerve cords of H. glaberrima and Lytechinus variegatus. Furthermore, immunoreactivity with anti-calbindin 1 and anti-parvalbumin was obtained to a fragment of calbindin-D32k of H. glaberrima. Our findings suggest that calbindin-D32k is present in invertebrates and its sequence is more similar to the vertebrate calbindin 2 than to calbindin 1. Thus, characterization of calbindin-D32k in echinoderms provides an important view of the evolution of this protein family and represents a valuable marker to study the nervous system of invertebrates.

Background: Determining the type and source of cells involved in regenerative processes has been one of the most important goals of researchers in the field of regeneration biology. We have previously used several cellular markers to characterize the cells involved in the regeneration of the intestine in the sea cucumber Holothuria glaberrima., Results: We have now obtained a monoclonal antibody that labels the mesothelium; the outer layer of the gut wall composed of peritoneocytes and myocytes. Using this antibody we studied the role of this tissue layer in the early stages of intestinal regeneration. We have now shown that the mesothelial cells of the mesentery, specifically the muscle component, undergo dedifferentiation from very early on in the regeneration process. Cell proliferation, on the other hand, increases much later, and mainly takes place in the mesothelium or coelomic epithelium of the regenerating intestinal rudiment. Moreover, we have found that the formation of the intestinal rudiment involves a novel regenerative mechanism where epithelial cells ingress into the connective tissue and acquire mesenchymal phenotypes., Conclusions: Our results strongly suggest that the dedifferentiating mesothelium provides the initial source of cells for the formation of the intestinal rudiment. At later stages, cell proliferation supplies additional cells necessary for the increase in size of the regenerate. Our data also shows that the mechanism of epithelial to mesenchymal transition provides many of the connective tissue cells found in the regenerating intestine. These results present some new and important information as to the cellular basis of organ regeneration and in particular to the process of regeneration of visceral organs.

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.

Metazoan immunity is mainly associated with specialized cells that are directly involved with the immune response. Nevertheless, both in vertebrates and invertebrates other organs might respond to immune activation and participate either directly or indirectly in the ongoing immune process. However, most of what is known about invertebrate immunity has been restricted to immune effector cells and little information is available on the immune responses of other tissues or organs. We now focus on the immune reactions of the intestinal tissue of an echinoderm. Our study employs a non-conventional model, the echinoderm Holothuria glaberrima, to identify intestinal molecules expressed after an immune challenge presented by an intra-coelomic injection of lipopolysaccharides (LPS). The expression profiles of intestinal genes expressed differentially between LPS-injected animals and control sea water-injected animals were determined using a custom-made Agilent microarray with 7209 sea cucumber intestinal ESTs. Fifty (50) unique sequences were found to be differentially expressed in the intestine of LPS-treated sea cucumbers. Seven (7) of these sequences represented homologues of known proteins, while the remaining (43) had no significant similarity with any protein, EST or RNA database. The known sequences corresponded to cytoskeletal proteins (Actin and alpha-actinin), metabolic enzymes (GAPDH, Ahcy and Gnmt), metal ion transport/metabolism (major yolk protein) and defense/recognition (fibrinogen-like protein). The expression pattern of 11 genes was validated using semi-quantitative RT-PCR. Nine of these corroborated the microarray results and the remaining two showed a similar trend but without statistical significance. Our results show some of the molecular events by which the holothurian intestine responds to an immune challenge and provide important information to the study of the evolution of the immune response.

Background: Among deuterostomes, the regenerative potential is maximally expressed in echinoderms, animals that can quickly replace most injured organs. In particular, sea cucumbers are excellent models for studying organ regeneration since they regenerate their digestive tract after evisceration. However, echinoderms have been sidelined in modern regeneration studies partially because of the lack of genome-wide profiling approaches afforded by modern genomic tools.For the last decade, our laboratory has been using the sea cucumber Holothuria glaberrima to dissect the cellular and molecular events that allow for such amazing regenerative processes. We have already established an EST database obtained from cDNA libraries of normal and regenerating intestine at two different regeneration stages. This database now has over 7000 sequences., Results: In the present work we used a custom-made microchip from Agilent with 60-mer probes for these ESTs, to determine the gene expression profile during intestinal regeneration. Here we compared the expression profile of animals at three different intestinal regeneration stages (3-, 7- and 14-days post evisceration) against the profile from normal (uneviscerated) intestines. The number of differentially expressed probes ranged from 70% at p < 0.05 to 39% at p < 0.001. Clustering analyses show specific profiles of expression for early (first week) and late (second week) regeneration stages. We used semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) to validate the expression profile of fifteen microarray detected differentially expressed genes which resulted in over 86% concordance between both techniques. Most of the differentially expressed ESTs showed no clear similarity to sequences in the databases and might represent novel genes associated with regeneration. However, other ESTs were similar to genes known to be involved in regeneration-related processes, wound healing, cell proliferation, differentiation, morphological plasticity, cell survival, stress response, immune challenge, and neoplastic transformation. Among those that have been validated, cytoskeletal genes, such as actins, and developmental genes, such as Wnt and Hox genes, show interesting expression profiles during regeneration., Conclusion: Our findings set the base for future studies into the molecular basis of intestinal regeneration. Moreover, it advances the use of echinoderms in regenerative biology, animals that because of their amazing properties and their key evolutionary position, might provide important clues to the genetic basis of regenerative processes.

