Pleiotropic and diverse expression of ZFHX1B gene transcripts during mouse and human development supports the various clinical manifestations of the “Mowat–Wilson” syndrome

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Abstract

ZFHX1B encodes Smad-interacting protein 1, a transcriptional corepressor involved in the transforming growth factors β (TGFβ) signaling pathway. ZFHX1B mutations cause a complex developmental phenotype characterized by severe mental retardation (MR) and multiple congenital defects. We compared the distribution of ZFHX1B transcripts during mouse and human embryogenesis as well as in adult mice and humans. This showed that this gene is strongly transcribed at an early stage in the developing peripheral and central nervous systems of both mice and humans, in all neuronal regions of the brains of 25-week human fetuses and adult mice, and at varying levels in numerous nonneural tissues. Northern blot analysis suggested that ZFHX1B undergoes tissue-specific alternative splicing in both species. These results strongly suggest that ZFHX1B determines the transcriptional levels of target genes in various tissues through the combinatorial interactions of its isoforms with different Smad proteins. Thus, as well as causing neural defects, ZFHX1B mutations may also cause other malformations.

Introduction

Transforming growth factors β (TGFβ) form a superfamily of signaling molecules that act on membrane receptors with a cytoplasmic serine/threonine kinase domain. They play numerous roles in many aspects of embryogenesis and control crucial aspects of neural development. For example, activin A and Vg1 are mesoderm inducers in Xenopus embryos. Furthermore, the chick homologue of Vg1 (cVg1) is involved in the induction of Hensen's node in avian embryos (Shah et al., 1997). The bone morphogenetic proteins (BMPs), including BMP2, BMP4, and nodal, which are major members of the TGFβ superfamily, play important roles in cell fate specification and patterning in mammalian embryos. The BMPs are particularly involved in the posteriorization of the mesoderm in vertebrates as well as in the dorsalization of the neural tube and somites (Liem et al., 2000).

The Smad proteins form a family of intracellular effector molecules that act downstream of the TGFβ receptors. The activation of these receptors triggers the phosphorylation of a family of cytoplasmic molecules: R-SMADs (SMAD1, 2, 3, 5, 8). These R-SMADs form heterodimeric complexes with the common SMAD4, resulting in the translocation of the complex into the nucleus of the activated cells.

ZFHX1B, or the Smad-interacting protein-1 (SIP1), is a novel member of the δEF1/Zfh1 family of two-handed zinc-finger/homeodomain transcription factors, which play a major role in the TGFβ signaling pathway. ZFHX1B has been identified as an R-SMAD-binding protein in vitro that acts as a co-transcriptional repressor. Several ZFHX1B target genes have been reported. The overexpression of SIP1 in Xenopus prevents the expression of the endogenous Xbra gene, which codes for a mesodermal marker (Verschueren et al., 1999). Other studies have identified the E-cadherin gene (which codes for a major adhesion molecule involved in developmental processes such as neurulation) as another target gene (Comijn et al., 2001). An early developmental arrest occurring at embryonic day (E) 9.5 was recently reported in homozygous ZFHX1B-deficient mice, whereas heterozygous mice develop normally. This severe phenotype is associated with a major anomaly of neurulation and neural crest migration (Van de Putte et al., 2003).

Mutations in the ZFHX1B gene were independently identified by Wakamatsu et al. (2001) and our group (Cacheux et al., 2001) in patients with a syndromic form of Hirschsprung disease (HSCR) associated with mental retardation (MR), microcephaly, and distinct facial features (HSCR–mental retardation syndrome, MIM 235730). Further reports extended the phenotypic spectrum associated with ZFHX1B mutations, resulting in complex developmental phenotypes with a wide range of clinically heterogeneous congenital abnormalities. Indeed, most affected patients show MR, microcephaly, characteristic facial dysmorphism, epilepsy, corpus callosum agenesis, combined with variable HSCR, cardiac defects, and anomalies of the hair, skeleton, and urogenital system Amiel et al., 2001, Wilson et al., 2003. Other studies highlighted the variable multiple congenital anomalies (MCAs) associated with this developmental disorder (Yamada et al., 2001), and identified MR and the characteristic facial dysmorphism described by Mowat et al. (1998) as being the most prevalent clinical features (Zweier et al., 2002). Thus, the recognizable clinical entity caused by mutations in the ZFHX1B gene prompted these authors to propose the term “Mowat–Wilson” (MWS) syndrome Garavelli et al., 2003, Zweier et al., 2002. All identified mutations are de novo nonsense or frameshift mutations, indicating that they lead to haploinsufficiency (Mowat et al., 2003 for a review). The recent identification of an apparently non-syndromic MR of late infantile-onset in a 48-year-old patient carrying a 3-bp non-frameshift deletion (Yoneda et al., 2002) further supports the hypothesis that ZFHX1B is crucial for normal brain development.

We used northern blotting and in situ hybridization to determine the distribution of ZFHX1B mRNA during human and mouse embryogenesis, and in adult mouse and human brains. This is an important step towards understanding the relationships between the expression of ZFHX1B and the clinical features associated with the syndrome and may help us to unravel the molecular mechanisms underlying this syndrome.

Section snippets

Tissue preparation

Twelve morphologically normal human embryos (aged 20–44 days) were obtained from legal abortions induced by Mifepristone (RU 486). This procedure was approved by the Ethical Committee of the Hôpital Necker-Enfants Malades (Paris). The developmental stage of each embryo was estimated according to the Carnegie classification (O'Rahilly and Müller, 1987) and ranged from stages 10 to 18. We also obtained the brains of three 25-week-old fetuses and the eyes of three 17.5-week-old fetuses. Normal

Northern blot analysis of Zfhx1b transcripts in embryonic and adult mouse tissues

To determine whether the Zfhx1b gene is expressed during mouse embryogenesis, we performed northern blotting using poly(A)+ RNA from whole mouse embryos at different stages of development (Fig. 1A). Zfhx1b mRNA was first detected at 7 days post-coitum (d.p.c.) and could still be detected at 11, 15, and 17 d.p.c. Both M3 and M2 antisense probes recognized four species of Zfhx1b mRNA of different sizes (Table 1A). The 10.5-, 6.6-, 5.2-, and 2.9-kb species were readily detectable by the M3 and the

Discussion

To determine whether the Zfhx1b/ZFHX1B gene is expressed during mouse and human embryogenesis as well as in adult mice and humans, we compared the distribution of Zfhx1b/ZFHX1B transcripts by northern blotting analysis and in situ hybridization, using antisense oligonucleotide probes, in various embryonic, fetal, and adult tissues from both species.

By northern blot analysis, we observed an early Zfhx1b/ZFHX1B expression during mouse embryogenesis that persists throughout mouse development as

Acknowledgements

This work was supported in part by grants from the Ministère de la Recherche, de L'Enseignement et de La Technologie, RETINA-France, Association GENESPOIR, Fondation pour la Recherche Médicale (FRM), and Association Française contre les Myopathies (AFM).

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    These authors contributed equally to the work.

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