Binary Gene Expression Patterning of the Molt Cycle: The Case of Chitin Metabolism

<div><p>In crustaceans, like all arthropods, growth is accompanied by a molting cycle. This cycle comprises major physiological events in which mineralized chitinous structures are built and degraded. These events are in turn governed by genes whose patterns of expression are presumably linked to the molting cycle. To study these genes we performed next generation sequencing and constructed a molt-related transcriptomic library from two exoskeletal-forming tissues of the crayfish <i>Cherax quadricarinatus</i>, namely the gastrolith and the mandible cuticle-forming epithelium. To simplify the study of such a complex process as molting, a novel approach, binary patterning of gene expression, was employed. This approach revealed that key genes involved in the synthesis and breakdown of chitin exhibit a molt-related pattern in the gastrolith-forming epithelium. On the other hand, the same genes in the mandible cuticle-forming epithelium showed a molt-independent pattern of expression. Genes related to the metabolism of glucosamine-6-phosphate, a chitin precursor synthesized from simple sugars, showed a molt-related pattern of expression in both tissues. The binary patterning approach unfolds typical patterns of gene expression during the molt cycle of a crustacean. The use of such a simplifying integrative tool for assessing gene patterning seems appropriate for the study of complex biological processes.</p></div

A Novel Chitin Binding Crayfish Molar Tooth Protein with Elasticity Properties

<div><p>The molar tooth of the crayfish <i>Cherax quadricarinatus</i> is part of the mandible, and is covered by a layer of apatite (calcium phosphate). This tooth sheds and is regenerated during each molting cycle together with the rest of the exoskeleton. We discovered that molar calcification occurs at the pre-molt stage, unlike calcification of the rest of the new exoskeleton. We further identified a novel molar protein from <i>C</i>. <i>quadricarinatus</i> and cloned its transcript from the molar-forming epithelium. We termed this protein Cq-M13. The temporal level of transcription of <i>Cq-M13</i> in an NGS library of molar-forming epithelium at different molt stages coincides with the assembly and mineralization pattern of the molar tooth. The predicted protein was found to be related to the pro-resilin family of cuticular proteins. Functionally, <i>in vivo</i> silencing of the transcript caused molt cycle delay and a recombinant version of the protein was found to bind chitin and exhibited elastic properties.</p></div

Normalized read count of key chitin metabolism-related genes transcripts.

<p>Read count of key chitin metabolism-related genes transcripts from the gastrolith-forming epithelium (left) and the mandible cuticle-forming epithelium (right). Numbers on the X axis represent the four molt stages, 1 inter-molt (pool of animals, n = 1), 2 early pre-molt (pool of animals, n = 1), 3 late pre-molt (two single animals and one pool, n = 3) and 4 post-molt (all single animals, n = 2). Presented transcripts are (a) chitin synthase, (b) chitinase, (c) chitin deacetylase, (d) GlcN-6P synthase and (e) GlcN-6P deaminase. Letters represent statistical groups which are significantly different (<i>p</i>-value <0.05), error bars represent standard error.</p

Chitin-binding ability of rCq-M13.

<p>Following incubation with chitin powder, equal amounts of Negative control—BSA (top), Positive control—GAP 65 (middle, using gastrolith protein extract with known chitin binding abilities [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127871#pone.0127871.ref002" target="_blank">2</a>], and rCq-M13 (bottom) underwent three consecutive washes prior to SDS-PAGE. Lane 1 represent the first wash with DDW, lane 2 represent the second wash with NaCl (0.2 M) and lane 3 represent the third wash with denaturation buffer (DB, containing SDS and 2-mercaptoethanol).</p

Relative levels of key chitin metabolism-related genes transcript.

<p>Relative levels of key chitin metabolism-related genes transcript from the gastrolith-forming epithelium (left), the mandible cuticle-forming epithelium (middle) and the hepatopancreas (right), as determined by qPCR. Numbers on the X axis represents the four molt stages, 1 inter-molt (n = 5), 2 early pre-molt (n = 6), 3 late pre-molt (n = 6) and 4 post-molt (n = 6). Presented transcripts are (a) chitin synthase, (b) chitinase, (c) chitin deacetylase, (d) GlcN-6P synthase and (e) GlcN-6P deaminase. Letters represent statistical groups which are significantly different (<i>p</i>-value <0.05), error bars represents standard error.</p

Duplicates of the amino sugar metabolism pathway, modified from KEGG.

<p>The upper panel shows gene expression patterns in the gastrolith-forming epithelium, while the lower panel shows gene expression patterns in the mandible cuticle-forming epithelium. Enzymes that were found in the transcriptomic library are highlighted in blue for pattern 1111, red for pattern 0110, orange for pattern 1001, green for pattern 0111 and grey for enzymes that did not show any pattern. Enzymes discussed in the text are marked as I for chitin synthase, II for uridylyltransferase, III for chitinase, IV for chitin deacetylase, V for GlcN-6P synthase and VI for GlcN-6P deaminase.</p

Enrichment analysis test results for different tissues.

<p>Enrichment analysis test results of contigs in patterns 1111, 0110 and 0111 (a, b, and c, respectively) from the gastrolith- (left) and mandible cuticle-forming (right) epithelia. Black bars represent the observed number of hits in the sample, while grey bars represent the expected number of hits if the sample was chosen randomly. Significant differences between observed and expected number of hits are indicated by * (FDR <0.05) or ** (<i>p</i>-value <0.05).</p

Two representative examples of the binary patterns code used in our study.

<p>Top graph—a 0110 pattern in which the quantity of reads in early and late pre-molt is significantly higher, as compared to inter-molt and post-molt (<i>p</i>-value <0.05). Bottom graph—a 1001 pattern in which there is a significant increase of reads in inter-molt and post-molt, as compared to early and late pre-molt (<i>p</i>-value <0.05). The number 1 in the code represents a high number of reads as compared to a low number of reads, represented with 0. High quantity of reads have a minimum of 10³ reads.</p

Number of contigs in each binary pattern.

<p>(a) Gastrolith-forming epithelium samples and (b) mandible cuticle-forming epithelium samples. Patterns are arranged by abundance, from most to least abundant. A representative example of the binary pattern code is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122602#pone.0122602.g001" target="_blank">Fig 1</a>.</p

Development of a new molar tooth during an induced molt cycle in an ecdysone-injected male <i>C</i>. <i>quadricarinatus</i> and spatial and temporal expression patterns of the <i>Cq-M13</i> transcript <i>in vitro</i> and <i>in silico</i>.

<p><b>A</b>. Changes in molt mineralization index (MMI) during the induced molt cycle following repetitive injections of ecdysone. The x axis is normalized to days from ecdysis. A dashed line on an isolated mandible represents a cut through which a transverse plane is visible (A-top). <b>B</b>. Visualization of the new molar tooth assembly process in the crayfish mandible on days -9, -8, -7, -4, -3 and 0, following ecdysone injection by <i>ex vivo</i> micro-computed tomography. The top and bottom series of images show a view of the transverse and posterior planes of the mandibles correspondingly. White arrows point to the newly formed molar tooth. <b>C</b>. <i>In silico</i> transcriptomic analysis of <i>Cq-M13</i> read counts from four different molt stages. Different letters above columns represent statistically significant differences (p < 0.05 ± SE). <b>D</b>. Agarose gels showing RT-PCR products demonstrating spatial and temporal expression patterns of the <i>Cq-M13</i> transcript in cuticle-, molar-, basal segment (B.S.)-, maxillae- and gastrolith-forming tissues, as well as in hepatopancreas (Hepato) and testis.</p