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Disruption of heparan sulfate (HS) synthesis in vertebrate development causes malformations that are composites of those caused by mutations of multiple HS binding growth factors and morphogens. We previously reported severe developmental defects of the forebrain and the skull in mutant mice bearing a targeted disruption of the heparan sulfate-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (Ndst1). Here, we further characterize the molecular mechanisms leading to frontonasal dysplasia in Ndst1 mutant embryos and describe additional malformations, including impaired spinal and cranial neural tube fusion and skeletal abnormalities. Of the numerous proteins that bind HS, we show that impaired fibroblast growth factor, Hedgehog, and Wnt function may contribute to some of these phenotypes. Our findings, therefore, suggest that defects in HS synthesis may contribute to multifactor types of congenital developmental defects in humans, including neural tube defects. Developmental Dynamics 236:556–563, 2007. © 2006 Wiley-Liss, Inc.
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Heparan sulfate (HS) is produced by most mammalian cells as part of membrane and extracellular matrix proteoglycans (Esko and Lindahl, 2001). The chain grows by exostosin (Ext) copolymerization of GlcAβ1,4 and GlcNAcα1,4 and is modified by one or more of the four N-deacetylase/GlcN N-sulfotransferase (Ndst) isozymes; the N-deacetylase activity of Ndsts removes acetyl groups from GlcNAc residues, which are then converted to GlcNS through the N-sulfotransferase activity. Subsequent modifications of the HS chain by O-sulfotransferases and a GlcA C5-epimerase depend on the presence of GlcNS residues, making the Ndsts responsible for the generation of sulfated ligand binding sites in HS (Lindahl et al., 1998). Ndst1 and Ndst2 mRNA are expressed in all embryonic and adult tissues examined, whereas Ndst3 and Ndst4 transcripts are predominantly expressed during embryonic development and in the brain (Aikawa et al., 2001).
Many growth factors and morphogens bind to HS. In some cases, HS proteoglycans are thought to act as coreceptors for these ligands. Studies in Drosophila melanogaster demonstrated that HS is crucial for embryonic development (Perrimon and Bernfield, 2000) and that the fly Ndst ortholog Sulfateless affects signaling mediated by Wingless (Wg), Hedgehog (Hh), and fibroblast growth factor (Fgf; Lin et al., 1999; Lin and Perrimon, 1999; The et al., 1999). As those factors also play critical roles in morphogenesis, growth regulation, and differentiation, defective HS synthesis affects multiple aspects of vertebrate development. Mice deficient in Ext1, Ndst1, 2-Ost, and GlcA C5-epimerase function have defective brain morphogenesis, axon guidance defects, craniofacial defects, renal agenesis, and eye defects due to the simultaneous inhibition of multiple HS binding factors (Bullock et al., 1998; Inatani et al., 2003; Li et al., 2003; McLaughlin et al., 2003; Grobe et al., 2005). Here, we report additional developmental defects of the skeleton and the developing head in Ndst1-deficient embryos resulting from impaired function of Hh, Fgf, and possibly Wnt. Thus, multiple developmental processes and signaling pathways depend on HS, underlining the crucial role of regulated HS synthesis during development.
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HS is known to bind numerous growth factors and morphogens in different tissues during development. Of interest, Ndst1 mutant mice display developmental defects that mainly resemble those found in embryos made deficient for two families of growth factors: the Fgfs and Hhs (Table 1). Previously, we showed genetic interaction between Shh and Ndst1 and reduced Ptc expression in facial Ndst1−/− mesenchyme as well as strongly reduced MAPK activity after Fgf-2 stimulation of Ndst1−/− mesenchymal fibroblasts (Grobe et al., 2005). In this work, we confirmed impaired Fgf function in the developing mutant skull in agreement with the established role of Fgf-8 in skull and facial development (Meyers et al., 1998; Trumpp et al., 1999; Abu-Issa et al., 2002; Frank et al., 2002). Notably, Fgf-8 mutants also share neural and limb defects similar to those found in Ndst1 mutant embryos. Moreover, impaired function of various Fgf proteins during eye development was recently found (Pan et al., 2006), confirming that Ndst1 regulates Fgf signaling. However, the striking finding of unilateral eye loss in Ndst1 mutant embryos cannot be explained by the impaired function of single HS binding factors but instead are likely to result from impaired function of numerous factors involved in the development of the vertebrate skull and eyes.
