Expression profiles during dedifferentiation in newt lens regeneration revealed by expressed sequence tags

Animals

One hundred Japanese newts, Cynopspyrrhogaster, were collected in the northern part of Okayama prefecture. All animal procedures were approved by animal care board in Center for Developmental Biology, Riken Kobe. Newts were euthanized by anesthesia (soaking in 0.1% of MS-222 [Sigma-Aldrich, Tokyo, Japan] for 15 min) followed by decapitation.

mRNA extraction

Both dorsal and ventral irises 8 days after lentectomy (when dedifferentiation events of PECs are ongoing, i.e., initiation of depigmentation and proliferation [6]) were collected for the cDNA library of lens-regenerating iris. mRNA was purified using Dynabeads Oligo(dT)₂₅ (Dynal Biotech, Oslo, Norway) according to the manufacturer’s instructions. Briefly, Dynabeads Oligo (dT) was added to homogenized iris sample. mRNA hybridized to Dynabeads Oligo(dT) was isolated by magnetic separation, washed, and eluted by incubation with 10 mM Tris (2-amino-2-hydroxymethylpropane-1,3-diol)-HCl buffer, pH 7.5 at 75 ºC for 2 min.

Construction of the cDNA library

The cDNA library was constructed using the ZAP-cDNA synthesis kit (Stratagene Japan, Kunitachi, Japan). Reverse transcription reaction was performed using oligo-dT primer, and the synthesized cDNAs were directionally inserted into a Uni-ZAP XR Vector (Stratagene Japan). The vector-containing cDNA was packaged in a lambda phage, using a Gigapack III gold cloning kit (Stratagene Japan). pBluescript phagemid was prepared by in vivo excision using ExAssist helper phage  (Stratagene Japan) and XLI-Blue MRF strain (Stratagene Japan). XLI-Blue MRF strain cells were transformed with the phagemid and plated on L-Broth-ampicilin plate.

Sequencing of cDNA

Each colony was picked using an automated colony picker Qpix (Genetix K.K., Toyo, Japan), and template DNA for sequencing was amplified using the TempliPhi DNA sequencing template amplification kit (GE Healthcare Life Sciences, Piscataway, NJ). The sequencing reaction was performed using the BigDye terminator v. 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA). The primer used in the sequence reaction was the T3 primer (Stratagene Japan). The sequence reaction products were analyzed using a 3730xl DNA analyzer (Applied Biosystems). The GenBank accession numbers for ESTs are FS290155-FS300559.

Assembly of sequence data

To remove possible low-quality fragments at both edges of individual reads, 20 bases were trimmed off each end of all sequences. Then, potential vector contamination was removed or masked. Individual reads from all tissues were assembled using CAP3 software with default settings [14].

Functional annotation

Functional annotation of the assembled sequences was performed using the Blast2GO program with default settings [15]. Putative “gene names” and functions are also assigned to sequences with GO annotations as part of the annotation process. The annotations were augmented by the Annotation Expander (ANNEX) software [16].

Sequencing, assembly, and functional annotation of newt expression sequence tags

As mentioned previously dedifferentiation events occur in the ventral iris as well as the dorsal iris. Therefore dorsal and ventral irises 8 days after lentectomy, when dedifferentiation of PECs is ongoing, were collected, cDNA libraries were constructed using mRNA from the regenerating iris, and ESTs from the iris libraries were obtained. The initial set of cDNA sequence reads were 10,449 (Table 1). The total length of these sequences was 9.85 Mb, which was reduced to 4.82 Mb by assembling overlapping reads, using CAP3 software. This assembly was composed of 4,725 unique sequences (1,368 contigs and 3,357 singlets). Of 1,219 contigs and 3,357 singlets, 780 contigs and 1,666 singlets were annotated by Blast2GO.

Expression profiles of iris ongoing dedifferentiation

Functional annotation of the contigs and singlets was performed using the Blast2GO program. The 15 most frequently counted biologic process terms, cellular component terms, and molecular function terms are shown in Figure 1.

Tyrosinase, a possible marker for differentiated PECs, was not found in the EST. No detection of tyrosinase in this EST might reflect changing of the original PEC gene expression 8 days after lentectomy. Nucleostemin, a nucleolar protein mainly expressed in stem cell populations in mammals [5], was found in the ESTs (Table 2). This result was consistent with a previous report in which nucleostemin emerged in nucleoli during newt dedifferentiation [6]. Crystallins, which are known to be lens differentiation markers, were not found in the EST list, suggesting that these ESTs consist of cDNAs involved in the dedifferentiation stage before lens differentiation.

