Nutrition and oral health.

The link between nutrition and oral health can be overt (vitamin B deficiency) or subtle (exacerbation of already existing periodontal disease). Medical, social, and clinical examinations can be useful tools for uncovering those patients at risk for nutrition deficiencies and can be used to guide these patients to healthy eating. Along with routine home care instruction, the practicing dentist and hygienist can provide a service for patients through nutrition counseling and by pointing out reputable sources of nutrient supplements.

Environmental signals and cell fate specification in premigratory neural crest.

Neural crest cells are multipotent progenitors, capable of producing diverse cell types upon differentiation. Recent studies have identified significant heterogeneity in both the fates produced and genes expressed by different premigratory crest cells. While these cells may be specified toward particular fates prior to migration, transplant studies show that some may still be capable of respecification at this time. Here we summarize evidence that extracellular signals in the local environment may act to specify premigratory crest and thus generate diversity in the population. Three main classes of signals-Wnts, BMP2/BMP4 and TGFbeta1,2,3-have been shown to directly influence the production of particular neural crest cell fates, and all are expressed near the premigratory crest. This system may therefore provide a good model for integration of multiple signaling pathways during embryonic cell fate specification.

Direct regulation of nacre, a zebrafish MITF homolog required for pigment cell formation, by the Wnt pathway.

We have shown that Wnt signals are necessary and sufficient for neural crest cells to adopt pigment cell fates. nacre, a zebrafish homolog of MITF, is required for pigment cell differentiation. We isolated a promoter region of nacre that contains Tcf/Lef binding sites, which can mediate Wnt responsiveness. This promoter binds to zebrafish Lef1 protein in vitro, and a nacre reporter construct is strongly repressed by dominant-negative Tcf in melanoma cells. Mutation of Tcf/Lef sites abolishes Lef1 binding and reporter function in vivo. Wnt signaling therefore directly activates nacre, which in turn leads to pigment cell differentiation.

Maternal and embryonic expression of zebrafish lef1.

Transcription factors of the TCF/LEF family interact with the Wnt signaling pathway to control transcription of downstream genes (Clevers, H., van de Wetering, M., 1997. TCF/LEF factor earn their wings. Trends Genet. 13, 485-489). We were interested in cloning family members which were expressed in zebrafish neural crest, because Wnt signaling modulates specification of neural crest fate (Dorsky, R.I., Moon, R.T., Raible, D.W., 1998. Control of neural crest cell fate by the Wnt signalling pathway. Nature 396, 370-373). We cloned a zebrafish homolog of lef1 and localized its chromosomal position by radiation hybrid mapping. lef1 is expressed in the neural crest as well as the tailbud and developing mesoderm, and is maternally expressed in zebrafish, unlike mouse and Xenopus homologs. In addition, we cloned two tcf3 genes and a homolog of tcf4, neither of which were strongly expressed in premigratory neural crest.

Control of neural crest cell fate by the Wnt signalling pathway.

Environmental signals are important in the development of neural crest, during which process multipotent progenitor must choose from several fates. However, the nature of these environmental signals is unknown. A previous fate map of zebrafish cranial neural crest showed that lineage-restricted clones of pigment cells arise from medial cells near the neural keel, and that clones of neurons arise from lateral cells farther from the neural keel. Wnt-1 and Wnt-3a are candidate genes for influencing neural crest fate, as they are expressed next to medial, but not lateral, crest cells. Here we determine the role of Wnt signals in modulating the fate of neural crest by injecting messenger RNAs into single, premigratory neural crest cells of zebrafish. Lineage analysis of injected cells shows that activation of Wnt signalling by injection of mRNA encoding cytoplasmic beta-catenin promotes pigment-cell formation at the expense of neurons and glia. Conversely, inhibition of the Wnt pathway, by injection of mRNAs encoding either a truncated form of the transcription factor Tcf-3 or a dominant-negative Wnt, promotes neuronal fates at the expense of pigment cells. We conclude that endogenous Wnt signalling normally promotes pigment-cell formation by medial crest cells and thereby contributes to the diversity of neural crest cell fates.

Inductive competence, its significance in retinal cell fate determination and a role for Delta-Notch signaling.

