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First published online November 21, 2008
doi: 10.1242/10.1242/dev.026807

Development 135, 4153-4164 (2008)
Published by The Company of Biologists 2008
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A differential developmental pattern of spinal interneuron apoptosis during synaptogenesis: insights from genetic analyses of the protocadherin-{gamma} gene cluster

Tuhina Prasad1, Xiaozhong Wang2, Paul A. Gray3 and Joshua A. Weiner1,4,*

1 Department of Biology, The University of Iowa, Iowa City, IA 52242, USA.
2 Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.
3 Department of Anatomy and Neurobiology, Washington University School of Medicine, St Louis, MO 63110, USA.
4 Neuroscience Graduate Program, The University of Iowa, Iowa City, IA 52242, USA.


Figure 1
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  		Fig. 1. Dorsal-ventral disparity in spinal interneuron apoptosis in Pcdh-{gamma} null mutant mice. (A-D) TUNEL (A), IB4 staining (C), and immunostaining with antibodies against cleaved caspase 3 (B) or Gfap (D) all demonstrate increased signs of apoptosis and neurodegeneration primarily in the ventral horn of Pcdh-{gamma} del/del spinal cords, as compared with littermate controls, at P0. Arrowheads in C mark blood vessels that, in addition to microglia, are stained by IB4. (E,F) In E12 (E) and P0 (F) Pcdh-{gamma} fus spinal cords, anti-GFP immunostaining demonstrates uniform expression of {gamma}-Pcdh-GFP fusion proteins. (G) In situ hybridization using a riboprobe against the Pcdh-{gamma} constant exons also yields uniform labeling of dorsal and ventral horns. (H) RT-PCR analysis of E16 spinal cord RNA demonstrates that all 22 variable exon-constant exon spliced transcripts can be detected. RT, reverse transcriptase. (I) Schematic of the mouse Pcdh-{gamma} genomic locus, indicating the 22 variable exons (A, B and C subfamilies, blue) and three constant exons (ce, red). Scale bar: 100 µm.

 

Figure 2
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  		Fig. 2. Loss of molecularly defined spinal interneuron populations in Pcdh-{gamma} null mutant mice. (A) Schematic of spinal cord domains and the molecular markers used in this study to identify discrete interneuron populations. (B-H) Hemicords from control and Pcdh-{gamma}del/del P0 mice immunostained using antibodies against the indicated markers and counterstained with DAPI (blue). At this age, dorsal interneuron populations are not reduced in the Pcdh-{gamma} del/del spinal cord (B,G). By contrast, most ventral interneuron populations are reduced, to varying extents (B-E), with the exception of calbindin-positive putative Renshaw cells in the deep ventral horn (F) and Chat-positive cholinergic interneurons at the border of the intermediate gray matter and the ventral horn (H). Scale bar: 100 µm.

 

Figure 3
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  		Fig. 3. Quantification of differential loss of molecularly defined spinal interneuron populations in Pcdh-{gamma} null mutant mice. Bars (in this and all subsequent bar charts) show mean ±s.e.m. of cell counts from 18 sections per molecular marker, taken from three animals per genotype. The survival of each cell population in E17 Pcdh-{gamma}del/del spinal cords is expressed as a percentage of that in controls.

 

Figure 4
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  		Fig. 4. Dorsal interneurons that migrate into the ventral horn are lost in Pcdh-{gamma} null mutant mouse embryos. (A-B') E11.5 control hemicords immunostained with antibodies as indicated. Many dI2-derived FoxD3-positive cells that migrate ventrally coexpress FoxP2 (A; A' shows a magnified view); double-labeled V1 interneurons are also observed. These dorsally derived FoxP2-positive interneurons do not coexpress Pax2 (B,B'); V0/V1-derived double-positive interneurons are, however, observed ventrally. (C) Schematic (left) showing the settling patterns of different populations of FoxP2-positive interneurons. Staining of sections (right) from spinal cords of E17 control and Pcdh-{gamma} del/del mice shows that ~50% of the dI2 population (FoxP2-positive Pax2-negative) is lost in mutants. (D) Isl1-positive dI3 interneurons also migrate ventrally, and ~50% are lost in Pcdh-{gamma} del/del embryos; note that there is no reduction in Isl1-positive motoneuron pools in mutants. Scale bar: 100µm.

