Hemicentin 2 and Fibulin 1 are required for epidermal–dermal junction formation and fin mesenchymal cell migration during zebrafish development

Abstract

Summary Hemicentin 1 (Hmcn1) and Hemicentin 2 (Hmcn2) belong to the fibulin family of extracellular matrix (ECM) proteins that play pivotal roles during development and homeostasis of a variety of vertebrate tissues. Recently, we have shown that mutations in zebrafish Hmcn1, also called Fibulin 6, lead to massive fin blistering, similar to the defects caused by the Fraser syndrome gene Fras1. In contrast, the role of Hmcn2 during vertebrate development has thus far been uncharacterized. In zebrafish, hmcn2, like fibulin 1 (fbln1), another member of the fibulin family, is predominantly expressed in fin mesenchymal cells and developing somites, contrasting the strict epithelial expression of hmcn1. While antisense morpholino oligonucleotide (MO) – based knockdown of hmcn2 did not yield any discernable defects, hmcn2/fbln1 double knockdown fish displayed blistering in the trunk, pointing to an essential contribution of these proteins from mesodermal sources for proper epidermal-dermal junction formation. In contrast, and unlike hmcn1 mutants, epidermal-dermal junctions in the fin folds of hmcn2/fbln1 double knockdown fish were only moderately affected. Instead, they displayed impaired migration of fin mesenchymal cells into the fin folds, pointing to a crucial role of Hmcn2 and Fbln1 to remodel the ECM of the fin fold interepidermal space, which is a prerequisite for fibroblast ingrowth. TEM analyses suggest that this ECM remodeling occurs at the level of actinotrichia, the collageneous migration substrate of mesenchymal cells, and at the level of cross fibers, which resemble mammalian microfibers. This work provides first insights into the role of Hmcn2 during vertebrate development, identifying it as an evolutionary conserved protein that acts in functional redundancy with Fbln1C and/or Fbln1D isoforms to regulate tissue adhesion and cell migration, while extending current knowledge of the functions of vertebrate Fbln1.

An active role for basement membrane assembly and modification in tissue sculpting

“…Following knockdown of the hemicentin ortholog hmcn2 and the fibulin family member fbl1, blisters develop between the somite basement membrane and the epithelial basement membrane (right). Data from Morrissey et al and Feitosa et al (Morrissey et al, 2014;Feitosa et al, 2012) and have been adapted with permission.…”

“…4C; Carney et al, 2010;Feitosa et al, 2012). Mutations in hmcn1 result in a loss of attachment between the epidermal sheets of the fin fold (Carney et al, 2010).…”

“…At a later time, hmcn2 in combination with the fibulin fbln1 organize electron-dense cross fibers that might represent a more elaborate or altered B-LINK ( Fig. 4C; Feitosa et al, 2012). The hnmc2 gene also regulates a basement membrane connection in the zebrafish trunk.…”

An exclusively mesodermal origin of fin mesenchyme demonstrates that zebrafish trunk neural crest does not generate ectomesenchyme

“…Probes used were bmp1a (Asharani et al, 2012), hmcn2 (Carney et al, 2010) and fbln1 (Feitosa et al, 2012). Whole-mount embryo fluorescent immunostaining was performed as described (Asharani et al, 2012 …”

“…We have recently identified that in situ probes detecting bmp1a (Asharani et al, 2012), hemicentin 2 (hmcn2) (Carney et al, 2010) and fibulin 1 (fbln1) (Feitosa et al, 2012) mark fin mesenchyme cells from 48 hpf onwards (Fig. 1G,J,M).…”