Developmental Expression Pattern N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase expression during early mouse embryonic development ANA-MARISA SALGUEIRO 1,2,# , MÁRIO FILIPE 1,# and JOSÉ-ANTÓNIO BELO 1,2, * 1 Instituto Gulbenkian de Ciência, Oeiras and 2 Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, Faro Portugal ABSTRACT N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) is an enzyme which is known to help build up the GlcAβ1-3GalNAc(4,6-bisSO 4 ) unit of chondroitin sulfate E (CS- E). This enzymatic activity has been reported in squid cartilage and in human serum, but has never been reported as an enzyme required during early mouse development. On the other hand, CS- E has been shown to bind with strong affinity to Midkine (MK). The latter is a heparin-binding growth factor which has been found to play important regulatory roles in differentiation and morphogenesis during mouse embryonic development. We have analyzed the expression pattern of the GalNAc4S-6ST gene during early mouse embryonic development by whole mount in situ hybridization. The results show that GalNAc4S-6ST is differentially expressed in the anterior visceral ectoderm at stage E5.5 and later becomes restricted to the embryonic endoderm, especially in the prospective midgut region. During the turning process, expression of GalNAc4S- 6ST gene is detected in the forebrain, branchial arches, across the gut tube (hindgut, midgut and foregut diverticulum), in the vitelline veins and artery and in the splanchnopleure layer. These results open the possibility of a role for GalNAc4S-6ST during early mouse development. KEY WORDS: mouse development, AVE, endoderm development, gut tube N- Acetylgalactosamine 4-sulfate 6 -O- sulfotransferase (GalNAc4S-6ST; EC 2.8.2.33) is a Golgi-resident enzyme that transfers sulfate from 3´-phosphoadenosine 5´-phosphosulfate (PAPS) to the C-6 hydroxyl group of N-acetylgalactosamine 4- sulfate. N-acetylgalactosamine 4,6-bissulfate is a component sugar residue of the repeating disaccharide unit, GlcAβ1- 3GalNAc(4,6-bisSO 4 ), in chondroitin sulfate E (CS-E) (Suziki et al., 1968). CS-E was found in various mammalian cells as well as in squid cartilage (Habuchi et al., 1971) and shows some activity in mast cell maturation (Eliakim et al., 1986), regulation of procoagulant activity of monocytes (McGee et al., 1995), promo- tion of neurite outgrowth (Clement et al., 1999), neural adhesion through binding to Midkine (Ueoka et al., 2000) and enhancement of plasminogen activation (Sakaia et al., 2000). Interestingly, CS- E has been shown to bind with strong affinity to Midkine (MK) (Zou et al., 2003), that together with heparin-binding growth-associ- ated molecule (HB-GAM or pleiotrophin) forms a family of hep- arin-binding growth factors. These two proteins can be found during mouse embryonic development playing regulatory roles in differentiation and morphogenenetic processes (Mitsiadis et al., 1995). It has also been shown that the nucleotide sequence of human Int. J. Dev. Biol. 50: 705-708 (2006) doi: 10.1387/ijdb.062168as *Address correspondence to: Dr. José A. Belo. Centro de Biomedicina Molecular e Estrutural, FERN, Campus de Gambelas, Universidade do Algarve, 8005- 139, Portugal. Fax: +3512-8981-8419. e-mail: jbelo@ualg.pt # Note: These authors contributed equally to this work Abbreviations used in this paper: ADE, anterior definitive endoderm; AP, anterior-posterior; AVE, anterior visceral endoderm; CS-E, chondroitin sulfate; dpc, days post coitum; FGF, fibroblast growth factor; MK, midkine; RA, retinoic acid. 0214-6282/2006/$25.00 © UBC Press Printed in Spain www.intjdevbiol.com GalNAc4S-6ST is nearly identical to that of human B cell RAG- associated gene (hBRAG), which raises the interesting possibility that GalNAc4S-6ST might be involved in maturation of B cells by regulating the recombination activating gene 1 (RAG1) (Ohtake et al., 2001). RAG1 plays an important role in V(D)J recombination and expression analysis indicates that hBRAG is coordinately expressed with RAG1 in human pro- and pre-B cell lines (Verkoczy et al., 1999). The homology between human and mouse GalNAc4S- 6ST might suggest a similar function. In the present study, we show that the GalNAc4S-6ST gene is expressed in the anterior visceral endoderm (AVE) and is there- fore likely to have an active role during early mouse development. The AVE comprises a population of primitive endoderm cells located at the distal tip of the 5.5 dpc (days post coitum) embryo, which undergo differentiation and migration to the prospective anterior region and have been reported to be involved in the establishment of the anterior-posterior (AP) axis (Rossant and