The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish

Abstract

We have isolated a novel gene, charon, that encodes a member of the Cerberus/Dan family of secreted factors. In zebrafish, Fugu and flounder, charon is expressed in regions embracing Kupffer's vesicle,which is considered to be the teleost fish equivalent to the region of the mouse definitive node that is required for left-right (L/R) patterning. Misexpression of Charon elicited phenotypes similar to those of mutant embryos defective in Nodal signaling or embryos overexpressing Antivin(Atv)/Lefty1, an inhibitor for Nodal and Activin. Charon also suppressed the dorsalizing activity of all three of the known zebrafish Nodal-related proteins (Cyclops,Squint and Southpaw), indicating that Charon can antagonize Nodal signaling. Because Southpaw functions in the L/R patterning of lateral plate mesoderm and the diencephalon, we asked whether Charon is involved in regulating L/R asymmetry. Inhibition of Charon's function by antisense morpholino oligonucleotides (MOs) led to a loss of L/R polarity, as evidenced by bilateral expression of the left side-specific genes in the lateral plate mesoderm (southpaw, cyclops, atv/lefty1, lefty2 and pitx2) and diencephalon (cyclops, atv/lefty1 and pitx2), and defects in early (heart jogging)and late (heart looping) asymmetric heart development, but did not disturb the notochord development or the atv/lefty1-mediated midline barrier function. MO-mediated inhibition of both Charon and Southpaw led to a reduction in or loss of the expression of the left side-specific genes,suggesting that Southpaw is epistatic to Charon in left-side formation. These data indicate that antagonistic interactions between Charon and Nodal(Southpaw), which take place in regions adjacent to Kupffer's vesicle, play an important role in L/R patterning in zebrafish.

FGF-dependent left–right asymmetry patterning in zebrafish is mediated by Ier2 and Fibp1

“…We reproduced this effect with the aid of an FGF8 MO and showed that knockdown of Ier2 and Fibp1 phenocopies loss of FGF8 in this system. Either of these interventions also led to a loss of cha expression, providing an additional link to the establishment of laterality (39). Most critically, the effect of FGF8 knockdown on ciliogenesis and cha expression could be rescued by supplying Ier2 and Fibp1, indicating that FGF8 action in laterality establishment is mediated by these two factors.…”

“…Double label fluorescence in situ hybridization of ier2 with fgf8 and with charon (cha), a marker for Kupffer's vesicle, shows colocalization of fgf8 and ier2 in Kupffer's vesicle, with cha limited to a horseshoe-shaped caudal region, as reported in ref. 39 (Fig. 6 C and D).…”

Secreted and Transmembrane Wnt Inhibitors and Activators

“…Rather, Cerberus-related proteins are key regulators of Nodal signaling and play an important role in the establishment of left -right asymmetry in the vertebrate embryo (Rodriguez Esteban et al 1999;Yokouchi et al 1999;Zhu et al 1999;Hashimoto et al 2004;Marques et al 2004;Tavares et al 2007;Vonica and Brivanlou 2007).…”

A mutation in the zebrafish Na,K‐ATPase subunit atp1a1a.1 provides genetic evidence that the sodium potassium pump contributes to left‐right asymmetry downstream or in parallel to nodal flow

“…Probes used were preproinsulin, ipf1 (Milewski et al, 1998), glucagon, preprosomatostatin2 (called somatostatin2 in this study) (Argenton et al, 1999), trypsin (Biemar et al, 2001), islet-1 (Korzh et al, 1993), nkx2.2 (Barth and Wilson, 1995;Kudoh et al, 2001), pax6.2 (Nornes et al, 1998), foxa2 (Strahle et al, 1993;Odenthal and Nusslein-Volhard, 1998), sox32 (Dickmeis et al, 2001;Kikuchi et al, 2001), sox17 (Al-exander and Stainier, 1999), foxA2 (Strahle et al, 1993;Odenthal and Nusslein-Volhard, 1998), foxa1 (Odenthal and Nusslein-Volhard, 1998), prox1 (Glasgow and Tomarev, 1998), cha (Hashimoto et al, 2004), lefty1 (Bisgrove et al, 1999;Thisse and Thisse, 1999), spaw (Long et al, 2003), and pitx2 (Essner et al, 2000).…”

“…4, Table 2). charon is a cerberus/ dan family member and inhibits nodal signaling during left-right asymmetry, with the zebrafish Nodal gene southpaw as a target (Hashimoto et al, 2004). charon expression initiates around 2-3 somite stage in the hypoblast of the tailbud.…”