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First published online 13 December 2006
doi: 10.1242/dev.02717

Development 134, 357-366 (2007)
Published by The Company of Biologists 2007
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Lhx1 and Lhx5 maintain the inhibitory-neurotransmitter status of interneurons in the dorsal spinal cord

Andrea Pillai1,5, Ahmed Mansouri2, Richard Behringer3, Heiner Westphal4 and Martyn Goulding5,*

1 Biology Graduate Program, University of California, San Diego, La Jolla, CA 92037, USA.
2 Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, Gottingen 37077, Germany.
3 Department of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030, USA.
4 Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.
5 Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.


Figure 1
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  		Fig. 1. Lhx1 and Lhx5 expression in the embryonic spinal cord. (A-F) In situ expression of Lhx1. (G-L) In situ expression of Lhx5. At E10.5 and E11.5, Lhx1 and Lhx5 are co-expressed in postmitotic neurons that correspond to dI2, dI4, dI6 and V0-V1 neurons (A,B,G,H). Lhx1 is also present in a subset of the motor neurons ventrally (arrow in B). Expression of Lhx1 and Lhx5 begins to diverge at E12.5 (C,I). Lhx1 is strongly expressed in the laterally located neurons (arrow in C), whereas Lhx5 is more strongly expressed in the subventricular zone where newborn dILA cells are emerging (arrow in I). By E13.5, the expression of Lhx1 and Lhx5 in the dorsal horn is largely complementary (D,J). Lhx1 persists in the dorsal horn (E,F; see arrow in E), whereas Lhx5 is downregulated at E14.5 (K) and is completely absent at E17.5 (L).

 

Figure 2
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  		Fig. 2. Dorsal interneuron development in Lhx1 and Lhx5 single mutants. (A-C) Inactivation of either Lhx1 or Lhx5 does not alter the specification of dILA interneurons. dILA interneurons express Pax2 and Lhx5 in Lhx1-/- embryos (B), or express Pax2 and Lhx1 in Lhx5-/- embryos (C). (D-I) In both Lhx1 and Lhx5 single mutants, dILA cells retain their GABAergic identity, and express Viaat (D-F) and Gad1 (G-I). (J-O) Lhx1 expression is unchanged in the cord of Lhx5-/- mutants. There is no upregulation of Lhx1 mRNA at early (not shown) or later (arrows in J,L) stages. There is also no change in Lhx5 expression in the cord of Lhx1-/- mutants at E12.5 (arrows in M,N).

 

Figure 3
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  		Fig. 3. Generation and testing of conditional Lhx1; Lhx5 double-knockout animals. (A) Simplified schematic adapted from Kwan and Behringer (Kwan and Behringer, 2002Go) and Zhao et al. (Zhao et al., 1999Go) showing the targeted alleles of Lhx1 and Lhx5 that were used in this study. (B) PCR amplification of a DKO embryo carrying the NesCre;Lhx1loxP and Lhx5- alleles. The Cre transgene is detected as a 1 kb band. A 480-base-pair band is diagnostic for the Cre-deleted Lhx1loxP allele. Neo is present in both Lhx5 heterozygotes and Lhx5 mutants. The absence of a wild-type Lhx5 band of approximately 430 base pairs distinguishes Lhx5 mutants from Lhx5 heterozygous animals. This wild-type Lhx5-specific band is absent in DKO embryos. (C,D) Lhx1-protein expression is nearly completely abolished in the DKO cord at E12.5, with the exception of a few ventral Lhx1+ escapees. (E) Expression of lacZ and NeuN in E11.5 spinal cords of NestinCre;Rosa26lacZ+/- animals. (F,G) High magnification of newly postmitotic NeuN+ interneurons (green) in E (box) showing colocalization with ß-gal (red; asterisk in G).

 

Figure 4
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  		Fig. 4. Early inhibitory-interneuron cell types are correctly specified in the Lhx1; Lhx5 double-knockout cord. (A-H) Cross sections through E11.5 wild-type (wt) and Lhx1;Lhx5 mutant (DKO) cord at forelimb level. (A,B) In the DKO cord, expression of Brn3a is unchanged in dI1-dI3 (arrow in B) and in dI5 (arrowhead) neurons. (C,D) Isl1 expression in dI3 neurons (arrowhead) is also unchanged. (E,F) Lbx1 expression in dI4-dI6 neurons is normal in the DKO cord (F, arrowhead). (G,H) Pax2, which marks dI4 inhibitory neurons (arrowhead) as well as the more ventrally located dI6, V0 and V1 inhibitory neurons, is expressed in a normal pattern in the DKO cord (H). A small decrease in the level of Pax2 expression was observed.

