Analysis of Fz10 expression in mouse embryos (original) (raw)

Abstract

Wnt signaling molecules regulate the development of multiple organs in vertebrate embryos. We have isolated cDNA clones for frizzled10 (Fz10), which encodes a putative Wnt receptor, to further characterize the mechanisms of Wnt signaling in mouse embryos. Interestingly, Fz10 is expressed in the same regions as Wnt7a in the neural tube, limb buds, and Müllerian duct.

Similar content being viewed by others

Wnt genes encode secreted signaling molecules that regulate many developmental events in a variety of multicellular organisms (Wodarz and Nusse 1998). For example, mouse Wnt gene products play roles in such diverse processes as primary axis determination, neural tube patterning, kidney formation, and sex determination (McMahon and Bradley 1990; Thomas and Capecchi 1990; Stark et al. 1994; Liu et al. 1999; Vainio et al. 1999). We are interested in the Wnt7a gene product, which is required for normal development of the limbs (Parr and McMahon 1995; Yang and Niswander 1995), cerebellum (Hall et al. 2000), and reproductive tract (Miller and Sassoon 1998; Parr and McMahon 1998).

The identification of a Wnt7a receptor would help decipher the mechanisms of Wnt7a signaling. At least two polypeptides are required to receive Wnt signals. Members of the low-density lipoprotein receptor-related (LRP) family appear to function as broadly expressed Wnt co-receptors (Pinson et al. 2000; Tamai et al. 2000; Mao et al. 2001). Interestingly, a subset of defects observed in LRP6 mutant mice resembles the Wnt7a mutant phenotype (Pinson et al. 2000). However, it seems that much of the specificity of Wnt signaling arises from the binding of Wnt proteins to the seven transmembrane frizzled receptors (Bhanot et al. 1996; Wang et al. 1996). Recently, Nohno and colleagues have identified the chicken frizzled10 (Fz10) gene product as a possible Wnt7a receptor (Kawakami et al. 2000a, 2000b). They demonstrated that Wnt7a and Fz10 share similar expression patterns in chick embryos and that the two gene products synergistically induce the expression of Wnt-responsive genes in a Xenopus injection assay. We have cloned and analyzed the expression of the mouse orthologue of Fz10 to gain possible insights into Wnt7a signaling in a species where Wnt7a mutant embryos are readily available.

We isolated mouse Fz10 cDNA clones that are predicted to encode a protein of 582 amino acids (Fig. 1). The mouse Fz10 sequence is very similar to chick Fz10 (85.1% amino acid identity) and to a recently described human Fz10 sequence (93.5% amino acid identity). Thus, our clones likely encode the orthologue of these chick and human proteins.

Fig. 1

Fig. 1

The alternative text for this image may have been generated using AI.

Full size image

Comparison of the predicted amino acid sequences for human, chicken, and mouse Fz10 (accession number AY509002). Shaded residues are identical in all three species. We identified an EST clone as a putative mouse Fz10 cDNA based on sequence similarity to the chicken Fz10 gene product. The 5′ end of the Fz10 cDNA was isolated by the rapid amplification of cDNA ends (RACE) procedure (Frohman et al. 1988)

We conducted in situ hybridization experiments to visualize Fz10 mRNA expression during mouse development. During initial stages of organogenesis in the mouse embryo, strong Fz10 expression is detected in the limbs, neural tube, and Müllerian duct. Interestingly, the three main areas of Wnt7a mRNA expression at these stages are the dorsal ectoderm of the limb, the Müllerian duct epithelium, and the ventro-medial neural tube (Parr et al. 1993; Parr and McMahon 1998).

We initially detect Fz10 expression in the mouse embryo at embryonic day 8.5 (E8.5). From E9.0 to E11.5, Fz10 expression is detected primarily in the neural tube and limbs (Fig. 2). At E9.0–E9.5, Fz10 mRNA is present in a continuous domain along the length of the neural tube (Fig. 2A, B). Fz10 expression is not evident in the telencephalon, but can be detected in the diencephalon, midbrain, hindbrain, and spinal cord. The expression pattern in the neural tube remains similar throughout E11.5, although the intensity of staining is greatly decreased by E11.5 (data not shown). In situ hybridizations of transverse sections through embryos reveal that Fz10 RNA is localized to the dorsal half of the neural tube, including the roof plate (Fig. 2C). In contrast, Wnt7a RNA is located primarily in the ventral and medial portions of the neural tube at this time (Fig. 2D; also see Parr et al. 1993). Thus, the domains of Wnt7a and Fz10 expression intersect only in the medial region of the neural tube. It should be noted that other Wnt genes, such as Wnt1 and Wnt3a, also are expressed in the dorsal neural tube at this time (Parr et al. 1993).

Fig. 2A–G

Fig. 2A–G

The alternative text for this image may have been generated using AI.

