Localization of circadian clock protein BMAL1 in the photoperiodic signal transduction machinery in Japanese quail

Abstract

The circadian clock is a fundamental property of living organisms and is involved in seasonal (photoperiodic) time measurement. Among vertebrates, birds have multiple circadian pacemakers in the eye, the pineal gland, and the suprachiasmatic nucleus (SCN), and have highly sophisticated photoperiodic mechanisms. However, because the removal of these circadian pacemakers fails to abolish the photoperiodic response, the existence of another "photoperiodic clock" has been suggested. Recent studies have revealed that the mediobasal hypothalamus (MBH) and the adjacent pars tuberalis (PT) of the pituitary gland constitute key components of the photoperiodic signal transduction machinery. In the present study, we generated a polyclonal antibody against the chicken circadian clock protein BMAL1 to examine BMAL1 distribution in the Japanese quail brain by using immunohistochemistry. BMAL1-like immunoreactivity (lir) was confirmed in the pineal gland and the medial SCN, which are critical circadian pacemakers. We also observed strong immunoreactivity in the MBH, including the ependymal cells (ECs), the infundibular nucleus (IN), the median eminence (ME), and the adjacent PT. Furthermore, semiquantitative analysis suggested that BMAL1-lir shows daily fluctuation in these regions. It is possible that circadian clocks in the photoperiodic signal transduction machinery such as the PT and the EC may be involved in the regulation of photoperiodism.

Tissue-Specific Posttranslational Modification Allows Functional Targeting of Thyrotropin

“…Immunohistochemistry to detect TSHβ was performed using rabbit polyclonal antibody to mouse/rat TSHβ (1:10,000) (provided by Dr. Albert F. Parlow, the NIDDK), as previously reported (Ikegami et al, 2009) (Figure S1). For double-immunofluorescence staining, PT and PD slices were incubated with rabbit polyclonal antibody against TSHβ and goat polyclonal antibody against ST3GalIV (1:2,000) or GnT-V (1:2,000) (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature, and then reacted with Alexa Fluor 546-conjugated donkey anti-goat IgG (1:400) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:400) (Molecular Probes), and mounted in DAPI/mounting medium (LK-096A, Kreatech).…”

The saccus vasculosus of fish is a sensor of seasonal changes in day length

“…Antibodies were diluted for western blotting: rabbit antiTSHb serum, 1:5,000; anti-DIO2 antibody, 1:200; anti-SWS1 antibody, 1:200; and anti-OPN4 antibody, 1:200; and goat anti-b-actin antibody (Santa Cruz Biotechnology Inc., sc-1616), 1:2,000. Following incubation with anti-rabbit or goat IgG secondary antiserum, immunoreactive bands were visualized by enhanced chemiluminescent detection (ImmunoStar LD, WAKO, Japan), and observed using an LAS-3000 mini imager (Fujifilm, Tokyo, Japan) 38 .…”

“…Immunohistochemistry was performed using rabbit anti-TSHb serum (1:5,000), anti-DIO2 (1:200), anti-SWS1 (1:200) and anti-OPN4 (1:200) antibodies, and a Vectastain Elite ABC rabbit IgG kit (Vector Laboratories) with a standard protocol 38 . Immunoelectron microscopic study was performed using post-embedding immunogold labelling technique with gold-tagged (15 nm) goat anti-rabbit IgG polyclonal antibody (1:100) (British Biocell International Ltd.).…”

A mammalian neural tissue opsin (Opsin 5) is a deep brain photoreceptor in birds

“…Western blotting was performed as previously reported (48). Brain slices (3 mm) were generated by a mouse brain matrix (ASI Instruments), and the MBH, including the PVO (2.5-mm diameter), was punched out (6).…”

“…Immunohistochemistry was performed using antiquail Opsin 5 antibodies (1:1,000) and a Vectastain Elite ABC rabbit IgG kit (Vector Laboratories) with a standard protocol (48).…”