Li M et al. (2006), The role of early lineage in GABAergic and glut...

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J Comp Neurol 2006 Apr 20;4956:645-57. doi: 10.1002/cne.20900.

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The role of early lineage in GABAergic and glutamatergic cell fate determination in Xenopus laevis.

Li M , Sipe CW , Hoke K , August LL , Wright MA , Saha MS .

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Proper functioning of the adult nervous system is critically dependent on neurons adopting the correct neurotransmitter phenotype during early development. Whereas the importance of cell-cell communication in fate determination is well documented for a number of neurotransmitter phenotypes, the contributions made by early lineage to this process remain less clear. This is particularly true for gamma-aminobutyric acid (GABA)ergic and glutamatergic neurons, which are present as the most abundant inhibitory and excitatory neurons, respectively, in the central nervous system of all vertebrates. In the present study, we have investigated the role of early lineage in the determination of these two neurotransmitter phenotypes by constructing a fate map of GABAergic and glutamatergic neurons for the 32-cell stage Xenopus embryo with the goal of determining whether early lineage influences the acquisition of these two neurotransmitter phenotypes. To examine these phenotypes, we have cloned xGAT-1, a molecular marker for the GABAergic phenotype in Xenopus, and described its expression pattern over the course of development. Although we have identified isolated examples of a blastomere imparting a statistically significant bias, when taken together, our results suggest that blastomere lineage does not impart a widespread bias for subsequent GABAergic or glutamatergic fate determination. In addition, the fate map presented here suggests a general dorsal-anterior to ventral-posterior patterning progression of the nervous system for the 32-cell stage Xenopus embryo.

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Species referenced: Xenopus laevis
Genes referenced: ascl1 dlx1 dlx2 gad1.1 gad1.2 gsx2 nkx2-1 slc17a7 slc6a1 tlx3 XB5944457

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	Fig. 1. Schematic of the blastomeres of a 32-cell stage Xenopus embryo with each cell labeled according to Nakamura and Kishiyama (1971).
	Fig. 2. Temporal analysis of xGAT-1 expression during Xenopus development. The graph shows the mean fold change in xGAT-1 mRNA expression for the stages indicated as measured using quantitative real-time PCR. xGAT-1 expression is first detectable above background during late neurula stages (stage 20) and increases dramatically over the course of development. Threshold cycle values were normalized to control for input template amount and analyzed as described in Materials and Methods. ue, unfertilized egg.
	Fig. 3. Whole mount xGAT-1 expression in the developing Xenopus embryo. Insets show a stage-matched embryo hybridized with an antisense GAD probe. A: xGAT-1 expression is first detected in the spinal cord during late neurula stages (stage 21). B: xGAT-1 signal is present in the hindbrain and forebrain by stage 23. C: By stage 26, expression increases in the spinal cord and can be observed in all parts of the developing brain. D–F: xGAT-1 expression continues to strengthen during tailbud stages (D, stage 28; E, stage 32), and by swimming tadpole stages (F, stage 40) is present at high levels throughout the central nervous system and eye. G: Higher magnification of the head of a stage 33 embryo showing distinct domains of xGAT-1 expression in the forebrain, midbrain, and hindbrain. H: Dorsal view of a tailbud stage embryo (stage 32). fb, forebrain; hb, hindbrain; mb, midbrain; ol, olfactory placodes; sc, spinal cord. Scale bar 1 mm in A–H.
	Fig. 4. Histological analysis of xGAT-1 mRNA expression. A–H: Serial (anterior to posterior) 10- m transverse sections of a larval stage embryo (stage 34). Left insets show a stage-matched embryo hybridized with the antisense GAD probe. A: Anteriormost xGAT-1 signal is concentrated in the lateral regions of the forebrain. B: In the midbrain, expression is found in more ventral regions. Higher magnification (right inset) demonstrates that xGAT-1 signal is present immediately adjacent to the neurocoel. C,D: At the level of the hindbrain, xGAT-1 signal is again concentrated in lateral regions midway down the dorsal-ventral axis of the structure. E,F: This expression pattern is maintained in the anterior spinal cord. G,H: xGAT-1 mRNA becomes localized to more distinct regions of the spinal cord containing Kolmer-Agduhr cells and presumptive interneurons. I: By swimming tadpole stages, xGAT-1 mRNA expression has increased dramatically throughout the brain, and is detectable in the retina of the eye. Arrows indicate areas of xGAT-1 mRNA expression. asterisk, otic vesicle; cg, cement gland; e, eye; gu, gut; no, notochord. Scale bar 0.5 mm in A–I.
	Fig. 5. Summary of GABAergic and glutamatergic fate mapping experiments. Blastomere contributions to GABAergic (A) or glutamatergic (B) cells in specific regions of the nervous system. Major regions of the CNS are denoted in black as follows: FB, forebrain; MB, midbrain; HB, hindbrain; SC, spinal cord. The specific regions of the forebrain, midbrain, hindbrain, and spinal cord examined for expression of xGAT-1 or xVGlut1 are indicated by lower case letters: d, dorsal; i, intermediate; v, ventral. The peripheral structures examined include the olfactory placodes (OL), the pineal gland (PG), and the cranial ganglia: V, trigeminal; VII, geniculate; VIII, acousticus; IX, glossopharyngeal. The approximate percentage of GABAergic or glutamatergic tissue in a specific CNS region or peripheral structure contributed by a given blastomere is indicated as follows: boldface type, 26–50%; regular type, 10–25%; white type with black outline, 0–10%.
	Fig. 6. Colocalization of xGAT-1 and xVGlut1 signal with RLDX. Representative sections of a B1-injected embryo demonstrating colocalization of RLDX with xGAT-1 (A–D) or xVGlut1 (E–H) in situ hybridization signals in the hindbrain. A,E: Brightfield images of xGAT-1 or xVGlut1 expression with arrowheads indicating ISH staining. B,F: RLDX fluorescence distribution in the same section. C,G: Overlay of the brightfield and fluorescent images reveals overlap of ISH signal with RLDX. D,H: A false-color merged image showing ISH signal (blue), RLDX-labeled tissue (red), and colocalization of xGAT-1 or xVGlut1 with RLDX (yellow). Scale bar 0.25 mm in A (applies to A–D), E (applies to E–H).
	Fig. 7. Summary of GABAergic and glutamatergic fate mapping experiments by structure and blastomere. Blastomere contributions to tissue expressing either xGAT-1 (left column) or xVGlut1 (right column) in specific regions of the nervous system. The approximate percentage of GABAergic or glutamatergic tissue in a specific structure contributed by a given blastomere is denoted by shades of gray, with darker shades representing a higher percentage contribution. These percentages were calculated by averaging the contributions to dorsal, intermediate, and ventral regions (or, in the case of the cranial ganglia, each nerve) as described in Materials and Methods. FB, forebrain; MB, midbrain; HB, hindbrain; SC, spinal cord; CG, cranial ganglia.
	unnamed (hypothetical protein LOC100127682) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, lateral view, anterior right, dorsal up.
	unnamed (hypothetical protein LOC100127682) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.
	unnamed (hypothetical protein LOC100127682) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anterior right, dorsal up.