Roles of HNK-1 carbohydrate epitope and its synthetic glucuronyltransferase genes on migration of rat neural crest cells

Abstract

HNK-1 carbohydrate epitope is localized on the surface of avian neural crest cells (NCCs), and is necessary for their migration. However, it is still disputed whether the epitope works in similar ways in mammalian embryos. In this study, we found that HNK-1 carbohydrate epitope was specifically detected in some of the cranial ganglia, migrating trunk NCCs and some non-NCC derivatives in the rat embryo. Two genes encoding glucuronyltransferases that synthesize the HNK-1 epitope in vitro (GlcAT-P and GlcAT-D) were recently identified in the rat. Interestingly, the NCCs in the cranial ganglia expressed the GlcAT-D gene, whereas the migrating trunk NCCs expressed the GlcAT-P gene. To investigate in vivo functions of the GlcATs in the NCC migration further, we overexpressed GlcAT genes by electroporation in the cranial NCCs in cultured rat embryos. Transfection of both GlcAT genes resulted in efficient synthesis of the HNK-1 epitope in the NCCs. GlcAT-P overexpression increased distance of cranial NCC migration, whereas GlcAT-D overexpression did not show this effect. Our data suggest that the HNK-1 epitope synthesized by different GlcATs is involved in migration in the sublineages of the NCCs in the rat embryo, and that GlcAT-P and GlcAT-D mediate different effects on the NCC migration.

Fig. 1

Whole mount immunostaining using HNK-1 monoclonal antibody (A) and in situ hybridization with a probe of the CRABP-I gene (B) in the E11.5 rat embryo. (A) HNK-1 is localized in the trigeminal ganglia (TG) and the migrating trunk NCCs more caudally than somite 15 level (arrowheads). HNK-1 staining is also positive in the eye primordia (E), the midbrain neuroepithelium, the longitudinal fibres (arrows) in the myotome, the limb bud and the heart primordia (H). (B) CRABP-I transcripts are localized in the facial primodia and the NCCs in the trunk (arrowheads). Note that the CRABP-I-positive facial primordia are devoid of the HNK-1 epitope. Scale bar = 1 mm.

Fig. 2

Localization of the HNK-1 epitope and expressions of other NCCs markers and GlcAT genes in the coronal sections of E11.5 rat embryos. (A) The HNK-1 epitope is localized in the trigeminal and vestibulocochlear ganglia (arrowheads), the rhombomeric neuroepithelium and the neurons in rhombomere 7 (r7) (arrows). (B,C) CRABP-I and AP-2 transcripts are positive in the cranial ganglia (arrowheads). CRABP-I expression is additionally detected in the neuroepithelium and r7 neurons. (D) The GlcAT-P gene is specifically expressed in r7 neurons (arrows). (E) The GlcAT-D gene is specifically expressed in the cranial ganglia (arrowheads). (F–H) Close-up views of the HNK-1-positive cells in the trigeminal ganglia. (F) HNK-1 immunostaining. (G) Immunostaining using anti Islet-1 antibody, a marker for sensory neurons in the ganglia. (H) An overlay image of HNK-1/anti Islet-1 immunostaining. Note that the HNK-1-positive cells are localized in the proximal portion of the Islet-1-positive early neurons, suggesting that placodal-derived cells in the ganglia are HNK-1 negative. ov, otic vesicle. Scale bars: 200 µm (A–E), 100 µm (F–H).

Fig. 3

Localization of the HNK-1 epitope and expressions of other NCCs markers and GlcAT genes in the trunk. (A) Schematic drawing of E11.5 rat embryo showing sections of somite 5–10 level (B–D) and somite15–20 level (E–I) of the trunk. (B) HNK-1 staining is positive in the dermatome, myotome (corresponding to longitudinal fibres) and migrating myoblasts in the limb bud, but not in the NCCs in the rostral trunk. (C,D) CRABP-I and AP-2 transcripts are found in the dorsal root ganglia. (E) In the caudal trunk, the HNK-1 epitope is found in the migrating NCCs (arrows). (F,G) Expressions of CRABP-I and AP-2 genes are also found in the NCCs (arrows). Not all the CRABP-I/AP-2-positive NCCs are stained with HNK-1 antibody. (H) The GlcAT-P gene is expressed in the NCCs just starting their migration (arrowheads). (I) The GlcAT-D gene expression is not detected in the NCCs. Scale bar = 200 µm.

Fig. 4

Transfection of GlcAT genes in the cranial NCCs. (A) Structures of expression vectors (pCDM8-GlcAT-P/D). (B) Time course of the electroporation experiment. So: somite number; EP: electroporation; WEC: whole embryo culture. (C,D) A representative sample successfully transfected with the GlcAT-P gene in the NCCs, which are detected by GFP due to co-transfection of the pCAX-GFP gene. The samples are subsequently sectioned at the level of the second pharyngeal arch (white line). (E–G) Transfection of the GlcAT-P gene. (E) The migrating NCCs positive for GFP, showing successful electroporation. (F) HNK-1 immunostaining is positive in the migrating NCCs. (G) An overlay image showing co-localization of HNK-1 staining and GFP. (H–J) Transfection of the GlcAT-D gene. HNK-1 synthesis is qualitatively almost similar to GlcAT-P transfection.

Fig. 5

Altered migration of the second pharyngeal arch NCCs after electroporation with GlcAT genes. (A) Measurement of NCC migration. The distance of each NCC from the posterior edge of the neuroepithelium is measured in the lateral digital images (yellow lines). This distance is an approximate projection of the three-dimensional migratory course to the sagittal plane. (B) NCCs were counted in the four zones as follows: Zone A, from the neuroepithelium to the dorsal edge of the otic vesicle; Zone B, between the dorsal and ventral edges of the otic vesicle; Zone C, from the ventral edge of the otic vesicle to the base of the second pharyngeal arch; Zone D, within the second pharyngeal arch. (C) Average of the NCC migratory distance in the control group (GFP transfection only; white bars), the GlcAT-P transfection group (grey bars) and the GlcAT-D transfection group (black bars). NCCs in the GlcAT-P group migrate further than in the other groups. Asterisk: P < 0.001 by anova and Sheffe's test. (D) Distribution of NCC numbers along their migratory distance. NCCs show relatively shorter migration in the control group. Two peaks are observed at about 0.2 mm and 0.6 mm of migration in the GlcAT groups. The 0.2-mm peak is higher in the GlcAT-D group. (E) Percentages of NCCs in the four zones. Numbers are high in Zone A in the control group, in Zone C in the GlcAT-P group and in Zone B in the GlcAT-D group.