Chitin is endogenously produced in vertebrates

In our surveys of vertebrate genomes and transcriptomes, numerous “unknown” fish and amphibian sequences were detected that exhibited striking homology to bona fide invertebrate chitin synthase genes (BlastX, e-value < 10^-150). These sequences had likely been overlooked as coding for chitin synthases (CHS) during the ab initio annotation process. This list (Table S1) includes CHS genes of several fishes and a salamander, and two species (zebrafish and Xenopus) previously identified in a recent report on metazoan chitin synthase genes [1]; amniote CHS genes have not been identified. A phylogenetic tree showing the interrelationships among the identified vertebrate CHS orthologs is given in Fig. S1A.

To investigate whether fish endogenously produce chitin, we employed the zebrafish (Danio rerio) model system. The zebrafish genome assembly [2] revealed the existence of four CHS genes: three closely related and linked genes on chromosome 18 and a gene on chromosome 13 (Table S1). Published Affymetrix microarray experiments (Supplemental Information) indicated that the chromosome-13 gene (DrCHS-1) is preferentially expressed during embryonic/larval development. Whole mount in situ hybridization (ISH) showed expression of DrCHS-1 in the larval gut (Fig. 1A-C), and subsequent transverse sections revealed expression in cells within the epithelial wall and in mesenchymal cells adjacent to the gut (Fig. 1D) [3].

For detection of chitin in situ, we used affinity histochemistry with fluorescent probes containing a highly-specific chitin binding domain (CBD) (Supplemental Information) [4]. When applied to a zebrafish developmental series, chitin was detected as early as 3 days post-fertilization (dpf) as multiple fibers within the intestinal bulb (Fig. S2B,C); an endoderm population dorsal to the gut also showed chitin signals (Fig. S2C) which largely disappeared by 4 dpf (cf Fig. 1E). At 4-5 dpf, the fibers coalesce in the middle intestine and are distributed throughout the digestive tract (Fig. 1E-G; Supplemental movie). The bulk of this chitin is extracellular and distributed throughout the intestinal lumen, however, cellular signals were detected among cells within the lumen wall and mesenchymal cells ventral to the intestinal tract (Fig. 1F). The similarity in the DrCHS-1 ISH patterns and chitin localization (Fig. 1A-F) suggest that chitin is being synthesized by cells in and around the developing intestine and sequestered within the lumen. Validation of the robustness of our chitin detection assay is shown in Figs. S1D and S2A-C.

In order to demonstrate that chitin production is coupled with chitin synthase expression, we knocked down the DrCHS-1 gene. Two different splice-blocking morpholinos were designed to DrCHS-1 and microinjected into fertilized eggs along with their mismatch controls (Supplemental Information, Fig. S2E). The knockdowns resulted in marked reduction in chitin signals in the gut (Fig. S2F), and are consistent with the early expression profile of DrCHS-1.

We also assayed for chitin in zebrafish scales since our initial RT-PCR survey indicated expression of a zebrafish chromosome-18 CHS gene in this tissue (Fig. S2D). Fish scales are primarily comprised of a hard, partly mineralized collagenous matrix as well as partially overlying epithelium (skin). Unlike in the gut, most the chitin signals were observed intracellularly in epithelial goblet and club cells (Fig. 1H,I). The chitin in the goblet cells was largely restricted to the periphery of the cells, whereas it was distributed throughout the cytoplasm in the club cells. Our findings on zebrafish scales were corroborated via RT-PCR and chitin histochemistry on scales of juvenile Atlantic salmon, Salmo salar (Fig. S2D, Supplemental Movie).

Chitin synthase genes were also identified in two amphibian species, Xenopus and axolotl. Transcriptome data indicated that a chitin synthase gene was expressed in larval axolotls (regenerating limb, Table S1), thus appendage tissues from larvae (stage ~ 52-53) were obtained and used for chitin histochemistry. Chitin is found predominantly in superficial epidermal cells resembling squamous epithelia, Leydig epidermal cells and mesenchymal (fibroblast-like) cells (Fig. 1J-M). The strong signals on the tips of the forelimb digits (Fig. 1J) are due to superficial epidermal cells. These findings in axolotl, including chitinase validation (Fig. S2C) are similar to those seen for fish scales.

Ultimate support for the existence of vertebrate chitin is isolation of chitin per se from a vertebrate. For this experiment, we used fresh scales from Atlantic salmon and employed a fastidious chemical extraction strategy. From 60 grams of dried scales we extracted a precipitate of ~ 1.5 mg of chitinous material for analysis. This low yield and the intractability of insoluble chitin to most characterization methods, necessitated its analysis via microscopic Fourier transform infrared spectrometry (FTIR). Micrographs of the precipitate collected from salmon scales are shown in Fig. 2A-B,E-F and the FTIR spectrum of the insoluble granules confirms its identity as chitin (Fig. 2G). The fingerprint region in the FTIR spectrograph reveals a split of the amine I band into two bands at 1655 and 1626 cm^-1, resulting from the two types of hydrogen bonds in the antiparallel alignment of α-chitin, though further analyses will be required to definitively rule out other isomeric forms. Our chemical results (expanded upon in Supplemental Information) demonstrate that fish scale material contains chitin and are in keeping with molecular and histochemical data of the scale epithelia of salmon and zebrafish.

First isolated from fungi over 200 years ago, chitin has since been identified from a wide range of organisms [5, 6], including fossils [7, 8]. It is an integral structural component of invertebrate organisms and its tractability to industrial-scale chemical extraction and bioengineering has enabled its use in many applications in biomedicine and technology [5, 9]. The difficulty of its extraction from vertebrates, however, likely has contributed to its assumed absence and there has been but one report of the detection of a fish chitin some 30 years ago [10]. We show here the existence of endogenous chitin in fishes and amphibians, which collectively comprise over half of all extant vertebrates. Efforts can now be focused on elucidating the underlying biology and biochemistry of vertebrate chitin.