Introduction
For neurons as well as people, youth is a time for determining the identity that one will carry through life. In vertebrates, this process of neuronal differentiation has been most thoroughly studied in the spinal cord, where an elaborate interplay of morphogens, other signaling molecules and transcription factors directs the development of the neural tube into increasingly distinct neuronal subtypes1. Much is known about the genetic blueprint for differentiating dorsal cord versus ventral, motor neurons versus interneurons, and even subpopulations of motor neurons from each other2. However, molecular information on a fundamental aspect of neuronal fate—the specification of neurotransmitter—has been lagging. In this issue, Cheng and colleagues use a clever combination of mouse genetic and chick experimental manipulations to demonstrate that the transcription factors Tlx1 and Tlx3 in the dorsal spinal cord promote the glutamatergic, excitatory fate and repress the GABAergic, inhibitory fate3.
Most neurons in the vertebrate central nervous system use either GABA or glutamate as a neurotransmitter, but rarely both. In the dorsal spinal cord, GABA or glutamate are expressed by distinct subpopulations of interneurons. Cheng et al. focus on two subgroups of these interneurons that derive from the Mash1-expressing domain of the ventricular zone and migrate to the most superficial lamina of the dorsal horn.
Starting with embryonic mouse tissue, Cheng et al. found that nearly all neurons in the outermost portion of the dorsal horn express either Tlx3 or Pax2. Tlx3-positive cells express the glutamate transporter VGLUT2. Pax2-positive neurons co-label with markers of GABAergic cells, including two isoforms of glutamic acid decarboxylase (GAD) and the GABA transporter VIAAT. Tlx1/Tlx3 double mutants (Tlx mutants) had fewer cells expressing the glutamatergic marker, and a dramatic expansion of cells expressing Pax2 and the GABAergic markers. In contrast, Pax2 mutants lacked the GABAergic cells in this dorsolateral region, although neither Tlx genes nor VGLUT2 were expanded in the Pax2 mutants.
These results suggest that Tlx genes may be promoting the glutamatergic fate and repressing GABAergic fate in the manner of 'selector' genes, transcription factors that bind multiple targets to coordinate cell type–specific fate choices during development4. However, crucial to this argument would be to demonstrate that the Pax2-positive/GABAergic cells are not merely expanding their numbers in the absence of Tlx-positive cells, but that in the absence of Tlx genes the cells themselves are converted into the Pax2-positive/GABAergic phenotype. Although the authors do not have definitive evidence, such as expression of Pax2 along with a genetic marker that was knocked into the Tlx1 or Tlx3 locus, they use three lines of evidence to convincingly support their contention that such a conversion has indeed occurred. First, Tlx1 and Tlx3 are only expressed in postmitotic cells5, so the absence of an increase in cell death in the Tlx mutants suggests that cells that would normally express Tlx are still present. In addition, these mutants have a grossly normal distribution of later born dorsal horn cells, implying that the Tlx-/- cells have not simply migrated into a different field5.
The second line of evidence relies on the observation that in the dorsal spinal cord at E13.5, cells expressing the lim-homeodomain transcription factor Lmx1b co-label with Tlx3, but not with Pax2 (ref. 6). In the Tlx mutants, Lmx1b-expressing cells remain, but now more than 90% of them coexpress Pax2. To provide the final line of evidence that Tlx genes are selecting the glutamatergic fate for a subset of interneurons, the authors switched to the developing chick spinal cord where they misexpressed Tlx3 by electroporation in ovo. Regions of Tlx3 misexpression within the dorsal cord showed reduced Pax2 and GAD, and increased VGLUT2.
These results indicate that Tlx genes within the dorsal spinal cord are able to direct postmitotic cells to the glutamatergic fate, and to repress the alternative GABAergic fate (Fig. 1). But what about the specification of neurotransmitter phenotype elsewhere in the nervous system? This role for Tlx genes is highly region specific; Tlx genes cannot convert GABAergic cells into glutamatergic cells in the ventral spinal cord. In fact, in the hindbrain, Tlx is required for the differentiation of cells that express an unrelated neurotransmitter, the catecholamine norepinephrine7. In the forebrain, Tlx1 and Tlx3 are not expressed at all. Clearly, different tissues use different genes to confer the glutamatergic phenotype, and most of the genes that serve this role have yet to be identified. It is possible that the neurotransmitter phenotype may be specified by the context-dependent action of transcription factors that impart not only the ability to make the transmitter, but also other region and cell type–specific aspects of the cell's phenotype. In the case of Tlx3 within the dorsal horn of the spinal cord, it is required not only for expression of the glutamate transporter, but also for the GluR2 glutamate receptor.
Figure 1: Results from Cheng et al. regarding the specification of neurotransmitter fate in later-born interneurons that reside in the most superficial lamina of the dorsal horn of the spinal cord.
Cells born within the Mash1-expressing domain of the ventricular zone migrate into the mantle region. It is not clear how Tlx genes come to be expressed in a subgroup of these cells. In the mantle region, Tlx1/3 repress Pax2 expression and promote glutamatergic differentiation.
Full size image (13 KB)Two key questions remain. First what other aspects of these cells' phenotype, such as morphology and connectivity, are regulated by Tlx genes? The answer to this question may need to await the generation of conditional Tlx3 nulls as well as null mice in which a genetic marker such as green fluorescent protein has been knocked in to the Tlx3 locus.
Second, is this regulation direct or indirect? Does Tlx bind to and activate transcription of genes required for glutamate synthesis and transport? Alternatively, despite the finding that glutamatergic fate is not expanded in the Pax2 mutants, it is conceivable that Tlx functions primarily by permitting this fate for the glutamatergic lineage by repressing Pax2. Evidence for direct promotion of glutamatergic fate could include identification of functional Tlx binding domains in VGLUT2, or a failure of rescue of the Tlx1/3 phenotype in Tlx1/3/Pax2 triple mutants, or ability of Tlx3 to induce a glutamatergic fate when electroporated into the normally Pax2-positive domain of Pax2-/- mutants. Finally, could there still be a master regulatory gene required to induce the genes for glutamate uptake, storage and synthesis that is downstream of different transcription factors within different regions of the nervous system?
Regardless of these remaining issues, the key point of this paper, that Tlx genes act as a binary, pro-glutamatergic switch in postmitotic mammalian neurons, is remarkable and has important implications for understanding and perhaps manipulating neuronal fate. Strategies for the generation of large numbers of specified neurons from stem cells for therapeutic transplants can be thought of occurring along a line with two extremes. At one end, stem cells are manipulated by a series of factors that grossly recapitulate normal development to produce a cohort of specified cells for transplantation8, 9. This approach has tremendous promise but depends upon a very detailed knowledge of factors involved in that cell's development. At the other end, more generic precursors can simply be forced to overexpress the synthesizing enzyme for a given transmitter10, but this approach has obvious limitations without the co-regulation of proteins needed to promote and coordinate multiple aspects of the neurotransmitter phenotype, and to suppress competing phenotypes. In a middle approach, neuronal stem cells can be transfected with a single gene that drives multiple aspects of that cell's differentiation toward the wanted phenotype11. By providing an example in which transcriptional regulation in postmitotic cells drives multiple aspects of neurotransmitter-associated fate, this work adds further promise to this last strategy whereby transmitter-associated selector genes could coordinate the differentiation of plastic but neuronally committed cells toward a desired fate. The ability to use single genes to select the desired fate of postmitotic, undifferentiated neurons for transplantation studies may not be beyond our grasp.