Background: Regeneration of neurons and fibers in the mammalian spinal cord has not been plausible, even though extensive studies have been made to understand the restrictive factors involved. New experimental models and strategies are necessary to determine how new nerve cells are generated and how fibers regrow and connect with their targets in adult animals. Non-vertebrate deuterostomes might provide some answers to these questions. Echinoderms, with their amazing regenerative capacities could serve as model systems; however, very few studies have been done to study the regeneration of their nervous system., Results: We have studied nerve cord regeneration in the echinoderm Holothuria glaberrima. These are sea cucumbers or holothurians members of the class Holothuroidea. One radial nerve cord, part of the echinoderm CNS, was completely transected using a scalpel blade. Animals were allowed to heal for up to four weeks (2, 6, 12, 20, and 28 days post-injury) before sacrificed. Tissues were sectioned in a cryostat and changes in the radial nerve cord were analyzed using classical dyes and immunohistochemistry. In addition, the temporal and spatial distribution of cell proliferation and apoptosis was assayed using BrdU incorporation and the TUNEL assay, respectively.We found that H. glaberrima can regenerate its radial nerve cord within a month following transection. The regenerated cord looks amazingly similar in overall morphology and cellular composition to the uninjured cord. The cellular events associated to radial cord regeneration include: (1) outgrowth of nerve fibers from the injured radial cord stumps, (2) intense cellular division in the cord stumps and in the regenerating radial nerve cords, (3) high levels of apoptosis in the RNC adjacent to the injury and within the regenerating cord and (4) an increase in the number of spherule-containing cells. These events are similar to those that occur in other body wall tissues during wound healing and during regeneration of the intestine., Conclusion: Our data indicate that holothurians are capable of rapid and complete regeneration of the main component of their CNS. Regeneration involves both the outgrowth of nerve fibers and the formation of neurons. Moreover, the cellular events employed during regeneration are similar to those involved in other regenerative processes, namely wound healing and intestinal regeneration. Thus, holothurians should be viewed as an alternative model where many of the questions regarding nervous system regeneration in deuterostomes could be answered.

Repair and regeneration are key processes for tissue maintenance, and their disruption may lead to disease states. Little is known about the molecular mechanisms that underline the repair and regeneration of the digestive tract. The sea cucumber Holothuria glaberrima represents an excellent model to dissect and characterize the molecular events during intestinal regeneration. To study the gene expression profile, cDNA libraries were constructed from normal, 3-day, and 7-day regenerating intestines of H. glaberrima. Clones were randomly sequenced and queried against the nonredundant protein database at the National Center for Biotechnology Information. RT-PCR analyses were made of several genes to determine their expression profile during intestinal regeneration. A total of 5,173 sequences from three cDNA libraries were obtained. About 46.2, 35.6, and 26.2% of the sequences for the normal, 3-days, and 7-days cDNA libraries, respectively, shared significant similarity with known sequences in the protein database of GenBank but only present 10% of similarity among them. Analysis of the libraries in terms of functional processes, protein domains, and most common sequences suggests that a differential expression profile is taking place during the regeneration process. Further examination of the expressed sequence tag dataset revealed that 12 putative genes are differentially expressed at significant level (R > 6). Experimental validation by RT-PCR analysis reveals that at least three genes (unknown C-4677-1, melanotransferrin, and centaurin) present a differential expression during regeneration. These findings strongly suggest that the gene expression profile varies among regeneration stages and provide evidence for the existence of differential gene expression.