Table 1. Developmental Defects Displayeda
|Tissue or organ||Phenotype in Ndst1−/− embryos||Similarity to known mutants|
|Neuroectoderm derivatives||Hypoplastic forebrain (100%)||2-Ost (McLaughlin et al., 2003), Ext (Mitchell et al., 2001; Inatani et al., 2003), Fgf-2 (Dono et al., 1998; Raballo et al., 2000)|
|Brain|| || |
| ||Lack of commissures (100%)||Ext (Inatani et al., 2003)|
| ||Eye defects (100%)||2-Ost (Bullock et al., 1998), Epimerase (Li et al., 2003)|
| ||Infrequent exencephaly||Perlecan (Arikawa-Hirasawa et al., 1999)|
| ||Infrequent NTD||n.f.|
|Spinal cord|| || |
|Neural crest derivatives|| || |
| ||Tongue frequently missing||Shh (Jeong et al., 2004)|
|Craniofacial bones||Hypoplasia of NC derived bones (14%)||Shh (Chiang et al., 1996; Jeong et al., 2004)|
|Craniofacial prominences||Hypoplastic maxillae/mandible (100%)||Shh (Jeong et al., 2004), Fgf-8 (Trumpp et al., 1999), Gli2 (Mo et al., 1997)|
|1st arch derivatives||Frequent secondary cleft palate (75%)||Fgf-8 (Trumpp et al., 1999; Abu-Issa et al., 2002; Frank et al., 2002; Macatee et al., 2003; Rice et al., 2004), Perlecan (Arikawa-Hirasawa et al., 1999), Fgf-10/FgfR2b (Rice et al., 2004), Gli2/Gli3 (Mo et al., 1997), TGFβ3 (Taya et al., 1999), Shh (Rice et al., 2004)|
|Mesoderm derivatives|| || |
|Lateral plate mesoderm||Partially split sternum (100%)||n.f.|
|Sclerotome||Vertebrae fusion and delayed ossification (100%)||Perlecan (Arikawa-Hirasawa et al., 1999), Ext (Koziel et al., 2004), Gli2 (Mo et al., 1997)|
|Notochord||Hypoplastic nucleus pulposus (100%)||Gli2 (Mo et al., 1997), Ext (Koziel et al., 2004), Shh (Chiang et al., 1996)|
|Lung||Not functional (100%)||Perlecan (Arikawa-Hirasawa et al., 1999)|
|Limb bud mesenchyme||Infrequent syndactyly||Ext (Koziel et al., 2004)|
| ||Delayed ossification (100%)||Gli2 (Mo et al., 1997), Ext (Koziel et al., 2004)|
In contrast to Fgf and Hh function, canonical Wnt signaling was not found to be significantly changed during facial development in all Ndst1−/− embryos investigated. In total, these results indicate that Ndst1 deficiency impairs numerous soluble, HS-binding protein factors during the formation of the skull, among those, Fgfs and Hhs.