A close examination of the list revealed that several members of the bone morphogenetic protein and transforming growth factor pathways were present in our ESTs. We were unable to find any from the wingless-type mouse mammary tumor virus integration site family, fibroblast growth factor, or sonic hedgehog pathway. The reason for the presence or absence could be due to the stage of the iris that was isolated. The iris was at the dedifferentiation stage. It is possible that pathways that are important for lens differentiation, such as fibroblast growth factor or sonic hedgehog, are simply not active at that stage [17,18]. Alternatively, it is possible that certain factors were not cloned in our experiment.

It appears that a wide variety of cancer-related genes were expressed during dedifferentiation (Table 3). A total of 27 cancer-related genes were found in the ESTs, especially a large number of Ras-related genes and tumor necrosis factor-related genes. The expression of cancer-related genes might be related to the initiation of proliferation during dedifferentiation. Additionally, as c-Myc is one of four factors (cellular myelocytomatosis oncogene, octamer-binding transcription factor 4, sex determining region Y box 2 and kruppel-like factor 4) to reprogram somatic cells to pluripotent stem cells [19], there is a possibility that cancer-related genes play a role in reprogramming during dedifferentiation. In addition to cancer-related genes, eight apoptosis-related genes were found in the EST list (Table 2). The expression of cancer- and apoptosis-related genes could be one of the hallmarks during dedifferentiation in newt regeneration.

Candidate genes that regulate reprogramming during dedifferentiation

From our list of the lens-regenerating iris ESTs, possible candidate genes for participating in nuclear regulation during newt dedifferentiation were identified (Table 4). Epigenetic regulation, a range of heritable chromatin modifications, including histone modifications, DNA methylation, and chromatin remodeling, play a pivotal role in the control of differentiation and maintenance of cellular identity. It is therefore expected that epigenetic regulation plays an important role during newt dedifferentiation.

Histone acetylation is generally related to transcriptional activation and mediated by histone acetyltransferase. In the ESTs, there were two types of histone acetyltransferases, cyclic adenosine monophosphate response element-biding protein binding protein/p300 [20] and MYST3 [21], as well as two deacetylases, histone deacetylase 2 and 5 (HDAC2, HDAC5; Table 4) [22]. The balance between histone acetyltransferase and deacetylase might be strictly regulated during newt dedifferentiation.

To form heterochromatin, heterochromatin protein 1 is recruited to methylated histone H3K9 [23,24]. It has been demonstrated that the Jumonji domain-containing 2a (JMJD2A) is a histone demethylase against histone H3K9 and H3K36 and antagonizes heterochromatin formation via histone H3K9 [25]. It is thought that JMJD1B is a H3K9 histone demethylase because JMJD1B has a JmjC domain [26]. Interestingly, JMJD2A and JMJD1B were found in the ESTs (Table 4). This might indicate that these histone demethylases eliminate heterochromatin during dedifferentiation.

DNA methylation is a covalent modification of DNA and confers a heritable gene repression during and after development [27,28]. DNA (cytosine-5-)-methyltransferase 1 [28] and putative DNA methyltransferase Williams Beuren syndrome chromosome region 22 [29] were found in the ESTs (Table 4).

The high mobility group protein is a nonhistone chromatin protein. In vitro experiments have demonstrated that high mobility group protein box 2 (HMGB2) nonspecifically binds and bends DNA. It is suggested that HMGB2 facilitates cooperative interactions between cis-acting proteins by promoting DNA flexibility [30]. Like HMGB2, HMGB3 contains DNA-binding HMG box domains and is thought to be able to alter DNA structure [31]. Thus, HMGB2 and HMGB3 might promote genome-wide DNA flexing, which allows new sets of gene expression during dedifferentiation.

Transcriptional factors, nuclear factor-κB [32], and chicken ovalbumin upstream promoter transcription factor 1 [33] were found. Repressor element 1 silencing transcriptional factor (REST) binds to repressor element 1 and recruits a wide variety of chromatin modification enzymes, such as the histone deacetylases HDAC1 and HDAC2, histone H3K9 methylases G9a and SUV39H1, and a histone H3K4 demethylase lysine-specific demethylase 1 (LSD1) directly or indirectly with the CoREST complex or the mammalian switch independent 3 (mSin3) complex [34]. Interestingly, REST/RE1-silencing transcription factor and HDAC2 were found in the ESTs, suggesting cooperation of these molecules during dedifferentiation.

The oocyte has an ability to reprogram the somatic nucleus, which was demonstrated by the nuclear transfer into an oocyte [35]. Interestingly, most of the nuclear genes identified as candidates to regulate newt dedifferentiation were found in ovary ESTs as well (14,429 reads) but not in intact limb ESTs (1,098 reads; Table 4). Functional analysis of these genes might provide an advanced understanding of cellular plasticity, with possible future applications in regenerative therapies.