Background: All animals possess some type of tissue repair mechanism. In some species, the capacity to repair tissues is limited to the healing of wounds. Other species, such as echinoderms, posses a striking repair capability that can include the replacement of entire organs. It has been reported that some mechanisms, namely extracellular matrix remodeling, appear to occur in most repair processes. However, it remains unclear to what extent the process of organ regeneration, particularly in animals where loss and regeneration of complex structures is a programmed natural event, is similar to wound healing. We have now used the sea cucumber Holothuria glaberrima to address this question., Results: Animals were lesioned by making a 3-5 mm transverse incision between one of the longitudinal muscle pairs along the bodywall. Lesioned tissues included muscle, nerve, water canal and dermis. Animals were allowed to heal for up to four weeks (2, 6, 12, 20, and 28 days post-injury) before sacrificed. Tissues were sectioned in a cryostat and changes in cellular and tissue elements during repair were evaluated using classical dyes, immmuohistochemistry and phalloidin labeling. In addition, the temporal and spatial distribution of cell proliferation in the animals was assayed using BrdU incorporation. We found that cellular events associated with wound healing in H. glaberrima correspond to those previously shown to occur during intestinal regeneration. These include: (1) an increase in the number of spherule-containing cells, (2) remodeling of the extracellular matrix, (3) formation of spindle-like structures that signal dedifferentiation of muscle cells in the area flanking the lesion site and (4) intense cellular division occurring mainly in the coelomic epithelium after the first week of regeneration., Conclusion: Our data indicate that H. glaberrima employs analogous cellular mechanisms during wound healing and organ regeneration. Thus, it is possible that regenerative limitations in some organisms are due either to the absence of particular mechanisms associated with repair or the inability of activating the repair process in some tissues or stages.

The echinoderm nervous system is one of the least studied among invertebrates, partly because the tools available to study the neurobiology of this phylum are limited. We have now produced a monoclonal antibody (RN1) that labels a nervous system component of the sea cucumber Holothuria glaberrima. Western blots show that our antibody recognizes a major band of 66 kDa and a minor band of 53 kDa. Immunohistological experiments show that, in H. glaberrima, the antibody distinctly labels most of the known nervous system structures and some components that were previously unknown or little studied. A surprising finding was the labeling of nervous plexi within the connective tissue compartments of all organs studied. Double labeling with holothurian neuropeptides and an echinoderm synaptotagmin showed that RN1 labeled most, if not all, of the fibers labeled by these neuronal markers, but also a larger component of cells and fibers. The presence of a distinct connective tissue plexus in holothurians is highly significant since these organisms possess mutable connective tissues that change viscosity under the control of the nervous system. Therefore, the cells and fibers recognized by our monoclonal antibodies may be involved in controlling tensility changes in echinoderm connective tissue.

Background: Ependymin (Epd), the predominant protein in the cerebrospinal fluid of teleost fishes, was originally associated with neuroplasticity and regeneration. Ependymin-related proteins (Epdrs) have been identified in other vertebrates, including amphibians and mammals. Recently, we reported the identification and characterization of an Epdr in echinoderms, showing that there are ependymin family members in non-vertebrate deuterostomes. We have now explored multiple databases to find Epdrs in different metazoan species. Using these sequences we have performed genome mapping, molecular phylogenetic analyses using Maximum Likelihood and Bayesian methods, and statistical tests of tree topologies, to ascertain the phylogenetic relationship among ependymin proteins., Results: Our results demonstrate that ependymin genes are also present in protostomes. In addition, as a result of the putative fish-specific genome duplication event and posterior divergence, the ependymin family can be divided into four groups according to their amino acid composition and branching pattern in the gene tree: 1) a brain-specific group of ependymin sequences that is unique to teleost fishes and encompasses the originally described ependymin; 2) a group expressed in non-brain tissue in fishes; 3) a group expressed in several tissues that appears to be deuterostome-specific, and 4) a group found in invertebrate deuterostomes and protostomes, with a broad pattern of expression and that probably represents the evolutionary origin of the ependymins. Using codon-substitution models to statistically assess the selective pressures acting over the ependymin protein family, we found evidence of episodic positive Darwinian selection and relaxed selective constraints in each one of the postduplication branches of the gene tree. However, purifying selection (with among-site variability) appears to be the main influence on the evolution of each subgroup within the family. Functional divergence among the ependymin paralog groups is well supported and several amino acid positions are predicted to be critical for this divergence., Conclusion: Ependymin proteins are present in vertebrates, invertebrate deuterostomes, and protostomes. Overall, our analyses suggest that the ependymin protein family is a suitable target to experimentally test subfunctionalization in gene copies that originated after gene or genome duplication events.

We report the characterization of an ependymin-related gene (EpenHg) from a regenerating intestine cDNA library of the sea cucumber Holothuria glaberrima. This finding is remarkable because no ependymin sequence has ever been reported from invertebrates. Database comparisons of the conceptual translation of the EpenHg gene reveal 63% similarity (47% identity) with mammalian ependymin-related proteins (MERPs) and close relationship with the frog and piscine ependymins. We also report the partial sequences of ependymin representatives from another species of sea cucumber and from a sea urchin species. Conventional and real-time reverse transcriptase polymerase chain reaction (RT-PCRs) show that the gene is expressed in several echinoderm tissues, including esophagus, mesenteries, gonads, respiratory trees, hemal system, tentacles and body wall. Moreover, the ependymin product in the intestine is overexpressed during sea cucumber intestinal regeneration. The discovery of ependymins in echinoderms, a group well known for their regenerative capacities, can give us an insight on the evolution and roles of ependymin molecules.

