Temporal effects of Notch signaling and potential cooperation with multiple downstream effectors on adenohypophysis cell specification in zebrafish

Abstract

The adenohypophysis (AH) consists of six distinct types of hormone‐secreting cells. In zebrafish, although proper differentiation of all AH cell types has been shown to require Notch signaling within a period of 14–16 h postfertilization (hpf), the mechanisms underlying this process remain to be elucidated. Herein, we observed using the Notch inhibitor dibenzazepine (DBZ) that Notch signaling also contributed to AH cell specification beyond 16 hpf. Specification of distinct cell types was perturbed by DBZ treatment for different time frames, suggesting that AH cells are specified by Notch‐dependent and cell‐type‐specific mechanisms. We also found that two hes‐family genes, her4.1 and hey1, were expressed in the developing AH under the influence of Notch signaling. her4.1 knockdown reduced expression of proopiomelanocortin a (pomca), growth hormone (gh), and prolactin, whereas hey1 was responsible only for gh expression. Simultaneous loss of both Her4.1 and Hey1 produced milder phenotypes than that of DBZ‐treated embryos. Moreover, DBZ treatment from 18 hpf led to a significant down‐regulation of both gh and pomca genes only when combined with injection of a subthreshold level of her4.1‐morpholino. These observations suggest that multiple downstream effectors, including Her4.1 and Hey1, mediate Notch signaling during AH cell specification.

Neural stem cell quiescence and stemness are molecularly distinct outputs of the Notch3 signalling cascade in the vertebrate adult brain

“…To assess Hey1 function in the adult pallial VZ in vivo , we needed a conditional method and designed two fluorescein-tagged morpholinos (MOs) targeting the ATG or the second donor splice site of hey1 transcripts, and a control MO harbouring 5 mismatches compared with the hey1 ATG MO. We verified their efficiency and selectivity by showing that both hey1 MOs mimicked the effect of the hey1 mutation on pituitary development in 72 h post-fertilization (hpf) larvae ( Nakahara et al, 2016 ), whereas the control MO had no effect ( ).…”

“…S6H,I " type="url"/> ), indicating that the treatment itself would not interfere with Hey1 function. Further, hey1 −/− larvae do not display any morphological abnormalities ( Nakahara et al, 2016 ), contrasting with larvae in which Notch signalling has been inhibited, which suffer visible defects in hey1 -expressing organs, notably the vasculature and nervous system (e.g. Quillien et al, 2014 ; Itoh et al, 2003 ).…”

“…hey1 mutants were obtained from Dr Y. Kikuchi (Hiroshima University, Japan). The published hey1 ha7 allele harbours a 7 bp deletion, causing a frameshift at amino acid 7 ( Nakahara et al, 2016 ). Upon sequencing, we found a new different allele, harbouring an 11 bp deletion, leading to a frameshift and the production of a truncated protein ( …”

“…Whole adult brains or embryos were incubated at 65°C for 18 h in 2 ng/μl DIG-labelled mRNA probes for notch3 ( Itoh et al, 2003 ), her4.1 ( Takke et al, 1999 ), hey1 and hey2 (see below), or pomca and gh ( Nakahara et al, 2016 ), then with POD-conjugated anti-DIG (sheep, Roche, 1:500). Dissected juvenile brains were hybridized with a DIG-labelled probe, incubated with anti-DIG-AP Fab fragments (Roche) and then cryosectioned.…”

let-7 MicroRNA-Mediated Regulation of Shh Signaling and the Gene Regulatory Network Is Essential for Retina Regeneration

“…We performed a luciferase assay in zebrafish embryos co-injected with notch intracellular domain ( nicd ) mRNA along with mmp9 :GFP-luciferase vector. nicd mRNA could cause an upregulation of Her4.1 ( Nakahara et al., 2016 , Wilson et al., 2016 ), and the luciferase assay showed dose-dependent downregulation of mmp9 promoter activity ( Figure 4 S), while mutations in Her4-binding sites abolished this impact ( Figure S4 D; Table S2 ). In summary, these results suggest that active Notch-signaling-mediated induction of her4.1 restricts the span of mmp9 expression at the site of injury.…”