Draft Genome of the Sea Cucumber Holothuria glaberrima, a Model for the Study of Regeneration

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.

Antibiotics Modulate Intestinal Regeneration

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.

Transcriptomic Analysis of Early Stages of Intestinal Regeneration in Holothuria glaberrima

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 hour 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.

Draft Genome of the Sea Cucumber Holothuria glaberrima, a model for the study of regeneration

Regeneration is one of the most fascinating and yet least understood processes of animals. Echinoderms, one of the closest related invertebrate groups to humans, can contribute to our understanding of the genetic basis of regenerative processes. Amongst 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 have 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 2,960,762 scaffolds totaling 1.5 gigabases with an N50 of 15 kilobases. Our BUSCO assessment of the genome resulted in 882 (90.2%) complete and partial genes from 978 genes queried. We incorporated transcriptomic data from several different life history stages to annotate 41,076 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 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.

A Roadmap for Intestinal Regeneration

Regeneration of lost or injured organs is an intriguing process where numerous cellular events take place to form the new structure. Studies of this process during reconstitution of the intestine have been performed in echinoderms, particularly in holothurians. Many cellular events triggered during regeneration have been described using the sea cucumber Holothuria glaberrima as a research model. More recent experiments have targeted the molecular mechanism behind the process, a task that has been eased by the new sequencing technologies now available. In this review we present the studies involving cellular processes and the genes that have been identified to be associated with the early events of gut regeneration. We also present the ongoing efforts to perform functional studies necessary to establish the role(s) of the identified genes. A synopsis of the studies is given with the course of the regenerative process established so far.

Biología del desarrollo: la capacidad regenerativa de Holothuria glaberrima

La regeneración es un proceso llamativo que permite la reconstitución de un miembro u órgano afectado o perdido. La regeneración en los organismos depende de sus características filogenéticas, las cuales permiten la agrupación de ciertos seres que son conocidos por su gran capacidad de reconstitución anatómica como es el caso de los platelmintos y equinodermos. Dentro del grupo de los equinodermos, el pepino de mar Holothuria glaberrima es un organismo modelo poco común que tiene la capacidad de regenerar sus órganos internos. Es un organismo invertebrado con un desarrollo embriológico similar a los vertebrados y al igual que estos se clasifica como deuterostomado. El estudio de los distintos mecanismos que se producen a partir de su capacidad regenerativa permite obtener valiosa información en el campo de la biología celular y molecular. Gracias al estudio pionero de este organismo por parte del grupo de investigadores del Laboratorio de Neurobiología de la Universidad de Puerto Rico, se ha obtenido información concerniente a procesos celulares que permiten la regeneración de los órganos internos en Holothuria glaberrima. Entre los estudios realizados que se han realizado en Puerto Rico, la actividad de la ruta de señalización de Wnt durante el inicio del proceso de regeneración, ha permitido descifrar que tipo de genes están involucrados en la activación de mecanismos celulares durante la regeneración. Estos estudios permiten asociar procesos celulares que pueden activarse en los seres que no gozan de la misma capacidad regenerativa que Holothuria glaberrima y así avanzar en el desarrollo de tratamientos médicos.

Retinoic Acid Signaling Is Associated with Cell Proliferation, Muscle Cell Dedifferentiation, and Overall Rudiment Size during Intestinal Regeneration in the Sea Cucumber, Holothuria glaberrima

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.