Full size image

Frizzled10 (Fz10) mRNA expression from E9.0 through to E11.5. At E9.0 (A), Fz10 is expressed along the neural tube (arrowheads), with a rostral limit in the anterior diencephalon (arrow). At later stages (B), Fz10 mRNA continues to be expressed in the neural tube (arrowheads) and is also detected in the limb buds (arrows). Cross-sections through E10.5 embryos demonstrate that Fz10 is expressed in the dorsal half of the neural tube (C), while Wnt7a is expressed in the ventral half of the neural tube (D). The limits of strong Fz10 and Wnt7a expression in C and D are denoted by arrows. Fz10 also is expressed in the limb buds from E 9.5 (E) through to E11.5 (F). Weak Fz10 expression in the somites (arrow) is detected at E11.5 (G)

As the forelimb bud begins to emerge from the flank of the embryo at E9.5, Fz10 mRNA is detected in the ectoderm and mesenchyme immediately underlying the ectoderm throughout most of the limb bud (Fig. 2E). At E10.5–E11.5, Fz10 RNA is detected in the distal half of the limb bud and is confined to the lateral edge of the limb bud in both the ectoderm and mesenchyme (Fig. 2F, section in situ data not shown). In contrast to Wnt7a, Fz10 does not exhibit a dorsally restricted expression pattern in the limb bud at this stage. Fz10 expression in the limb buds of Wnt7a mutant embryos at E10.5 and E11.5 appears identical to Fz10 expression in wild-type embryos (data not shown). Kawakami et al. (2000b) observed that ectopic Wnt7a could induce Fz10 expression in chick limbs, suggesting that Fz10 gene expression might be regulated by the Wnt7a signaling pathway itself. Our results indicate that Fz10 expression patterns remain largely unaltered in Wnt7a mutant mice. Outside of the limb buds and neural tube, we can detect weak Fz10 expression in the somites by E11.5 (Fig. 2G).

In the reproductive tract, Fz10 expression is detected in the Müllerian duct in both male and female embryos beginning at E12.5 (Fig. 3). Fz10 expression in the Müllerian duct is restricted to the anterior half of the duct (nearest to the gonad) when it is first detected at E12.5 (Fig. 3A), but spreads along the entire length of the duct by E13.5. Sections in situ demonstrate that Fz10 is expressed in the Müllerian duct epithelium (Fig. 3B). Expression of Fz10 in the Müllerian duct occurs at comparable levels in male embryos and female embryos at E13.5 (data not shown). Subsequently, Fz10 is expressed at high levels in wild-type and Wnt7a mutant Müllerian ducts in female embryos at E14.5 (Fig. 3C, D). In males, Fz10 is expressed at high levels in Wnt7a mutant embryos, but at substantially decreased levels in wild-type embryos at E14.5 (Fig. 3E, F). Decreased Fz10 expression in wild-type male embryos at E14.5 is likely a consequence of the initial events of Müllerian duct regression. Müllerian duct regression is blocked in Wnt7a mutant embryos (Parr and McMahon 1998), so that Fz10 expression is maintained at high levels in both male and female embryos. It is interesting that Fz10 expression occurs in a gradient in the Müllerian ducts of wild-type males at E14.5, with little expression detected in the anterior duct adjacent to the gonad and strongest expression in the posterior duct furthest from the gonad (Fig. 3E). Fz10 expression in the Müllerian duct epithelium should provide a useful marker to monitor the early steps of Müllerian duct regression in male embryos.

Fig. 3A–F

Fig. 3A–F

The alternative text for this image may have been generated using AI.

Full size image

Fz10 mRNA expression in the Müllerian duct. The gonads and adjacent regions of the reproductive tract are displayed. Fz10 mRNA can be detected in the anterior part of the Müllerian duct (between the arrows) at E12.5 (A). At E13.5, cross-sections through the urogenital region reveal that Fz10 is expressed in the ductal epithelium (B). By E14.5, Fz10 expression again appears similar in wild-type (C) and Wnt7a mutant (D) female embryos. However, expression in wild-type E14.5 male embryos (E) is notably decreased compared to male mutant embryos (F) and is much weaker in anterior regions of the duct next to the testis compared to the posterior regions (arrows). md Müllerian duct, g gonad

If Fz10 functions as a Wnt7a receptor in Müllerian duct, Fz10 expression in the ductal epithelium predicts a complex model for Wnt7a regulation of Müllerian duct regression. Our previous results demonstrated that a Wnt7a signal from the ductal epithelium is required for expression of the MIS type II receptor in the adjacent mesenchyme cells (Parr and McMahon 1998). Since Fz10 is expressed in the epithelium, Wnt7a would have to signal between adjacent cells within the epithelium and/or function in an autocrine fashion. In this case, the effect of Wnt7a on MIS receptor expression would be indirect and require an additional signal from the epithelium to the mesenchyme.

References

Download references

Acknowledgements

We thank Kari McDonald for help with the initial characterization of the Fz10 clone. A.P.N. was supported by N.I.H. training grant GM07135 to the MCD Biology Department at the University of Colorado.

Author information

Authors and Affiliations

  1. MCD Biology, University of Colorado, 347 UCB, Boulder, CO 80309-0347, USA
    Amy P. Nunnally & Brian A. Parr

Authors

  1. Amy P. Nunnally
  2. Brian A. Parr

Corresponding author

Correspondence toBrian A. Parr.

Additional information

Edited by R.P. Elinson

Rights and permissions

About this article

Cite this article

Nunnally, A.P., Parr, B.A. Analysis of Fz10 expression in mouse embryos.Dev Genes Evol 214, 144–148 (2004). https://doi.org/10.1007/s00427-004-0386-4

Download citation

Keywords