Identification and Isolation of Gbx1 cDNA
In order to identify genes differentially expressed in the dorsal spinal cord at E13.5, a microarray-based genome wide expression analysis was performed (Britsch and Birchmeier, unpublished data). Among the genes specifically expressed in the developing dorsal neural tube, we identified an EST sequence (Affymetrix-ID 116752_at, corresponding to GenBank AI464517). Computational analysis with CELERA DISCOVERY SYSTEM indicated that the EST mapped to the 3′-UTR of the murine Gbx1 gene. To isolate the complete Gbx1 cDNA, we screened a cDNA library generated from E13.5 mouse spinal cord with a probe derived from the EST. We isolated two independent cDNA clones. One contained the entire ORF of the murine Gbx1 gene including 5′ and 3′ untranslated sequences.
Expression Analysis of Gbx1 and Gbx2 During Spinal Cord Development
We determined the expression of the Gbx1 gene in the developing mouse spinal cord by in situ hybridization. Gbx1 transcripts were first detected at E11.5 in the ventricular zone of the spinal cord (Fig. 1A). At E12.5–13.5, Gbx1 was broadly expressed in the mantle zone of the dorsal spinal cord (Fig. 1B,C). With the appearance of a discernable dorsal horn around E14, Gbx1 expression became more restricted (Fig. 1D). Perinatally, Gbx1 expression was observed in a narrow layer in the superficial dorsal horn (Fig. 1F). Gbx1 expression was still detectable in a small number of neurons in the superficial dorsal horn in the adult (not shown).
Figure 1. Expression analysis of Gbx1 and Gbx2 in the developing spinal cord. Vibratome sections from mouse cervical spinal cords at E11.5–E19.5 (A–J). Spinal cord tissues were hybridized with probes specific for Gbx1 (A–F) or Gbx2 (G–J). Note that both genes are transiently expressed in overlapping regions within the alar plate (B and H). ap, alar plate; vz, ventricular zone; mz, mantle zone.
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Gbx2 is closely related to Gbx1 and has been reported to be expressed in the neural tube (Rhinn et al., 2004). Functional analyses have demonstrated an essential role of Gbx2 during development of the mid-hindbrain boundary (Wassarman et al., 1997; for review, see Joyner et al., 2000). As yet, a detailed analysis of the expression of Gbx2 in the spinal cord has not been reported. We compared expression of Gbx1 and Gbx2 during spinal cord development. At E11.5, Gbx2 expression was detected in the dorsal ventricular zone and in the mantle zone within the ventral half of the neural tube (Fig. 1G). Strong expression of Gbx2 was observed at E12.5 in the ventricular zone and the mantle zone of the entire dorsal spinal cord. At this developmental stage, the expression domains of Gbx1 and Gbx2 overlapped (Fig. 1B and H). After E12.5, Gbx2 expression was rapidly downregulated (Fig. 1I,J). Gbx1 and Gbx2 appear thus to be transiently co-expressed in neurons of the dorsal spinal cord. In addition to its dorsal expression, Gbx2 is also expressed in the ventral spinal cord. In this region, no Gbx1 co-expression was detectable. Ventral expression of Gbx2 was observed between E11.5 and E14.5. In this part of the spinal cord, Gbx2 may identify a distinct population of ventrally located spinal interneurons.
Characterization of Gbx1+ Neurons in the Dorsal Spinal Cord
Dorsal interneurons are generated during two distinct waves of neurogenesis in the spinal cord (Gross et al., 2002; Muller et al., 2002, 2005). During the second phase of neurogenesis (E11.5–14.0), the majority of neurons generated from dorsal progenitors are class B neurons that express the homeodomain transcription factor Lbx1. During further development, these late-born neurons colonize the superficial dorsal horn (Gross et al., 2002; Muller et al., 2002). In situ hybridization indicated that Gbx1 is expressed in the neurons generated during the second phase of neurogenesis. To define this neuronal population, anti-Gbx1 antibodies were generated. Immunohistological analyses of the spinal cord at E12.5 demonstrated that Gbx1 protein was detected in cells located in the alar plate, but not in the ventricular zone or in the ventral spinal cord. In addition, when Gbx1 in situ hybridization and Gbx1 immunohistology were carried out on identical sections, both signals were found to co-localize. Together, this indicates that the antibody specifically recognizes Gbx1 protein (compare Fig. 1B,H and Fig. 2A,B; and not shown). Further analysis revealed that Gbx1+ cells co-express Lbx1 at E12.5. Gbx1+ cells correspond thus to late-born class B neurons (Fig. 2A). Lbx1 is a key determinant for the specification of class B neurons (Gross et al., 2002; Muller et al., 2002). We, therefore, tested if Gbx1 expression is detectable in the dorsal spinal cord of Lbx1 mutant mice, and found that Gbx1 was absent at E12.5 (Fig. 2C). In Lbx1 mutants, miss-specified neurons that arise in the dorsal spinal cord are eliminated by apoptosis during further development (Gross et al., 2002; Muller et al., 2002). At E12.5 Gbx1+ wildtype spinal neurons co-express the homeodomain factor Lhx1/5 (Fig. 2B). In Lbx1 mutants, Lhx1/5+ neurons are still detectable in similar numbers in the dorsal spinal cord (Fig. 2D), indicating that the lack of Gbx1 expression is not caused by the apoptotic loss of dorsal neurons in Lbx1 mutants. We conclude that Gbx1 expression depends on Lbx1 function and that the Gbx1+ neuronal cell population is not generated in the dorsal horn of Lbx1 mutant animals.