We have previously characterized the first invertebrate homolog of serum amyloid A (SAA) proteins in the sea cucumber Holothuria glaberrima, where its expression is associated with intestinal regeneration, suggesting a possible involvement of SAA proteins in intestinal morphogenesis. Here we show that bacterial lipopolysaccharides (LPS) trigger a coelomocyte-mediated immune response in H. glaberrima, inducing an approximately threefold increase in coelomocyte phagocytic activity. Furthermore, LPS induces an approximately fourfold increase in SAA mRNA levels in non-regenerating intestines. These results show that in H. glaberrima, LPS act as an immune activator and that SAA expression can be modulated by immune-associated processes.

The sea cucumber, Holothuria glaberrima, has the capacity to regenerate its internal organs. Intestinal regeneration is accomplished by the thickening of the mesenteric border and the invasion of this thickening by mucosal epithelium from the esophagus and the cloaca. Extracellular matrix (ECM) remodeling has been associated with morphogenetic events during embryonic development and regeneration. We have used immunohistochemical techniques against ECM components to show that differential changes occur in the ECM during early regeneration. Labeling of fibrous collagenous components and muscle-related laminin disappear from the regenerating intestine and mesentery, while fibronectin labeling and 4G7 (an echinoderm ECM component) are continuously present. Western blots confirm a decrease in fibrous collagen content during the first 2 weeks of regeneration. We have also identified five 1,10-phenanthroline-sensitive bands in collagen gelatin zymographs. The gelatinolytic activities of these bands are enhanced during early stages of regeneration, suggesting that the metalloprotease activity is associated with ECM remodeling. Inhibition of MMPs in vivo with 1,10-phenanthroline, p-aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamate or N-CBZ-Pro-Leu-Gly hydroxamate produces a reversible inhibition of intestinal regeneration and ECM remodeling. Our results show that significant changes in ECM content occur during intestine regeneration in the sea cucumber and that the onset of these changes is correlated to the proteolytic activities of MMPs.

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.

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.

We have studied the expression and development of neuropeptide Y-like immunoreactivity (NPY-LI) in the sympathoadrenal system of the chicken using single and double immunocytochemical techniques and radioimmunoassay. NPY-LI is expressed by neurons of the paravertebral sympathetic ganglia and by chromaffin cells of the adrenal gland in embryonic and adult chickens. The peptide is coexpressed with catecholaminergic properties in neurons. In chromaffin cells, it is also expressed with immunoreactivity to somatostatin and serotonin. We have used the expression of NPY-LI to analyze how cells that coexpress two or more neuroactive substances arrive at their final phenotype. Our results suggest that the ontogeny of coexpression in neurons of the avian paravertebral sympathetic ganglia occurs in a sequential pattern, where the expression of the peptide follows the initial expression of the "classical neurotransmitter". In contrast, in chromaffin cells, expression of the peptides occurs concomitantly with expression of catecholaminergic properties or soon after. Initially, coexpression of several neuroactive substances occurs, but this is followed by further specialization where the expression of one peptide prevails over the other. We believe that the two models of coexpression shown by our results can be used to describe the ontogeny of coexpression in other cells of the nervous system.

The appearance and development of somatostatin-like immunoreactivity (SLI) in the peripheral nervous system of quail embryos were studied using radioimmunoanalysis and immunocytochemistry. In vivo, no SLI is observed in neural crest cells before or during migration. SLI appears between days 3 and 4 of incubation in sympathetic ganglia, immediately following ganglion formation, and between days 4 and 5 of incubation in the adrenal gland, soon after the adrenal gland primordium first appears. The development of SLI in the adrenal gland differs from that in the sympathetic ganglia. While in the former the amount of SLI and the number of SLI-containing cells increase as the embryo ages, in the sympathetic ganglia the amount of SLI and the percentage of SLI-containing cells decrease. When migrating neural crest cells are obtained from the sclerotomal part of 3-day embryos and grown in culture, they first display SLI after 48 hr, and the amount of SLI increases thereafter. When the sympathoadrenal precursors are removed at 4 days of incubation and grown in vitro, SLI appears after 24 hr in culture and increases during the next few days. Our results demonstrate that SLI is present very early in the quail embryo and that its appearance parallels the differentiation of neural crest cells into autonomic sympathetic ganglionic cells. We also show that the differentiation of neural crest into SLI-containing cells can be reproduced in culture, thus permitting the study of peptide production and expression in vitro.