Osteoblasts pattern endothelium and somatosensory axons during zebrafish caudal fin organogenesis.

Skeletal elements frequently associate with vasculature and somatosensory nerves, which regulate bone development and homeostasis. However, the deep, internal location of bones in many vertebrates has limited in vivo exploration of the neurovascular-bone relationship. Here, we use the zebrafish caudal fin, an optically accessible organ formed of repeating bony ray skeletal units, to determine the cellular relationship between nerves, bones and endothelium. In adult zebrafish, we establish the presence of somatosensory axons running through the inside of the bony fin rays, juxtaposed with osteoblasts on the inner hemiray surface. During development we show that the caudal fin progresses through sequential stages of endothelial plexus formation, bony ray addition, ray innervation and endothelial remodeling. Surprisingly, the initial stages of fin morphogenesis proceed normally in animals lacking either fin endothelium or somatosensory nerves. Instead, we find that sp7+ osteoblasts are required for endothelial remodeling and somatosensory axon innervation in the developing fin. Overall, this study demonstrates that the proximal neurovascular-bone relationship in the adult caudal fin is established during fin organogenesis and suggests that ray-associated osteoblasts pattern axons and endothelium.

Rho-associated kinase regulates Langerhans cell morphology and responsiveness to tissue damage.

Skin damage requires efficient immune cell responses to restore organ function. Epidermal-resident immune cells known as Langerhans cells use dendritic protrusions to surveil the skin microenvironment, which contains keratinocytes and peripheral axons. The mechanisms governing Langerhans cell dendrite dynamics and responses to tissue damage are poorly understood. Using skin explants from adult zebrafish, we show that Langerhans cells maintain normal surveillance following axonal degeneration and use their dendrites to engulf small axonal debris. By contrast, a ramified-to-rounded shape transition accommodates the engulfment of larger keratinocyte debris. We find that Langerhans cell dendrites are populated with actin and sensitive to a broad-spectrum actin inhibitor. We show that Rho-associated kinase (ROCK) inhibition leads to elongated dendrites, perturbed clearance of large debris, and reduced Langerhans cell migration to epidermal wounds. Our work describes the dynamics of Langerhans cells and involvement of the ROCK pathway in immune cell responses.

Rho-associated kinase regulates Langerhans cell morphology and responsiveness to tissue damage.

Skin is often the first physical barrier to encounter invading pathogens and physical damage. Damage to the skin must be resolved quickly and efficiently to maintain organ homeostasis. Epidermal-resident immune cells known as Langerhans cells use dendritic protrusions to dynamically surveil the skin microenvironment, which contains epithelial keratinocytes and somatosensory peripheral axons. The mechanisms governing Langerhans cell dendrite dynamics and responses to tissue damage are not well understood. Using skin explants from adult zebrafish, we show that Langerhans cells maintain normal surveillance activity following axonal degeneration and use their dynamic dendrites to engulf small axonal debris. By contrast, a ramified-to-rounded shape transition accommodates the engulfment of larger keratinocyte debris. We find that Langerhans cell dendrites are richly populated with actin and sensitive to a broad spectrum actin inhibitor. We further show that Rho-associated kinase (ROCK) inhibition leads to elongated dendrites, perturbed clearance of large debris, and reduced Langerhans cell migration to tissue-scale wounds. Altogether, our work describes the unique dynamics of Langerhans cells and involvement of the ROCK pathway in immune cell responses to damage of varying magnitude.

Dendritic atoh1a+ cells serve as transient intermediates during zebrafish Merkel cell development and regeneration.

Sensory cells often adopt specific morphologies that aid in the detection of external stimuli. Merkel cells encode gentle touch stimuli in vertebrate skin and adopt a reproducible shape characterized by spiky, actin-rich microvilli that emanate from the cell surface. The mechanism by which Merkel cells acquire this stereotyped morphology from basal keratinocyte progenitors is unknown. Here, we establish that dendritic Merkel cells (dMCs) express atonal homolog 1a (atoh1a), extend dynamic filopodial processes, and arise in transient waves during zebrafish skin development and regeneration. We find that dMCs share molecular similarities with both basal keratinocytes and Merkel cells, yet display mesenchymal-like behaviors, including local cell motility and proliferation within the epidermis. Furthermore, dMCs can directly adopt the mature, microvilliated Merkel cell morphology through substantial remodeling of the actin cytoskeleton. Loss of Ectodysplasin A signaling alters the morphology of dMCs and Merkel cells within specific skin regions. Our results show that dMCs represent an intermediate state in the Merkel cell maturation program and identify Ectodysplasin A signaling as a key regulator of Merkel cell morphology.

Dermal appendage-dependent patterning of zebrafish atoh1a+ Merkel cells.

Touch system function requires precise interactions between specialized skin cells and somatosensory axons, as exemplified by the vertebrate mechanosensory Merkel cell-neurite complex. Development and patterning of Merkel cells and associated neurites during skin organogenesis remain poorly understood, partly due to the in utero development of mammalian embryos. Here, we discover Merkel cells in the zebrafish epidermis and identify Atonal homolog 1a (Atoh1a) as a marker of zebrafish Merkel cells. We show that zebrafish Merkel cells derive from basal keratinocytes, express neurosecretory and mechanosensory machinery, extend actin-rich microvilli, and complex with somatosensory axons, all hallmarks of mammalian Merkel cells. Merkel cells populate all major adult skin compartments, with region-specific densities and distribution patterns. In vivo photoconversion reveals that Merkel cells undergo steady loss and replenishment during skin homeostasis. Merkel cells develop concomitant with dermal appendages along the trunk and loss of Ectodysplasin signaling, which prevents dermal appendage formation, reduces Merkel cell density by affecting cell differentiation. By contrast, altering dermal appendage morphology changes the distribution, but not density, of Merkel cells. Overall, our studies provide insights into touch system maturation during skin organogenesis and establish zebrafish as an experimentally accessible in vivo model for the study of Merkel cell biology.