Impaired Hh function may also underlie defects in skeletal development. We hypothesized that undersulfated HS in Ndst1 mutant embryos, by providing limited Ihh binding sites, may influence Ihh signaling in the embryonic spine and limb digits, resulting in delayed mineralization in the osteoblastic layer. This hypothesis was supported by four findings: First, mouse Gli2 mutants (a transcription factor that mediates intracellular hedgehog effects) have cleft palate, delayed ossification of the digits, no or little ossification of vertebrae, and lack of intervertebral discs (Mo et al., 1997), all of which are also commonly found in Ndst1 mutant embryos (Table 1). Second, our previous work showed that binding of a recombinant Shh fusion protein to HS derived from Ndst1 mutant embryos is reduced (Grobe et al., 2005), indicating direct hedgehog–HS interaction. Third, mouse embryos made deficient in Ndst2 or Ndst3 function did not show delayed ossification (not shown). Lastly, Ndst1 is the predominant Ndst expressed in multipotent C3H10T1/2 mouse mesenchymal precursor cells, which, under the induction of Hhs, differentiate into AP-producing osteoblasts (Kinto et al., 1997). The importance of Ndst1 in Hh-induced C3H10T1/2 differentiation was shown by siRNAi, and the general importance of HS in that system was confirmed by chlorate treatment or addition of heparin, which resulted in undersulfated cell surface HS (Safaiyan et al., 1999) or competing Hh binding sites in the medium, respectively, resulting in reduced availability of soluble Hh to cell surface HS. Because Ndst1 mutant embryos display some but not all defects seen in Shh mutant embryos (Grobe et al., 2005), Ndst1 does not seem to be always necessary for Hh signaling, but becomes crucial in the absence of other, possibly compensating Ndsts. In contrast, strong expression of all Ndst isoforms in other systems may allow for sufficient HS sulfation in the absence of Ndst1. Consistent with this possibility, Ndst1;Ndst2 and Ndst1;Ndst3 double mutants exhibited much more severe phenotypes than either single mutant (Grobe et al., 2002; and unpublished observations). However, lack of unique HS epitopes generated by Ndst1 (as shown by general loss of 10E4 reactivity in the Ndst1 mutant embryo at all stages) indicates that compensation for Ndst1 activity may only be partial.
Ndst1 mutant mice also have several informative low-frequency malformations, including cleft formation of the primary and secondary palate and median clefts. Cleft lip and cleft palate are common multifactor birth defects. Notably, among those HS binding factors, their receptors and transcription factors, deficient function of members of the Fgf- and Hh families often results in cleft formation that can also be observed in HS-deficient embryos (Frank et al., 2002; Rice et al., 2004; reviewed in Jiang et al., 2006). Mild posterior cleft deformities could be detected in 75% of all Ndst1 mutant mice investigated and 3% of Ndst1 mutant mice also display median cleft lip, a rare abnormality in humans related to defective Shh signaling. A second group of low-frequency midline defects found in Ndst1 mutants are neural tube closure defects (NTDs). Although NTDs are the second most common human birth defect, with an incidence of approximately 1/1,000 live births, the underlying genetic basis of NTDs is poorly understood. More than 90 mutations in a variety of genes have been identified and linked to rodent NTDs, mostly demonstrating variable low penetrance and complex inheritance patterns. NTDs originate in impaired rising of elevation zones that depends on the highly conserved Wnt/frizzled signal transduction pathway (for a review, see Copp et al., 2003). HS is known to bind to Wnt/Wg (Ai et al., 2003), and notably, reduced Wnt signaling was observed in the presumptive midbrain, hindbrain, and spinal cord of 30% of the Ndst1−/− embryos. This finding indicates that NTDs in Ndst1-deficient embryos may (partially) be based on impaired Wnt signaling. Additionally, Shh mutations as well as mutations for the hedgehog-dependent transcription factor Gli3 have been described to be associated with the NTD exencephaly (anencephaly; Harris and Juriloff, 1997). However, due to their low penetrance, molecular mechanisms leading to clefting and NTDs were not assessed in more detail.
Taken together, we conclude that Ndst1 plays a significant role in the synthesis of HS necessary for neural tube closure, fusion of the primary and secondary palate, bone formation, and development of the face/skull. However, incomplete penetrance and strong variability for some of these developmental defects is observed, possibly because developmental processes in the Ndst1 mutant mouse may be (partially) rescued by the expression of compensatory Ndst isoforms. Among the multiple heparan sulfate-binding factors potentially affected, impaired Fgf signaling could be demonstrated in the developing face, impaired Hh signaling in osteoblast differentiation and, in approximately 30% of Ndst1 mutant embryos, also reduced Wnt signaling in the presumptive mid/hindbrain area.