High Mobility Group Box-1 (HMGB1; Amphoterin) Is Required for Zebrafish Brain Development

Abstract

Hmgb1 (high mobility group box-1; amphoterin) is highly expressed in brain during early development of vertebrate and nonvertebrate species. However, its role in brain development remains elusive. Here we have cloned the zebrafish Hmgb1 and specifically manipulated Hmgb1 expression using injection of morpholino antisense oligonucleotides or Hmgb1 cRNA. The HMGB1 knockdown morphants produced by injection of three different morpholino oligonucleotides display a characteristic phenotype with smaller size, smaller brain width, and shorter distance between the eyes. Closer examination of the phenotype reveals severe defects in the development of the forebrain that largely lacks catecholaminergic neural networks. The HMGB1 morphant is deficient in survival and proliferation of neural progenitors and displays fewer cell groups expressing the transcription factor Pax6a in the forebrain and aberrant Wnt8 signaling. The mechanism of HMGB1-dependent progenitor survival involves the neuronal transmembrane protein AMIGO (amphoterin-induced gene and orf), the expression of which is regulated by HMGB1 in vivo. Our data demonstrate that HMGB1 is a critical factor for brain development, enabling survival and proliferation of neural progenitors that will form the forebrain structures.The high mobility group box-1 protein (HMGB1 3 ; also designated as HMG1 and amphoterin) is an abundantly occurring parental form of the HMG proteins (for review, see Ref. 1). HMGB1 is an exceptional member in the family of HMG-box proteins; depending on the cell type and its activation state, HMGB1 displays a nonnuclear localization and is secreted from cells, in contrast to most HMG-box proteins that are strictly bound to the cell nuclei (for recent reviews, see Refs. 2 and 3). During the last few years, the extensive literature dealing with HMGB1 functions has mainly focused on extracellular regulation of cells by HMGB1. HMGB1 can be passively released from injured cells, but it is also actively secreted due to several types of stimuli such as cell contact with extracellular matrix and cytokine stimulation of cells (2). Acetylation of lysine residues of HMGB1 has been shown to act as a signal leading to extracellular export via a non-classical secretory pathway (4). HMGB1 functions are currently mainly associated with binding to the cell surface receptor RAGE (receptor for advanced glycation end products), but Toll-like receptors have been increasingly suggested as membrane receptors of HMGB1 (for review, see Ref. 2).Compared with the extensive recent literature dealing with the pathophysiological roles of HMGB1 in inflammation, much less attention has been paid to its physiological roles. The Hmgb1 knock-out mouse survives until early postnatal age, and problems in glucose homeostasis have been suggested to cause multiorgan failure in these mice (5).HMGB1 was isolated from developing rat brain using neurite outgrowth in embryonic forebrain neurons as a readout in protein fractionation (6). These studies provided the initial evidence of HMGB1 as a...

Amigo Adhesion Protein Regulates Development of Neural Circuits in Zebrafish Brain

“…Animals-An outbred zebrafish (Danio rerio) strain from a local resource, the Turku line, was used in this study for its steady yield of embryos (12). Fish feeding, breeding, and maintenance were done according to an established protocol (13).…”

“…Mouse monoclonal anti-␤-actin antibody (A2228, 1:1000; Sigma-Aldrich) was used as the control of sample loading. The zebrafish tissue preparation and Western blotting were carried out as described previously (12,13).…”

“…Total RNA from 1-8-dpf larvae was extracted and reverse-transcribed. The subsequent PCR was performed as described previously (12). RNA quality and amount were analyzed spectrophotometrically by a NanoVue Plus spectrophotometer (GE Healthcare).…”

“…The qPCR primers of amigo3b were 5Ј-ACA CTG GCT TCA CCA CAC T-3Ј and 5Ј-AGC GAC AGG GAG TCA AGT AG-3Ј. The zebra fish ␤-actin (AF057040.1, GI:3044209), elongation factor 1 ␣1 (zgc:109885, GI:90652818), and ribosomal protein L13a (BC047855.1, GI:28838761) were set as template quantity controls, and the same primers were used as described previously (12). The PCRs were processed with the Bio-Rad CFX96 realtime PCR machine using the CFX96 TM real-time PCR detection system.…”

“…Immunocytochemistry-Mouse monoclonal anti-tyrosine hydroxylase (TH) antibody (Diasorin, Stillwater, MN) was used for the whole mount staining of the catecholaminergic system in 3 dpf larval brains as described earlier (12). The mouse anti-3A10 (1:100; Developmental Studies Hybridoma Bank), chicken anti-Amigo1 antibodies (5 g/ml), and anti-HNK-1 antibodies (1:100) were used for whole mount immunostaining of zebrafish larvae.…”