 

Figure 5
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  		Fig. 5. Loss of spinal interneurons in Pcdh-{gamma} null mutant mice is due to apoptosis, not aberrant specification. (A-C) P0 hemicords from control, Pcdh-{gamma} del/del and Pcdh-{gamma} del/del;Bax-/- mice immunostained as indicated. The reductions in ventral populations observed in Pcdh-{gamma} del/del mutants are rescued in the Pcdh-{gamma} del/del; Bax-/- double mutants, confirming that they are due to increased apoptosis. (D-F) Fragmented ventral interneurons in P0 Pcdh-{gamma} del/del spinal cord can be double-labeled (insets) with antibodies against cleaved caspase 3, a marker of apoptotic cells. (G) Quantitative analysis of ventral interneuron populations at E14, E17 and P0 demonstrates that a normal number of postmitotic interneurons are produced in Pcdh-{gamma} del/del embryos, but that many are lost between E14 and E17, coincident with the initial period of synaptogenesis; further loss is apparent by P0. Scale bar: 100 µm.

 

Figure 6
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  		Fig. 6. Spinal interneurons undergo a normal developmental pattern of apoptosis that proceeds in a ventral-to-dorsal temporal gradient. (A-C) Multiple molecularly defined interneuron populations were quantified in wild-type (WT) mouse spinal cords at E14, E17, P0, P2 and P5 (at least six sections per marker per animal, three sets of animals). Means are graphed in A and B as a percentage of population size at E14. Ventral interneuron populations are lost, to differing extents, primarily between E14 and P0 (B), whereas dorsal interneuron populations are lost primarily after P0 (A). The increased apoptosis observed in Pcdh-{gamma} null mutant mice is likely to reflect an exacerbation of this normal pattern, as there is a near-perfect correlation between the extent of cell loss in each population during WT development (y-axis in C) and the extent to which apoptosis is increased in that population in mutants as compared with controls (x-axis in C).

 

Figure 7
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  		Fig. 7. Analysis of Bax-/- mice confirms that spinal interneuron apoptosis in Pcdh-{gamma} mutant mice represents an exacerbation of an underlying normal developmental pattern. (A-C) P0 hemicords from control and Bax-/- mice immunostained as indicated. (D) Quantification demonstrates that the sizes of these and other ventral interneuron populations (but not dorsal populations) are increased in the Bax-/- spinal cord at P0, indicating that they undergo a normal period of developmental apoptosis. (E) Quantification of dorsal horn interneurons in P5 Bax-/- spinal cords confirms that they do undergo a normal period of developmental apoptosis, but only after birth. (F) The increased apoptosis observed in Pcdh-{gamma} null mutants represents an exacerbation of an underlying developmental pattern, as the percentage decrease in each interneuron population correlates strongly with its percentage increase in Bax-/- neonates. Scale bar: 100 µm.

 

Figure 8
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  		Fig. 8. Cell type-restricted Pcdh-{gamma} mutation reveals a non-cell-autonomous requirement of {gamma}-Pcdh function for neuronal survival. (A,B) FoxP2-positive Pax2-negative dI2 interneurons were quantified in both Wnt1-Cre; Pcdh-{gamma} fcon3/fcon3 mice, in which they are mutant, and in Pax2-Cre; Pcdh-{gamma} fcon3/fcon3 mice, in which they are not, at P0. There is no reduction in this population in Wnt1-Cre; Pcdh-{gamma} fcon3/fcon3 mice (A, see C), but a loss of ~30% is observed in Pax2-Cre; Pcdh-{gamma} fcon3/fcon3 mice compared with controls (B, see C). (C) Quantitative analysis of ventral interneuron populations in Wnt1-Cre; Pcdh-{gamma} fcon3/fcon3, Pax2-Cre; Pcdh-{gamma} fcon3/fcon3 and Hb9-Cre; Pcdh-{gamma} fcon3/fcon3 spinal cord at P0. Data for mutants are expressed as a percentage of control values. *P<0.05; **P<0.01. Scale bar: 100 µm.

 

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© The Company of Biologists Ltd 2008