FAST-2 is a mammalian winged-helix protein which mediates transforming growth factor beta signals.

The mechanisms by which transforming growth factor beta (TGF-beta) and related ligands regulate transcription remain poorly understood. The winged-helix (WH) transcription factor fork head activin signal transducer 1 (FAST-1) was identified as a mediator of activin signaling in Xenopus embryos (X. Chen, M. J. Rubock, and M. Whitman, Nature 383:691-696, 1996). We have cloned a novel WH gene from the mouse which shares many properties with FAST-1. We find that this gene, which we call FAST-2, is able to mediate transcriptional activation by TGF-beta. FAST-2 also interacts directly with Smad2, a cytoplasmic protein which is translocated to the nucleus in response to TGF-beta, and forms a multimeric complex with Smad2 and Smad4 on the activin response element, a high-affinity binding site for FAST-1. Analysis of the sequences of FAST-1 and FAST-2 reveals substantial protein sequence divergence compared to known vertebrate orthologs in the WH family. This suggests that FAST-2 represents a new WH gene related to FAST-1, which functions to mediate TGF-beta signals in mammals. We have also examined the structure of the FAST-2 gene and find that it overlaps with a kinesin motor protein gene. The genes are transcribed in opposite orientations, and their transcripts overlap in the 3' untranslated region.

Inhibition of neurite growth by the NG2 chondroitin sulfate proteoglycan.

The chondroitin sulfate proteoglycans (CSPGs) have been implicated as both positive and negative modulators of axonal growth; however, the functional properties of only a few specific CSPGs have been investigated. Here we demonstrate that NG2, an integral membrane CSPG expressed on the surfaces of glial progenitor cells, inhibits neurite growth from neonatal rat cerebellar granule neurons when presented to the cells as a component of the substrate. Growth inhibition occurred when NG2 was mixed with either laminin or L1, two potent promoters of axonal extension. Moreover, when given a choice between surfaces coated with NG2 and laminin or L1, the axons of the cerebellar neurons extended preferentially on laminin or L1 and avoided areas of the substrate containing NG2. The NG2 proteoglycan inhibited neurite growth after digestion with chondroitinase ABC, demonstrating that the inhibitory activity is a property of the core protein and not the covalently attached chondroitin sulfate glycosaminoglycan chains. NG2 also inhibited neurite growth from embryonic rat dorsal root ganglia neurons on substrates containing laminin. However, when the sensory neurons were plated onto surfaces containing the L1 glycoprotein and NG2, neurite growth was not inhibited. These results demonstrate that the NG2 proteoglycan provides an unfavorable substrate for axonal growth. Cells that express this proteoglycan in vivo may participate in axonal guidance by defining areas of the developing CNS that are nonpermissive for axonal extension from specific classes of developing neurons.

Identification of a neuronal cell surface receptor for a growth inhibitory chondroitin sulfate proteoglycan (NG2).

The NG2 chondroitin sulfate proteoglycan inhibits neurite outgrowth from neonatal rat cerebellar granule neurons when presented to the neurons as a component of the substrate. To begin to understand the cellular mechanisms by which this inhibition occurs, we investigated the hypothesis that cerebellar granule neurons express cell surface receptors for NG2 and that these receptors are linked to cellular signaling pathways. Here, we show that the NG2 core protein binds specifically and with high affinity to cerebellar granule neurons. Using protein cross-linking techniques and immunoprecipitation, a 280-kDa membrane cell surface protein of granule neurons was identified as an NG2-binding site. Treatment of the neurons with pertussis toxin reversed the growth inhibition, suggesting a role for pertussis toxin-sensitive G proteins in the inhibitory response. Treatment of the neurons with pharmacological agents that increase either intracellular calcium or intracellular cyclic AMP levels partially reversed the growth inhibition induced by NG2. These results suggest that the growth-inhibitory actions of NG2 proteoglycan are due to an interaction with a specific cell surface receptor that is linked, either directly or indirectly, to intracellular second messenger systems.

Differential effects of glycosaminoglycans on neurite growth on laminin and L1 substrates.

Glycosaminoglycans (GAGs), the carbohydrate moieties of proteoglycans, are thought to be positive and negative regulators of axonal growth. The physiological role of GAGs is controversial as some studies have shown that GAGs inhibit cell adhesion and neurite elongation (Exp Neurol 109:111, 1990) whereas other studies have reported a growth stimulatory effect of GAGs (Development 114:17, 1992). These and other studies have examined the effects of GAGs using different types of neurons and different substrate conditions thereby making a direct comparison of the experimental data difficult. To resolve the controversy concerning the ability of exogenous GAGs to modulate neurite growth, we examined the effects of a panel of structurally different GAGs on the growth of postnatal rat cerebellar granule neurons and embryonic rat dorsal root ganglia (DRG) neurons on substrates of either laminin or the L1 glycoprotein. Here we show that chondroitin 4-sulfate (CS4), chondroitin 6-sulfate (CS6), and keratan sulfate (KS) inhibit neurite growth from both cerebellar and DRG neurons on laminin-coated surfaces. On L1 surfaces, however, these GAGs are either extremely weak inhibitors of neurite extension or, in the case of CS4, a modest stimulator of neurite growth. Heparan sulfate (HS) and dermatan sulfate (DS) inhibited the growth of cerebellar neurons but not the growth of DRG neurons on L1-coated surfaces. On laminin surfaces, DS and HS had no effect on neurite growth from both cerebellar and DRG neurons. These results demonstrate a cellular and a substrate specificity to the effects of exogenous GAGs on neurite extension in vitro. They suggest that while CS and KS GAGs may not exert strong negative influences over axonal growth in regions of the developing CNS where the L1 glycoprotein is abundant, these GAGs are capable of inhibiting the growth of axons that extend within an environment rich in laminin.

Dual role of brain factor-1 in regulating growth and patterning of the cerebral hemispheres.

Brain factor-1 (BF-1) is a winged-helix (WH) transcription factor with a restricted pattern of expression in the neural tube. In the embryo, BF-1 is localized to the progenitor cells of the most rostral neural tube, the telencephalic neuroepithelium. Expression of BF-1 persists in the adult brain in the structures derived from the telencephalon, including the cerebral cortex, the hippocampus, the olfactory bulbs and the basal ganglia. Targeted disruption of the BF-1 gene in mice results in hypoplasia of the cerebral hemispheres. Proliferation of the telencephalic neuroepithelium is decreased and neuronal differentiation occurs prematurely. The forebrain of the BF-1 (-/-) mutant also displays dorsal-ventral patterning defects. Development of the ventral (basal) region of the telencephalon is more severely affected than the dorsal region. These anomalies are associated with the ectopic expression of BMP4 in the dorsal telencephalic neuroepithelium and the loss of shh in the ventral telencephalon. These results raise the possibility that BF-1 may modulate both progenitor cell proliferation and regional patterning by regulating the expression or activity of inductive signals which act on the telencephalic neuroepithelium.