 

Figure 5
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  		Fig. 5. Lhx1;Lhx5 double-knockout mutants fail to maintain inhibitory-neurotransmitter gene expression in the dorsal horn. (A-D) Dorsal quadrants of the spinal cord at E12.5 and E13.5 showing the expression of Viaat. In the DKO cord, a normal pattern of Viaat expression in dILA neurons is observed up to E13.5. (E,F) Beginning at E14.5, Viaat is downregulated in the most-dorsolateral cells in the DKO cord (arrow in F). (G,H) E17.5 DKO animals show a marked loss of Gad1 expression in the dorsal regions (arrow in H). (I,J) VGluT2 expression is not altered in the DKO dorsal horn at E17.5.

 

Figure 6
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  		Fig. 6. Lhx1 and Lhx5 regulate late aspects of development in Pax2 inhibitory interneurons. (A-D) Dorsal horn quadrants showing the downregulation of Pax2 in dILA neurons. This downregulation begins at E12.5 in DKO animals (asterisk, B) and is more pronounced at E14.5 (asterisk, D). (E-H)Lmx1b+ dILB neurons are specified normally and settle in the superficial dorsal horn in DKO animals at similar stages to wild type. (I-L) VGluT2 is not upregulated in the superficial dorsal horn (arrows) indicating that GABAergic Pax2+ cells do not adopt a dILB glutamatergic phenotype in the DKO cord. (M,N) E17.5 DKO animals exhibit a loss of Pax2 expression both dorsally and ventrally at E17.5. (O) Cell counts of Pax2+ and Lmx1b+ cells at E14.5 show a >60% reduction in the number of dorsal Pax2+ cells without any increase in the number of Lmx1b+ dILB cells.

 

Figure 7
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  		Fig. 7. BrdU pulse-chase and histological analysis of late neuron development in DKO spinal cord. (A,B) Late-born interneurons were pulsed with BrdU at E12.5, and were analyzed at E14.5 when the downregulation of Gad1 is first observed in the dorsal horn. The distribution of BrdU+ cells in wild-type (wt) and DKO spinal cord is similar, suggesting that there are no major defects in cell birth or cell migration in the DKO cord. (C-F) Anatomical analysis indicates no marked loss of neurons in the superficial dorsal horn. The dorsal funiculus is reduced in size (arrowheads) in the lumbar spinal enlargement of the DKO cord. E and F: enlargements of the boxed regions in C and D.

 

Figure 8
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  		Fig. 8. Pax2 maintains Lhx1 and Lhx5 expression in dILA interneurons. (A-D) Expression of Lhx1 and Lhx5 in the Pax2-/- cord. Lhx1 and Lhx5 antibody stainings showing Lhx1 and Lhx5 expression in the dorsal neurons is Pax2-independent at E11.5 (A,B) and E12.5 (C,D). (E-H) In situ hybridization analysis of Lhx1 (E,F) and Lhx5 (G,H) at E14.5 shows that both genes depend on Pax2 for maintenance in the dILA population. (I-N) Viaat expression is correctly initiated in the Pax2-/--mutant cord (I,J), but begins to be downregulated dorsally at E12.5. The dILA population shows a downregulation of Viaat at E12.5 (arrow in L). Viaat expression in the dorsal horn is largely missing by E14.5 (arrow in N) (see also Cheng et al., 2004Go). Notice that Viaat is downregulated dorsally, but remains largely intact in the ventral cord.

 

Figure 9
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  		Fig. 9. Expression of Pax5 and Pax8 is downregulated in DKO and Pax2-/- spinal cords. (A-C) Pax5 expression is lost in the Pax2-/- and DKO cord at E12.5. Some ventral Pax5+ cells persist in the DKO cord. (D-F) At E17.5, Pax5 expression is partially lost in the DKO cord but, unlike Pax8, it is completely abolished in Pax2-/- cord. (G-I) At E12.5, Pax8 expression is gradually downregulated dorsally but maintained ventrally at reduced levels in both the Pax2 and Lhx1; Lhx5 DKO-mutant cord. (J-L) At E17.5, there is a complete loss of Pax8 expression dorsally. Some Pax8 expression is retained ventrally in the Pax2-/- cord. The DKO cord shows a partial loss of Pax8 expression in both dorsal and ventral regions.

 

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© The Company of Biologists Ltd 2007