Figure 2. Lbx1 controls expression of Gbx1 in the dorsal spinal cord. Immunofluorescence analysis of the developing cervical spinal cord from wildtype (A, B) and Lbx1−/− (C, D) embryos at E12.5 using antibodies directed against Gbx1 (A–D), Lbx1 (A, C), and Lhx1/5 (B, D). Within the alar plate of control embryos, Gbx1+ cells co-express Lbx1 and Lhx1/5 (A, B). In homozygous mutants, expression of either Lbx1 or Gbx1 is no longer detectable, demonstrating that Gbx1 expression in dorsal spinal neurons depends on Lbx1 function (C, D). Lhx1/5 expressing cells are still present on E12.5 in homozygous mutants, indicating that the loss of Gbx1 expression is not caused by a depletion of the corresponding cells.
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Late-born class B neurons comprise initially two neuron populations, dILA and dILB, which are born in an apparent salt and pepper pattern in the dorsal spinal cord. dILA neurons express Lbx1, Pax2, and Lhx1/5, whereas dILB cells express Lbx1, Lmx1b, and Tlx3 (Muller et al., 2002). At E12.5, only a subpopulation of the Lbx1+ cells co-expressed Gbx1 (Fig. 2A). We, therefore, characterized the Gbx1+ neuronal population further. At E12.5 and 14.5, Gbx1+ neurons co-express the transcription factors Lhx1/5 and Pax2, but are negative for Lmx1b and Tlx3. This indicates that Gbx1+ neurons correspond to the dILA neuronal subtype (Figs. 2B, 3A–D, and not shown). We observed at no developmental stage that all Pax2+ or Lhx1/5+ cells expressed Gbx1, and many Pax2+ or Lhx1/5+ cells that do not express Gbx1 were detectable. Thus, Gbx1 expression distinguishes a subpopulation of dILA cells (Figs. 2B, 3A,C, and not shown). We analyzed Gbx1-expressing cells also during the maturation of the dorsal spinal cord. At E16.5, most Gbx1+ cells were located in the superficial dorsal horn (Fig. 3E). At this stage, only few Gbx1+ neurons co-expressed Lbx1; however, most of the Gbx1+ cells co-expressed Pax2 or Lhx1/5 (Fig. 3E, and not shown). It has been shown previously that dILA neurons undergo GABAergic differentiation (Cheng et al., 2004). We have, therefore, tested if markers for GABAergic differentiation are expressed in Gbx1+ cells. When GABA+ or Gad67+ neurons are first detectable in the dorsal spinal cord, the GABA+ or Gad67+ neurons co-express Gbx1. Gbx1+ cells can thus differentiate into GABAergic neurons (Fig. 3F, and not shown). Recent studies demonstrated that Pax2 is essential for GABAergic differentiation in the dorsal spinal cord, since GABAergic differentiation does not occur correctly in Pax2 mutant mice (Cheng et al., 2004). Gain-of-function studies demonstrated that Pax2, when over-expressed in the chick spinal cord, is however not sufficient to induce GABAergic differentiation (Cheng et al., 2004). This suggests that additional factors might be involved in this process.
Figure 3. Characterization of Gbx1 expressing dorsal spinal neurons. Co-expression analysis of Gbx1+ dorsal spinal neurons at E12.5 (A, B), E14.5 (C, D), E16.5 (E, F), and P28 (G, H). At E12.5–E14.5, Gbx1+ cells co-express Pax2 (A, C) but exclude expression of Lmx1b and Tlx3 (B, D) indicating that Gbx1 expressing dorsal neurons share molecular characteristics of dILA cells. Note that only part of Pax2+ cells co-express Gbx1. At E16.5, Gbx1+ neurons are detectable in the superficial dorsal horn and only few of them co-express Lbx1 (E). With the onset of GABA expression, both GABA and Gbx1 are co-expressed in the superficial dorsal horn (F). At postnatal day 28 (P28), Gbx1-expressing cells are distributed throughout the entire superficial dorsal horn (G), and most of the Gbx1+ cells co-express GABA (G, H).
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In order to further determine the fate of Gbx1+ cells, we have analyzed Gbx1 expression in the early postnatal (P7) and in the adult spinal cord (Fig. 3G and H). Here, Gbx1 identifies a small population of interneurons that are scattered throughout the entire superficial dorsal horn. Very few Gbx1+ cells are located in the deeper layers of dorsal horn, close to the central canal (Fig. 3G and not shown). GABA expression was found to be abundant in the adult superficial dorsal horn. Within this region, the great majority of Gbx1+ neurons co-express GABA, further indicating that Gbx1 comprises a distinct subpopulation of GABAergic dorsal spinal neurons (Fig. 3G and H).
We have analyzed here the expression pattern of the homeodomain transcription factor Gbx1 during development of the mouse spinal cord. We show that Gbx1 expression is restricted to the dorsal spinal cord and that it coincides with the generation of a late neurogenic cell population. Immunohistological analyses identified Gbx1+ neurons as a subpopulation of dILA neurons that require the homeodomain transcription factor Lbx1 for development. As development proceeds, Gbx1+ interneurons differentiate into a subpopulation of GABAergic neurons and colonize the superficial layers of the dorsal horn. Physiological experiments have indicated that GABAergic neurons are not homogeneous, but GABAergic subpopulations are ill defined on a molecular level (Heinke et al., 2004). Further work is required to define the functional properties of the Gbx1+ population of GABAergic neurons, and to determine the role of Gbx1 in the development of this neuronal lineage.