Localisation of specific heparan sulfate proteoglycans during the proliferative phase of brain development.

Early brain development is characterised by the proliferation of neural precursor cells. Several families of signalling molecules such as the fibroblast growth factors (FGFs) and Wnts are known to play important roles in this early phase of brain development. Accumulating evidence demonstrates that signalling of these molecules requires the presence of heparan sulfate chains attached to a proteoglycan core protein (HSPG). However, the specific identity of the HSPG components in the developing brain is unknown. To determine which HSPGs might be involved at this early phase, we analysed the expression of the major cell surface HSPG families in the developing brain at a time of most active proliferation. Syndecan-1 and glypican-4 were the most highly expressed in the developing brain during the time of peak proliferation and localise to ventricular regions of the brain, where the precursor cells are proliferating. Syndecan-4, although less abundant, also localises to cells in the ventricular zone. We have also examined HSPG involvement in brain development using cultures of embryonic neural precursor cells. We find that FGF2 stimulation of proliferation is inhibited in the presence of sodium chlorate, an inhibitor of heparan sulfate synthesis, and is rescued by addition of exogenous heparan sulfate. These data support a requirement for heparan sulfate in FGF signalling for proliferation of brain precursor cells. The expression of these specific HSPGs within the proliferative zone of the brain suggests that they may be involved in regulation of early brain development, such as FGF-stimulated proliferation.

Neural crest cell lineage segregation in the mouse neural tube.

Neural crest (NC) cells arise in the dorsal neural tube (NT) and migrate into the embryo to develop into many different cell types. A major unresolved question is when and how the fate of NC cells is decided. There is widespread evidence for multipotential NC cells, whose fates are decided during or after migration. There is also some evidence that the NC is already divided into subpopulations of discrete precursors within the NT. We have investigated this question in the mouse embryo. We find that a subpopulation of cells on the most dorsomedial aspect of the NT express the receptor tyrosine kinase Kit (previously known as c-kit), emigrate exclusively into the developing dermis, and then express definitive markers of the melanocyte lineage. These are thus melanocyte progenitor cells. They are generated predominantly at the midbrain-hindbrain junction and cervical trunk, with significant numbers also in lower trunk. Other cells within the dorsal NT are Kit-, migrate ventrally, and, from embryonic day 9.5, express the neurotrophin receptor p75. These cells most likely only give rise to ventral NC derivatives such as neurons and glia. The p75+ cells are located ventrolateral to the Kit+ cells in areas of the NT where these two cell types are found. These data provide direct in vivo evidence for NC lineage segregation within the mouse neural tube.

Variant heparan sulfates synthesized in developing mouse brain differentially regulate FGF signaling.

Heparan sulfates (HSs) exert critical regulatory actions on many proteins, including growth factors, and are essential for normal development. Variations in their specific sulfation patterns are known to regulate binding and signaling of fibroblast growth factors (FGFs) via tyrosine kinase receptors (FGFRs). We previously reported differences in sulfation patterns between HS species expressed by embryonic day 10 (E10) and E12 mouse neural precursor cells. We have examined the abilities of the different HS species to support signaling of the relevant FGF-FGFR combinations expressed early during brain development. For FGF8, which only functions early (E8-E11), E10 HS showed preferential activation. The most potent signaling for FGF8 was via FGFR3c, for which E10 HS was strongly active and E12 HS had no activity. For FGF2, which functions from E10 to E13, HS from both stages showed similar activity and were more potent at activating FGFR1c than the other receptors. Thus, we find a stage-specific correlation with activation. To explore the potential mechanisms for the generation of these stage-specific HS species, we investigated the expression of the HS sulfotransferase (HSST) isozymes responsible for creating diverse sulfation motifs in HS chains. We find that there are stage-specific combinations of HSST isozymes that could underlie the synthesis of different HS species at E10 and E12. Collectively, these data lead us to propose a model in which differential expression of HSSTs results in the synthesis of variant HS species that form functional signaling complexes with FGFs and FGFRs and orchestrate proliferation and differentiation in the developing brain.