Expression of pluripotency factors in echinoderm regeneration

Abstract

Cell dedifferentiation is an integral component of post-traumatic regeneration in echinoderms. Since dedifferentiated cells become multipotent, we asked if this spontaneous broadening of developmental potential is associated with the action of the same pluripotency factors (known as Yamanaka factors) that were used to induce pluripotency in specialized mammalian cells. In this study, we investigate expression of orthologs of the four Yamanaka factors in regeneration of two different organs, the radial nerve cord and the digestive tube, in the sea cucumber Holothuria glaberrima. All four pluripotency factors are expressed in uninjured animals, although their expression domains do not always overlap. In regeneration, the expression levels of the four genes were not regulated in a coordinated way, but, instead, showed different dynamics for individual genes and also were different between the radial nerve and the gut. SoxB1, the ortholog of the mammalian Sox2, was drastically downregulated in the regenerating intestine, suggesting that this factor is not required for dedifferentiation/regeneration in this organ. On the other hand, during the early post-injury stage, Myc, the sea cucumber ortholog of c-Myc, was significantly upregulated in both the intestine and the radial nerve cord, and is, therefore, hypothesized to play a central role in dedifferentiation/regeneration of various tissue types.

A START-domain-containing protein is a novel marker of nervous system components of the sea cucumber Holothuria glaberrima

“…After demonstrating that RN1 can immunoprecipitate proteins, the proteins obtained from the IP were resolved by SDS-PAGE, stained and the detected bands corresponding to the 53 and 66 kDa species were digested with trypsin for protein identification using tandem mass spectrometry analysis. The spectra obtained were mapped onto the sequences of translated cDNA libraries of the sea cucumber’s intestinal and radial nerve tissues (Ortiz-Pineda et al 2009, Mashanov et al 2014, Mashanov et al 2015). At least 6 peptides were identified by MS/MS with a high confidence level and identified in the spectra from both the 53 and 66 kDa fractions (Figure 1b).…”

“…The samples were analyzed in an LTQ mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Identification was performed using Thermo Proteome Discoverer version 1.2.0.208 (Thermo Fisher Scientific, Waltham, MA) that correlates MS–MS spectra with sequences from translated cDNA libraries of radial nerve and regenerating intestine databases using SEQUEST database search algorithms (Ortiz-Pineda et al 2009, Mashanov et al 2014, Mashanov et al 2015). All the sequences from these libraries were translated using the EMBOSS sixpack algorithm.…”

“…cDNA was prepared from RNA isolated from radial nerve tissue extractions (see Mashanov et al 2014, 2015). Using this template, we amplified the START domain gene with the primer pair RNIF and RN1R2.…”

“…One of the peculiarities of the echinoderm nervous system is its adult radial symmetry that differs from the bilateral symmetry of other deuterostome animals (Hyman 1955, Mashanov et al 2015). The echinoderm central nervous system is composed of an anterior nerve ring connected to radial nerve cords that extend to the posterior end of the animals.…”

Expression of stem cell factors in the adult sea cucumber digestive tube

“…Quantitative real-time PCR was performed as previously described (Mashanov et al, 2015a) to determine the relative abundance of transcripts in the five anatomical regions of the sea cucumber digestive tube. Briefly, RNA was extracted from small tissue samples (not exceeding 100 mg) dissected from four different H. glaberrima individuals (biological replicates).…”