Nomo (pM5) is a major binding partner of Nicalin. (A) Myc-tagged Nicalin was immunoprecipitated from Sw/Ncl cell membrane preparations and the bound proteins were visualized by Coomassie staining. Bands of molecular weight 60 and 130 kDa were specifically enriched in precipitates from Sw/Ncl cells. Peptide mass fingerprints generated by MALDI analysis identified these bands as Nicalin and pM5, which we refer to as Nomo (bands denoted with ^* represent immunoglobulin heavy and light chains). (B) FLAG-tagged Nomo was transfected into Sw/Ncl cells and immunoprecipitated. Western blot analysis of bound (b) and unbound (s) material revealed co-precipitation of Nicalin in Nomo-overexpressing cells (+), but not in control cells (−). (C) Northern blot analysis using full-length Nicalin and Nomo probes and 2 μg each of mRNA from various human tissues. Molecular weight markers in kilobases are shown on the left.

Coexpression of ncl1 and nomo impairs forebrain patterning leading to cyclopia. 36 hpf morphology of wild-type embryos (A, A′) and embryos coinjected with ncl1 and nomo capped RNAs (150 ng/μl each) at the one-cell stage (B, B′). Note the prominent cyclopia of the injected embryo (arrows).

Nomo antagonizes Nodal signaling. Expression of the anterior mesendodermal marker hgg1 in embryos injected with 1 ng capped lefty RNA (A) or with lefty RNA and nomo-grip (B). Activation of Nodal signaling by Lefty reduces the number of hgg1-positive cells dramatically (compare to Figure 5E). Coinjection of the nomo-grip rescues this effect. (C–F) Model of the effects of lefty and nomo on Nodal signaling. In wild-type embryos, increasing levels of Nodal account for the generation of mesendodermal derivatives of progressively more anterior character (ppcp and hg) (C). Their formation is enhanced upon downregulation of nomo, indicating upregulated Nodal signaling (D). The endoderm, which requires highest Nodal activity, is not affected (not represented). Ectopic expression of lefty leads to inhibition of Nodal signaling and the formation of primarily posterior structures (n) (E). This effect can be reversed by simultaneously downregulating nomo (F). hg: presumptive hatching gland; n: notochord; ppcp: posterior part of the prechordal plate.

Nicalin is distantly related to Nicastrin and part of a high-molecular-weight complex unrelated to γ-secretase. (A) Phylogenetic tree showing that the Nicalins are more closely related to Nicastrins than to transferrin receptors and active aminopeptidases. NAALADL: N-acetyl-alpha-linked acidic dipeptidase-like protein; PSMA: prostate-specific membrane antigen; TFR: transferrin receptor; NCL: Nicalin; NCT: Nicastrin; HS: Homo sapiens; RN: Rattus novegicus; MM: Mus musculus; DM: Drosophila melanogaster; CE: C. elegans; AT: Arabidopsis thaliana. (B) Western blot of postnuclear (pns) and membrane (mem) protein fractions of cells expressing endogenous Nicalin (Sw and SH-SY5Y) or myc-tagged Nicalin (Sw/Ncl). Immunodetection with α-myc or α-Nicalin antibodies reveals a 60 kDa protein enriched in membrane fractions. (C) Immunoblot of Blue-Native gels of Sw and Sw/Ncl cell membrane protein extracts. Nicalin immunoreactivity is seen in a high-molecular-weight complex with a size similar to γ-secretase, which is visualized using an α-Presenilin 1 antibody (α-PS1). (D) Nicastrin and Nicalin were immunoprecipitated from Sw/Ncl cells and the bound material was analyzed by Western blotting. In Nicastrin immunoprecipitates (IP Nct), Presenilin N-terminal fragments (PS1-NT) and APH-1a were present, but Nicalin could not be detected. In Nicalin immunoprecipitates (IP Ncl), neither Nicastrin nor PS1-NT or APH-1a could be detected (band denoted with ^* is unspecific).

Zebrafish ncl1 and nomo are expressed in largely overlapping domains at somitogenesis stages. Expression of ncl1 (A–C) and nomo (D–F) revealed by whole-mount in situ hybridization (blue staining) at the stages indicated (anterior left, dorsal up). ncl1 expression is ubiquitous, with higher levels in the presumptive tectum (t), retina (r) and somites (s) (white arrows) and in the endoderm (black arrowhead) at 20 somites. nomo is transcribed at lower levels during earlier stages, with the most prominent expression in the anterior mesendoderm (black arrows) and endoderm (black arrowhead). At the 20-somite stage, nomo is expressed in a pattern very similar to ncl1.

Nomo activity controls the extent of anterior mesendoderm. (A–B′) Morphology at 24 hpf of wild-type embryos (A, A′) and embryos injected with an antisense gripNA oligonucleotide blocking translation of endogenous nomo (nomo-grip) (B, B′). All embryos are anterior left, dorsal up; (A′) and (B′) are higher magnifications of the territories boxed in (A) and (B), respectively. Blocking Nomo activity leads to the formation of enlarged hatching glands, the most anterior mesendodermal derivative (arrows). (C–J) Expression of the markers indicated (bottom right of each panel) at the end of gastrulation (tail-bud stage) in wild-type (C, E, G, I) or nomo-grip-injected embryos (D, F, H, J). All panels are dorsal views, anterior left. The expression of anterior mesendodermal markers (gsc: presumptive hatching gland+prechordal plate; hgg1: presumptive hatching gland; hlx1: prechordal plate) is enlarged in injected embryos (C–H, arrows to the enlarged domains), while znot expression in the anterior notochordal domain is absent (I, J, arrows). (K, L) The number of hatching gland precursors, labeled with hgg1, is restored when nomo-grip is coinjected with a capped RNA encoding Nomo (nomo^*) (L, compare with K and E, F). (M, N) Expression of the endodermal marker sox17 is similar in wild-type (M) and nomo-grip-injected embryos (N).

Nicalin and Nomo inhibit Nodal- and Activin-induced signaling in 293T cells. Transient transfection assays were performed with the A3-lux luciferase reporter plasmid containing three tandem copies of a Nodal-responsive element. Luciferase activity is shown in arbitrary units with each bar representing four measurements from two independent transfections. (A) Strong induction of luciferase activity was measured in cells transfected with expression plasmids encoding Nodal, Cripto and FAST2. Adding increasing amounts of plasmids encoding Nicalin or Nomo led to a dose-dependent reduction of the luciferase signal, which corresponded to Nicalin and Nomo protein levels as demonstrated by immunoblotting. Transfection of Nicastrin (Nct) had no effect. (B, C) Luciferase activity was induced by transfection of FAST2 and treatment of the cells with 20 ng/ml recombinant, purified Activin for 18–22 h. Again, Nicalin and Nomo cause a dose-dependent reduction of luciferase activity (B). The reduction of Activin-induced luciferase activity is enhanced in cells transfected with both Nicalin and Nomo, suggesting a synergistic effect (C).