Notch signalling in the paraxial mesoderm is most sensitive to reduced Pofut1 levels during early mouse development

Abstract

Background: The evolutionarily conserved Notch signalling pathway regulates multiple developmental processes in a wide variety of organisms. One critical posttranslational modification of Notch for its function in vivo is the addition of O-linked fucose residues by protein O-fucosyltransferase 1 (POFUT1). In addition, POFUT1 acts as a chaperone and is required for Notch trafficking. Mouse embryos lacking POFUT1 function die with a phenotype indicative of global inactivation of Notch signalling. O-linked fucose residues on Notch can serve as substrates for further sugar modification by Fringe (FNG) proteins. Notch modification by Fringe differently affects the ability of ligands to activate Notch receptors in a context-dependent manner indicating a complex modulation of Notch activity by differential glycosylation. Whether the context-dependent effects of Notch receptor glycosylation by FNG reflect different requirements of distinct developmental processes for O-fucosylation by POFUT1 is unclear.

Results: We have identified and characterized a spontaneous mutation in the mouse Pofut1 gene, referred to as "compact axial skeleton" (cax). Cax carries an insertion of an intracisternal A particle retrotransposon into the fourth intron of the Pofut1 gene and represents a hypomorphic Pofut1 allele that reduces transcription and leads to reduced Notch signalling. Cax mutant embryos have somites of variable size, showed partly abnormal Lfng expression and, consistently defective anterior-posterior somite patterning and axial skeleton development but had virtually no defects in several other Notch-regulated early developmental processes outside the paraxial mesoderm that we analyzed.

Conclusion: Notch-dependent processes apparently differ with respect to their requirement for levels of POFUT1. Normal Lfng expression and anterior-posterior somite patterning is highly sensitive to reduced POFUT1 levels in early mammalian embryos, whereas other early Notch-dependent processes such as establishment of left-right asymmetry or neurogenesis are not. Thus, it appears that in the presomitic mesoderm (PSM) Notch signalling is particularly sensitive to POFUT1 levels. Reduced POFUT1 levels might affect Notch trafficking or overall O-fucosylation. Alternatively, reduced O-fucosylation might preferentially affect sites that are substrates for LFNG and thus important for somite formation and patterning.

Figure 1

External phenotype and skeletal and somite defects in cax mutant mice. (A-C) Examples of homozygous mutants demonstrating the variable phenotype in the backcross progeny. (D-G) Skeletal preparations of E15.5 embryos showing that even externally apparently normal mice have skeletal defects (D), and demonstrating various defects such as split vertebrae (asterisks in D, F, G), rib fusions and bifurcations (arrowheads in F), reduced or missing pedicles (arrows in G), and axial truncations (E). White and black boxes in (E) indicate the regions shown enlarged in (F) and (G), respectively. (H, I) Sections of E9.5 cax mutant embryos showing distinct somite borders (indicated by arrowheads) and somites of variable size.

Figure 2

The cax mutation is an allele of Pofut1. (A) Genetic map position of chromosome 2 around the cax mutation based on analysis of 1339 backcross progeny. Markers and genetic distances are indicated below and above the map, respectively. (B) Physical map of the Pofut1 genomic region and location of D2Mit195. (C) Normal external (a) and skeletal (b) phenotype of E15.5 heterozygous cax/+ mice, and shortened body axis (c) and defective axial skeleton (d) in Pofut1^tm1Pst/cax double heterozygous mice. (D) Reduced levels of Pofut1 mRNA in Pofut1^cax/cax mutants (b, d) compared with wild type embryos (a, c) on E9 (a, b) and E9.5 (c, d) detected by in situ hybridization under identical hybridization and staining conditions. Red lines in c and d indicate the PSM and somites.

