Delta/Jagged-mediated Notch signaling induces the differentiation of agr2-positive epidermal mucous cells in zebrafish embryos

Abstract

Teleosts live in aquatic habitats, where they encounter ionic and acid-base fluctuations as well as infectious pathogens. To protect from these external challenges, the teleost epidermis is composed of living cells, including keratinocytes and ionocytes that maintain body fluid ionic homeostasis, and mucous cells that secret mucus. While ionocyte progenitors are known to be specified by Delta-Notch-mediated lateral inhibition during late gastrulation and early segmentation, it remains unclear how epidermal mucous cells (EMCs) are differentiated and maintained. Here, we show that Delta/Jagged-mediated activation of Notch signaling induces the differentiation of agr2-positive (agr2+) EMCs in zebrafish embryos during segmentation. We demonstrated that agr2+ EMCs contain cytoplasmic secretory granules and express muc5.1 and muc5.2. Reductions in agr2+ EMC number were observed in mib mutants and notch3 MOs-injected notch1a mutants, while increases in agr2+ cell number were detected in notch1a- and X-Su(H)/ANK-overexpressing embryos. Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of agr2+ EMCs. Increased agr2+ EMC numbers were also observed in jag1a-, jag1b-, jag2a- and dlc-overexpressing, but not jag2b-overexpressing embryos. Meanwhile, reductions in agr2+ EMC numbers were detected in jag1a morphants, jag1b mutants, jag2a mutants and dlc morphants, but not jag2b mutants. Reduced numbers of pvalb8-positive epidermal cells were also observed in mib or jag2a mutants and jag1a or jag1b morphants, while increased pvalb8-positive epidermal cell numbers were detected in notch1a-overexpressing, but not dlc-overexpressing embryos. BrdU labeling further revealed that the agr2+ EMC population is maintained by proliferation. Cell lineage experiments showed that agr2+ EMCs are derived from the same ectodermal precursors as keratinocytes or ionocytes. Together, our results indicate that specification of agr2+ EMCs in zebrafish embryos is induced by DeltaC/Jagged-dependent activation of Notch1a/3 signaling, and the cell population is maintained by proliferation.

Jag2b‐Notch3/1b‐mediated neuron‐to‐glia crosstalk controls retinal gliogenesis

“…DeltaB interacts with Notch3 to maintain MG quiescence, thereby acting as a negative regulator of neuron regeneration in the light‐damaged zebrafish retina (Campbell et al , 2021). DeltaC interacts with Notch1a/3 receptors on adjacent epidermal cells to prevent deltaC ( dlc ) expression from those cells, resulting in generating keratinocytes (Lu et al , 2021). Interestingly, previous studies also indicated individual ligands could mediate unique functions not entirely due to the receptor they activate (D'Souza et al , 2008; Choi et al , 2009).…”

Esrrγa regulates nephron and ciliary development by controlling prostaglandin synthesis

“…Next, we designed genetic interaction studies to explore the relationship between esrrγa and ppargc1a . Suboptimal morpholinos (SOMOs) have been a consistent and well-established strategy to test whether multiple genes operate synergistically in a pathway in multiple species, including zebrafish and Xenopus laevis ( Chambers et al, 2020b ; Choi et al, 2015 ; DiBella et al, 2009 ; Epting et al, 2022 ; Fowler et al, 2021 ; Janssens et al, 2013 ; Kallakuri et al, 2015 ; Lefkopoulos et al, 2020 ; Lu et al, 2021 ; Maerker et al, 2021 ; Tingler et al, 2022 ; Wagle et al, 2011 ). Therefore, we injected SOMO doses of both esrrγa and ppargc1a , based on previously published doses ( Chambers et al, 2018 , 2020b ).…”

“…S2B ) or previously published doses ( Chambers et al, 2018 , 2020b ). Suboptimal doses were determined by using half of the optimal dose, as prescribed by previous studies ( Chambers et al, 2018 , 2020b ; Epting et al, 2022 ; Lefkopoulos et al, 2020 ; Lu et al, 2021 ). esrrγa was targeted with two morpholinos: a start site (ATG) morpholino: 5′-CAATGTGGCGGTCCTTGTTGGACAT-3′ (667 µM optimal), and a splice-blocking morpholino 5′-AGGGTAAAAGCCAACCTTGAATGGT-3′ (400 µM optimal, 200 µM suboptimal).…”

Distinct pathways drive anterior hypoblast specification in the implanting human embryo

“…Following thaw, blastocysts were briefly treated with acidic Tyrode's solution (T1788, Sigma) to remove the zona pellucida and placed in pre-equilibrated human IVC1 in eight-well µ-slide tissue culture plates (80826, Ibidi) in approximately 400 µl volume per embryo per well. Half medium changes were done every 24 h. For small-molecule experiments, human IVC1 was supplemented with either 2 µM A83-01 (72022, STEMCELL Technologies) 80,81 , 25 ng ml −1 Activin-A (Qk001, QKINE) [82][83][84] , 200 nM LDN (S2618, SelleckChem) 85,86 , 50 ng ml −1 BMP6 (SRP3017, Sigma Aldrich) 85,86 , 20 µM DAPT (72082, STEMCELL Technologies) [87][88][89][90] , 10 µM Compound-E (ab142164, Abcam) [91][92][93] , 20 µM MK-0752 (S2660, Selleck Chemicals) [94][95][96] or dimethyl sulfoxide (DMSO) for 48 h. In all cases, these concentrations fall within a range of those used for either vertebrate embryos or complex human ES cell-derived models of the embryo. Within these ranges, a low-to-intermediate concentration was selected to avoid non-specific cytotoxic effects while still considering the higher concentration needed for embryo permeation compared with minimal 2D cell culture to achieve inhibitor action.…”