Making maxillary barbels with a proximal-distal gradient of Wnt signals in matrix-bound mesenchymal cells

Abstract

The evolution of specific appendages is made possible by the ontogenetic deployment of general cell signaling pathways. Many fishes, amphibians and reptiles have unique skin appendages known as barbels, which are poorly understood at the cellular and molecular level. In this study, we examine the cell arrangements, cell division patterns, and gene expression profiles associated with the zebrafish maxillary barbel, or ZMB. The earliest cellular organization of the ZMB is an internal whorl of mesenchymal cells in the dermis of the maxilla; there is no epithelial placode, nor any axially-elongated epithelial cells as expected of an apical ectodermal ridge (AER). As the ZMB develops, cells in S-phase are at first distributed randomly throughout the appendage, gradually transitioning to a proliferative population concentrated at the distal end. By observing ZMB ontogenetic stages in a Wnt-responsive transgenic reporter line, TCFsiam, we identified a strongly fluorescent mesenchymal cell layer within these developing appendages. Using an in vitro explant culture technique on developing barbel tissues, we co-localized the fluorescent label in these cells with the mitotic marker EdU. Surprisingly, the labeled cells showed little proliferation, indicating a slow-cycling subpopulation. Transmission electron microscopy of the ZMB located these cells in a single, circumferential layer within the barbel's matrix core. Morphologically, these cells resemble fibroblasts or osteoblasts; in addition to their matrix-bound location, they are identified by their pancake-shaped nuclei, abundant rough endoplasmic reticulum, and cytoplasmic extensions into the surrounding extracellular matrix. Taken together, these features define a novel mesenchymal cell population in zebrafish, the "TCF(+) core cells." A working model of barbel development is proposed, in which these minimally mitotic mesodermal cells produce collagenous matrix in response to ectodermally-derived Wnt signals deployed in a proximal-distal gradient along the appendage. This documents a novel mechanism of vertebrate appendage outgrowth. Similar genetic signals and cell behaviors may be responsible for the independent and repeated evolution of barbel structures in other fish species.

Figure 1. Evolutionary and developmental context of barbel development in several species of ray-finned fishes (Actinopterygii)

A) Simplified diagram of actinopterygian phylogeny. Two barbelled species within this clade are the zebrafish (Danio rerio) and the channel catfish (Ictalurus punctatus). B) Comparative schematic of barbel development in D. rerio and I. punctatus. The zebrafish, left, develops two pairs of barbels as a juvenile, approximately 4–6 weeks after fertilization. The catfish, right, develops four pairs of barbels while still an embryo (figure modified from Hawkins, 2011). meb = mental barbels; mnb = mandibular barbels; mxb = maxillary barbels; nb = nasal barbels.

Figure 2. Ectodermal and mesodermal arrangements in the maxillary barbel bud

All images are 3D renderings of confocal Z-stacks from fixed tissues. All cells are stained with DAPI (gray) to show nuclear morphology and cell arrangement. In all panels, anterior/proximal is to the left. A) Lateral view of the surface of a developing maxilla from a juvenile zebrafish (standard length < 10 mm) at the prospective site of barbel outgrowth. Taste buds appear as cellular rosettes. B) Deep confocal slice of the same specimen. The presumptive barbel is marked by a cellular condensation of dermal mesenchyme. C) Optical section through an early maxillary barbel bud (~100 μm). The proximal-distal axis is dotted, with distal to the lower right. In this transgenic specimen, epithelial cells (e) express membrane-bound EGFP (white). The epithelial layer of the barbel bud (e) contains 2–3 layers of cuboidal or slightly flattened cells. The mesenchymal cells (m) form a whorl around the bud center. D) Optical section through an elongating barbel bud (~300 μm). At this stage, the distalmost epithelial cells (e) are more squamous, with tangentially flattened nuclei. e = epithelial cells; m = mesenchymal cells

Figure 3. Patterns of cell division in the developing zebrafish maxillary barbel

Whole-mount in vitro mitotic labeling of explanted maxillary barbel buds. Nuclei are stained blue; cells in S-phase (EdU+) are also red. Panels A and B are depth-sensitive full-thickness image renderings, in which superficial nuclei appear brighter than deeper ones. Panel C is a similar rendering; for clarity, the membrane-GFP signal has been removed from the superficial layers. A) Mitotic labeling of a wildtype juvenile barbel (~300 μm). Dividing cells are scattered throughout the appendage. B) Mitotic labeling of a slightly longer barbel appendage (~500 μm). Clusters of dividing cells on the ventral side of the barbel correspond to the developing taste buds. C) Mitotic labeling of an elongated juvenile barbel (~1 mm) from a membrane-EGFP transgenic zebrafish. Labeled nuclei are found in both the epithelial and mesenchymal layers throughout the appendage.