Figure 3

Pofut1^cax contains an IAP insertion. (A) Map of the region flanking exon5 and PCR-amplified DNA fragments. Mutant DNAs did not produce fragment 4/11 (g, h) but all others (c, d, k, l, o, p) as wild type (wt) (a, b, e, f, i, j, m, n). (B) Map of the 3' region of Pofut1, and Southern blot of wt and mutant DNA hybridized with a cDNA probe containing exon5, 6, and a 5' portion of exon7. Restriction sites and fragments (arrows, labelled by Roman numerals in the scheme and blot) are indicated above and below the map. Asterisks indicate mutant-specific fragments. (C) Long range PCR amplifying fragment 4/11 gave a mutant product (c) larger than wt (a, b). (D) Insertion site map, and junction fragments (1 and 2) amplified with mutant (c, d, g, h) but not wt (a, b, e, f) DNA. Co: water, M: 1 kb ladder. (E) Relative Pofut1 mRNA levels in E10.5 wt (n = 6; white boxes) and mutant embryos (n = 12; gray boxes) determined by exon 6–7 and exon 1–2 amplification. Boxed areas contain 50% of all values. Stippled lines indicate Median expression, whiskers of the boxed areas the total range of values. (F) POFUT1 protein (arrowhead) detected in E9.5 wt embryo extracts was reduced in cax mutants, and absent from Pofut1^{tm1Pst/tm1Pst}. Due to the lower amount of protein in a single growth retarded Pofut1^{tm1Pst/tm1Pst} embryo this lane shows a longer exposure using the non-specific 55 kDa band as an adjustment. Bars indicate molecular weight markers (kDa).

Figure 4

Disturbed A-P somite polarity in Pofut1^cax/cax embryos. (A) WISH of E9.5 embryos with Notch targets. Hes1, Hey1 and HeyL expression is abnormal in the paraxial mesoderm of Pofut1^cax/cax embryos (b, f; n, r; z, zd) compared to wild type (a, e; m, q; y, zc), largely normal in other regions (compare j, v, zh with i, u, zg), and overall severely reduced in Pofut1^{tm1Pst/tm1Pst} embryos (d, h, l; p, t, x; zb, zf, zj). In Pofut1^cax/tm1Pst embryos expression in the paraxial mesoderm is more severely disrupted (c, g; o, s; za, ze), Hey1 and HeyL expression in the branchial bars reduced (w, zi) and Hes1 expression in the optic vesicle apparently unaffected (k). (B) WISH on E9.5 embryos detecting A-P somite polarity. Dll1, Tbx18 and Uncx4.1 show abnormal expression in Pofut1^cax/cax (b, f, j) and Pofut1^cax/tm1Pst (c, g, k) somites compared to wild type (a, e, i). Stripes are weaker (arrows in b), irregularly spaced (arrows in f and j) or blurred with stronger abnormalities in heteroallelic embryos (c, g, k). Pofut1^{tm1Pst/tm1Pst} embryos show increased expression of Dll1 (d) or Uncx4.1 (l) in the neural tube and no segment polarity (h, l). Instead of distinct stripes detected in wild type (m, q, u) Pofut1^cax/cax and Pofut1^cax/tm1Pst mutants exhibit blurry Cer1, Mesp2, and Papc expression (red lines in n, r, v, o, s, w). Pofut1^{tm1Pst/tm1Pst} embryos show weaker and fuzzy expression (p, t, x). Red lines indicate regions of fuzzy gene expression, arrows point to stripes of abnormal expression.

Figure 5

Expression of cyclic Notch targets in the PSM. Hes7 expression in Pofut1^cax/cax mutant E10.5 embryos (d-f) was found in patterns similar to wild type embryos (a-c). In contrast, only 9/33 E9.5 embryos showed Lfng expression patterns (j-l) that could clearly be assigned to the distinct phases of expression seen in wild type embryos (g-i). The remainder showed either expression throughout the psm (m) or in one broad stripe anteriorly (n).

Figure 6

Apparently normal Notch-dependent early developmental processes. Expression of Nodal in Pofut1^cax/cax mutants (B, E) is identical to wild type (A, D) at E8 (A-C) and 8.5 (D-F) indicating undisturbed left-right determination in contrast to Pofut1^{tm1Pst/tm1Pst} embryos (C, F) that lack nodal expression completely. (G-L) Whole mount in situ hybridization of wild type (G, J), Pofut1^cax/cax (H, K), and Pofut1tm1Pst^/tm1Pst (I, L) embryos with NeuroD at E9.5 (G-I) and Neurogenin1 at E9 (J-L) as well as whole mount immunohistochemistry with anti NF160 antibody at E10.5 (S-V) indicates apparently normal neuronal differentiation in Pofut1 ^cax/cax embryos in contrast to Pofut1tm1Pst^/tm1Pst embryos that show upregulation of NeuroD (I) and Neurogenin1 (L) indicative of enhanced neuronal differentiation. (M-R) Whole mount immunohistochemistry with anti-PECAM antibody at E9.5 showing the limb bud region (M-O) and intersomitic vessels (P-R). In Pofut1^cax/cax mutant embryos the vascular network was virtually identical to wild type (N) and showed only minor irregularities in intersomitic vessels (Q), whereas vessels in Pofut1^{tm1Pst/tm1Pst} embryos were severely disorganized (O, R).