The retina is a favorite model for studying determination and differentiation in the central nervous system because of its ready accessibility, diversity of cell types and regular organization. Many molecules which act as potential inducers of cell fate have been identified by exposing progenitors to them and assaying their differentiation. In addition, heterochronic transplants demonstrate that regulation of cellular competence (i.e. the ability of progenitors to respond to inducers) plays an important role in differentiation. The neurogenic genes Delta and Notch acting as ligand and receptor, respectively, play a role in regulating cell competence by normally inhibiting progenitors from differentiating. Misexpression of an activated form of Notch 'freezes' progenitors in an undifferentiated, neuroepithelial state. Conversely, progenitors failing to be inhibited, either by their own overexpression of Delta, or by a dominant-negative Delta construct which blocks signaling, adopt the earliest fates generated in the retina (i.e. cones and ganglion cells). We suggest that retinal progenitors use lateral inhibition mediated by Delta-Notch to regulate their competence to respond to inductive cues in a changing environment. Such signaling is essential for formation of the proper cell types in appropriate numbers at the right stage of development to make functional circuits.

Xath5 participates in a network of bHLH genes in the developing Xenopus retina.

We examined the function of basic-helix-loop-helix (bHLH) transcription factors during retinal neurogenesis. We identified Xath5, a Xenopus bHLH gene related to Drosophila atonal, which is expressed in the developing Xenopus retina. Targeted expression of Xath5 in retinal progenitor cells biased the differentiation of these cells toward a ganglion cell fate, suggesting that Xath5 can regulate the differentiation of retinal neurons. We examined the relationship between the three bHLH genes Xash3, NeuroD, and Xath5 during retinal neurogenesis and found that Xash3 is expressed in early retinoblasts, followed by coexpression of Xath5 and NeuroD in differentiating cells. We provide evidence that the expression of Xash3, NeuroD, and Xath5 is coupled and propose that these bHLH genes regulate successive stages of neuronal differentiation in the developing retina.

Regulation of neuronal diversity in the Xenopus retina by Delta signalling.

To generate the variety of mature neurons and glia found in the developing retina, the competence of pluripotent progenitor cells to respond to extracellular signals must be controlled. Delta, a ligand of the Notch receptor, is a candidate for regulating progenitor competence on the grounds that activation of the pathway involving Notch and Delta can inhibit cellular differentiation. Here we test this possibility in the developing Xenopus retina by misexpression of Delta messenger RNA. We find that Delta-misexpressing cells with wild-type neighbours adopt earlier fates, primarily becoming ganglion cells and cone photoreceptors. Progenitors transfected with Delta later in development also produce rod photoreceptors, but not the latest-generated cell types, demonstrating the importance of timing in Delta function. We conclude that Delta signalling in the vertebrate retina is a basic regulatory mechanism that can be used to generate neuronal diversity.

Xotch inhibits cell differentiation in the Xenopus retina.

The neurogenic gene Xotch acts to divert cellular determination during gastrulation in Xenopus embryos. We examined the role of Xotch in the developing retina, where cell signaling events are thought to affect differentiation. Xotch is expressed in undifferentiated precursor cells of the ciliary marginal zone and late embryonic central retina. It is not expressed in stem cells or in differentiated neurons and glia. Expression in the retina is spatially restricted even in the absence of cell division. The final Xotch-positive precursor cells in the central retina mostly differentiate as Müller glia, suggesting that this is the last available fate of cells in the frog retina. Transfection of an activated form of Xotch into isolated retinal cells causes them to retain a neuroepithelial morphology, indicating that the continued activation of Xotch inhibits cell differentiation.

XASH1, a Xenopus homolog of achaete-scute: a proneural gene in anterior regions of the vertebrate CNS.

The pro-neural achaete-scute complex (ASC) of Drosophila encodes four homologous proteins, each containing a basic helix-loop-helix (bHLH) domain, characteristic of a large family of transcription factors. We have isolated XASH1, a Xenopus homolog of achaete-scute. The XASH1 protein is very similar to the ASC proteins of Drosophila and the rat homolog, MASH1. XASH1 is expressed in the embryonic anterior central nervous system in a dynamic sequence, first in the midbrain, then in the forebrain, and then in the eye and hindbrain. In the larva, XASH1 expression correlates with regions of continued neurogenesis in the CNS, revealing the pattern of rhombomeres in the hindbrain, and other proliferative zones in the eye and midbrain. As a heterodimer with the bHLH protein E12, XASH1 binds specifically to an enhancer sequence derived from the promoter of the proneural achaete gene of Drosophila. This binding is inhibited by the extramacrochaete protein, a negative regulator of ASC gene function and neurogenesis in Drosophila. The combined evidence described in this paper strongly suggests that XASH1 plays a role in Xenopus neurogenesis similar to that played by the ASC genes in Drosophila.