Cerebral hypoplasia and craniofacial defects in mice lacking heparan sulfateNdst1gene function

Abstract

Materials and methods Targeted recombination of the Ndst1 geneThe thymidine-kinase/neomycin containing targeting vector was constructed by insertion of loxP-sites in intron-sequences surrounding exon 2 (the first coding exon) of Ndst1. The final targeting vector was linearized using SalI before transfection of ES cells. R1 ES cells were grown, transfected and subjected to neomycin G418 selection. Homologous recombinants were identified by Southern blotting and PCR and transfected with a Cre-expressing vector, followed by ganciclovir selection. Four type II recombinants were chosen and injected in C57Bl/6J blastocysts. Two mouse lines, derived from two different ES cell clones, were obtained, and backcrossed into a C57Bl/6 background for more than 10 generations. The primers employed for genotyping were: P1, 5′-CCCAGATGGCGAGACT-GAGG-3′; P2, 5′-CCAGGGCGTCAGGGCCTCCTG-3′; P3, 5′-CATCCTCTGAGGTGACCGC-3′; P4, 5′-GGTACCCGGGGAT-CAATTCG-3′; P5, 5′-CCAGAAGGCTAACACTGTAAAG-3′; P6, 5′-GAAAGTGAAGTCTCTGGGCGG-3′; P7, 5′-GCTTGGATGAT-TTGGTCACACT-3′.Histology and in situ detection of RNA and protein expression Embryos were fixed in 4% paraformaldehyde overnight, dehydrated, embedded in paraffin and sectioned. Sections were stained with Haematoxylin and Eosin for histological analysis. Cartilage and bone were stained with Alcian Blue and Alizarin Red in whole embryos. For whole-mount in situ hybridization a 700 bp riboprobe against the most variable N-terminal region of Ndsts was employed (DIG RNA Labeling Kit, Roche, Mannheim, Germany). Immunohistochemical analysis of Ndst1 expression and western blotting was performed using an anti-Ndst1 antiserum (Grobe and Esko, 2002) followed by detection with goat anti-rabbit HRP-conjugated antibodies (Zymed, San Francisco, USA).Quantitation of apoptosis was performed on paraffin sections of three mutant and three wild-type E15.5 embryos, using the TUNEL Assay Kit (Roche, Mannheim, Germany). BrdU-labelled nuclei were quantified using anti-BrdU antibodies (Zymed) on three mutant and wild-type E15.5 and E17.5 embryos. BrdU (70 μg/g mouse) was injected intraperitoneally and mothers were sacrificed after 1 hour. The number of BrdU-labelled cells relative to nonlabelled cells in the VZ was determined. Two different horizontal levels of the embryonic brains, 250 μm apart, were assessed in each embryo. Eight serial sections per level, each of three wild type and three mutant E17.5 embryos, were analyzed in anterior, posterior, median and lateral forebrain positions.Patched expression was detected using anti-Ptch1 antiserum (Acris Antibodies, Hiddenhausen, Germany) and secondary FITC-labelled goat anti rabbit antibodies (Dianova, Hamburg, Germany) on three mutant and wild-type embryos. Images were taken on a Zeiss Axiophot microscope employing a 10ϫ/0.3, a 20ϫ/0.5 and a 63ϫ/1.25 Zeiss objective, and a Leica DFC280 camera. Leica software was used for image capturing and Photoshop 7 software run on Macintosh computers for the generation of figures. Contrast and brightness were adjusted for whole images ...

Heparan sulfate Ndst1 gene function variably regulates multiple signaling pathways during mouse 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 Ϫ/Ϫ mesenchyme as well as strongly reduced MAPK activity after Fgf-2 stimulation of Ndst1 Ϫ/Ϫ 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).…”

“…Also, as we and others (Ledin et al, 2004;Grobe et al, 2005) found that (undersulfated) HS was still being made in Ndst1 mutant embryos, we conclude that Ndst1 is responsible for the generation of specific HS structures recognized by the monoclonal antibody 10E4 that are not synthesized by other Ndst isoforms.…”

“…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.…”

“…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).…”

“…Our previous studies showed that mitogen-activated protein kinase (MAPK) signaling was strongly impaired in fibroblasts derived from Ndst1 Ϫ/Ϫ embryos (Grobe et al, 2005), suggesting impaired Fgf-dependent proliferation as an underlying reason for the observed craniofacial defects. Indeed, the number of bromodeoxyuridine (BrdU) -labeled nuclei relative to total nuclei was found to be reduced to 53% Ϯ 7% (P Ͻ 0.001; n ϭ 5) in E17.5 mutant nasal epithelium and skin, confirming reduced cell proliferation in dysmorphic mutant heads (arrows, Fig.…”