Figure 4. TCFsiam:mCherry reporter expression in zebrafish maxillary barbel tissues

A) Overview of a juvenile zebrafish head. All nuclei are blue; TCF+ nuclei are also red. Strong nuclear mCherry signal was detected in cells of the olfactory pit (o) and Meckel’s cartilage (mc). A third small domain is in the area of the early maxillary barbel bud (mb). B) Transmitted light micrograph of an early maxillary barbel bud (mb). B′) The same specimen as B, showing a subepithielial cone of red fluorescent nuclei. C) An elongated juvenile maxillary barbel (mb). A “sleeve” of red nuclei occupies the distal two-thirds of the appendage. Signal is weaker in the proximal regions. D) An adult maxillary barbel. mCherry+ cells are highly concentrated at the distal tip just under the epidermis. Only scattered proximal cells express the label. E) Magnification of the distal tip of a juvenile barbel. The mCherry+ nuclei are found in a single layer underneath the epidermis. F) Volume rendering of the mCherry+ cells in a juvenile maxillary barbel. In this panel, the volume is rotated and cleaved to show an oblique cross-section. Red nuclei are arranged in a single, circumferential layer just underneath the barbel epithelium (blue), and directly surrounding the acellular barbel core (black).

Figure 5. Co-localization of a canonical Wnt reporter (TCFsiam:mCherry) with EdU-488 mitotic labeling using in vitro explant cultured maxillary barbels/

A) Two-channel rendering of a developing zebrafish maxillary barbel; the distal end, which is digitally truncated, points right. mCherry+ cells (red) occupy a cylindrical, mesenchymal sheath surrounding the barbel core. Numerous EdU+ cells (green) are superficial to this layer, indicating maximal cell division in the overlying epithelium. Only two ventral cells are double-labeled (mCherry+/EdU+). B) mCherry+ cells from a second double-labeled specimen. C) EdU+ cells (green) from the same region as B. D) Merged image of B and C. The two yellow nuclei (*) are double-labeled.

Figure 6. Transmission electron microcopy of the TCF+ ZMB core cells

A) Cross-sectional overview of the adult zebrafish maxillary barbel. The rectangular boundary indicates the region magnified in panel B. B) The maxillary barbel epidermal-dermal boundary. Ectodermal cells (ec) are electron-dense and rest on a prominent basement membrane (bm). Approximately 2 microns below this membrane lies the radially-flattened, pancake-shaped nucleus (n) of a TCF+ core cell. Adjacent to the cell are several collagen bundles (c). Deep to the cell are several axons (a) and the acellular matrix of the barbel’s central rod. C) Micrograph of a similar region. The electron-dense epithelial cells (ec) are connected by tight junctions (tj). Approximately 2–3 microns below the basement membrane is a layer of cell cytoplasm (*) that appears as a continuous, circumferential ring. D) A cytoplasmic projection (asterisk) extends from a matrix-bound cell through a dense field of collagen fibers (c). E) Several cross-cut collagen bundles (c) near a TCF+ cell. The heterochromatic cell nucleus (n) is at the upper right. Between the bundles are ribbons of cytoplasm enriched in rough endoplasmic reticulum (rer). The small, dark nodules are individual ribosomes. F) Magnification of a cytoplasmic ribbon shows extensive rough endoplasmic reticulum (rer) and intracellular vesicles. The double-wrapped ovoid organelles are mitochondria (m). Collagen fibers close to the cell appear well separated, showing individual ovoid cross-sections. In contrast, the collagenous matrix farther away from the cell appears hyperpolymerized (arrows), with no individual fibrils present. a = axon; bm = basement membrane; c = collagen bundle; ec = ectodermal cell; m = mitochondria; n = nucleus; rer = rough endoplasmic reticulum; tj = tight junction.

Figure 7. Making maxillary barbels with Wnt signals: location of a new dermal cell population, and alternative models of appendage outgrowth

Diagrammatic section through a developing maxillary barbel bud, distal end to the right. An outer later of stratified epithelium (blue) covers a convex dermal core. Within the core is a mass of acellular matrix (yellow), in which is embedded arranged a single layer of Wnt-responsive cells (the TCF+ core cells, red). These cells contact each other circumferentially via overlapping cytoplasmic bridges; smaller cytoplasmic projections extend radially into the surrounding collagenous material. Activated TCF+ cells divide rarely, but increase secretory activity, depositing collagenous matrix to widen and lengthen the appendage. A) Paracrine activation: Wnt ligands secreted by the overlying epidermis activate the TCF+ core cells. B) Autocrine activation: TCF+ cells maintain their own activation by secreting Wnt ligand(s) that are bound to the surrounding matrix. For simplicity, other cell types within the dermal compartment (e.g., blood vessels, peripheral nerves, and melanophores) are not shown.