Canopy1, a positive feedback regulator of FGF signaling, controls progenitor cell clustering during Kupffer's vesicle organogenesis

Abstract

The assembly of progenitor cells is a crucial step for organ formation during vertebrate development. Kupffer's vesicle (KV), a key organ required for the left-right asymmetric body plan in zebrafish, is generated from a cluster of ∼20 dorsal forerunner cells (DFCs). Although several genes are known to be involved in KV formation, how DFC clustering is regulated and how cluster formation then contributes to KV formation remain unclear. Here we show that positive feedback regulation of FGF signaling by Canopy1 (Cnpy1) controls DFC clustering. Cnpy1 positively regulates FGF signals within DFCs, which in turn promote Cadherin1-mediated cell adhesion between adjacent DFCs to sustain cell cluster formation. When this FGF positive feedback loop is disrupted, the DFC cluster fails to form, eventually leading to KV malformation and defects in the establishment of laterality. Our results therefore uncover both a previously unidentified role of FGF signaling during vertebrate organogenesis and a regulatory mechanism underlying cell cluster formation, which is an indispensable step for formation of a functional KV and establishment of the left-right asymmetric body plan.left-right patterning | ciliogenesis F ibroblast growth factor (FGF) signaling plays crucial roles in multiple morphogenetic processes of vertebrate development, including gastrulation movement, mesoderm formation, and leftright (LR) patterning (1-3). Because gain or loss of function of FGF signaling results in morphological changes in the embryo, some mechanism must ensure appropriate FGF signal levels in space and time for proper morphogenesis throughout development. FGF effectors acting as positive or negative regulators show a wide range of expression patterns and activities, contributing to the precise regulation of FGF signal activity (1, 4). Although most effectors identified to date act as negative regulators of FGF signaling, a few that positively regulate FGF activity have been reported (1, 4).We recently identified in zebrafish a positive regulator of FGF signaling named canopy1 (cnpy1), which is required for maintenance of the midbrain-hindbrain boundary (MHB) (5). Expression of cnpy1 was restricted to the MHB at late-somitogenesis stages, whereas cnpy1 was broadly distributed in earlier embryos (5) (SI Appendix, Fig. S1A), suggesting an additional role(s) for Cnpy1-mediated FGF signaling beyond the regulation of MHB formation. In this study, we characterize cnpy1 in detail during early zebrafish development and show that a Cnpy1-mediated positive feedback loop of FGF signaling promotes cell cluster formation between dorsal forerunner cells (DFCs) during gastrulation. We also demonstrate that the failure of DFCs to cluster when this FGF positive loop is disrupted eventually leads to Kupffer's vesicle (KV) malformation and randomization of LR asymmetric patterning. Results Positive Feedback Loop of FGF Signaling Mediated by Cnpy1 IsActivated Specifically in DFCs During Zebrafish Gastrulation. To reveal the role of Cnpy1-mediated FGF signaling in ...

Celf1 regulation of dmrt2a is required for somite symmetry and left-right patterning during zebrafish development

“…Whole-mount in situ hybridization was performed as described previously (Matsui et al, 2005;Matsui et al, 2011). cDNA fragments of celf1, dlx3, dmrt2a, her1, hgg1 (ctsl1b -Zebrafish Information Network), mlc2a (mgl7 -Zebrafish Information Nework), myod, ntl, pitx2a and uncx4.1 were used as templates for the antisense probes.…”

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

“…Of course, some genes may function in both the DFC/KV cells and the yolk 16 . Although we focus of MO-mediated loss-offunction analyses here, the DFC-targeted injection technique and associated control experiments can be used to assess a gain-of-function by injecting synthetic mRNA to overexpress a particular protein in the DFC/KV lineage 17,22,24,29 . One clever adaption of this approach used DFC-targeted mRNA injections to rescue global MO knockdown only in DFC/KV cells 30 .…”

Retinoic acid controls proper head-to-trunk linkage in zebrafish by regulating an anteroposterior somitogenetic rate difference

“…Single-or double-color whole-mount in situ hybridization was performed as described previously (Matsui et al, 2005;Matsui et al, 2011). cDNA fragments of fgf8a, her1, mespb, papc, ripply1, tbx24 (tbx6 -Zebrafish Information Network), uncx4.1 and Venus were used as templates for antisense probes.…”

“…The following antisense MO oligonucleotides against raldh2, deltad (deltaD -Zebrafish Information Network), fgf8a and a control MO were obtained from Gene Tools: control MO: 5′-CCTCTTACCTCAGTTACAATTTATA-3′; deltad MO: 5′-AACAGCTATCATTAGTCGTCCCATG-3′ (Kawamura et al, 2008) (a gift from Dr Shinji Takada); fgf8a MO: 5′-GAGTCTCATGTTTATAGCCTCAGTA-3′ (Matsui et al, 2011); raldh2 MO: 5′-GTTCAACTTCACTGGAGGTCATC-3′ (Begemann et al, 2001;Kawakami et al, 2005).…”