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First published online 29 May 2007
doi: 10.1242/jcs.002063

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Hrk/DP5 contributes to the apoptosis of select neuronal populations but is dispensable for haematopoietic cell apoptosis

Leigh Coultas^1,*, Susanna Terzano², Tim Thomas¹, Anne Voss¹, Kate Reid¹, Edouard G. Stanley^1,, Clare L. Scott¹, Philippe Bouillet¹, Perry Bartlett^1,, Jonathan Ham², Jerry M. Adams¹ and Andreas Strasser^1,¶

¹ The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia
² University College London, Gower Street, London, WC1E 6BT, UK

View larger version (26K): [in this window] [in a new window]   Fig. 1. Targeting of the Hrk locus. (A) Schematic diagram (to scale) of the Hrk-targeting construct, the wild-type (WT) Hrk locus and the targeted locus. Dashed lines demarcate regions of the WT locus used in the targeting construct. Location of 5' and 3' external probes for genomic Southern blot analysis and expected fragment sizes from indicated restriction digests are shown. B, BglII; Sc, ScaI; N, NcoI. (B) Confirmation by Southern blotting using 5' genomic probes of the loss of both wild-type Hrk alleles in Hrk–/– mice. (C) Northern blot analysis of polyA+ mRNA extracted from brains of neonatal wild-type (+/+), Hrk+/– (+/–) and Hrk–/– (–/–) littermates. Gapdh RNA was loaded as control.

View larger version (24K): [in this window] [in a new window]   Fig. 2. HRK is not required for cytokine withdrawal-induced death of haematopoietic progenitors. (A) Foetal liver cells from E13.5 wt, Hrk+/– and Hrk–/– embryos were plated in soft agar and colonies scored 7 days after the addition of cytokines. Cytokines were added to the cultures at the time of plating (0 hours) or after 24, 48 or 72 hours. Data are the mean ± s.d. from three or nine mice of each genotype. (B) Northern blot analysis of Hrk gene expression on polyA+ mRNA extracted from IL3-dependent haematopoietic cell lines and their derivatives over-expressing Bcl2. Each cell line was deprived of IL3 for the indicated times. PolyA+ mRNA from neonatal brain was used as a positive control. Northern blotting for the gene encoding Bim was included as a control to show that each cell line was stimulated to upregulate BH3-only gene expression. Gapdh RNA was loaded as control. (C) Northern blot analysis of Hrk gene expression on polyA+ mRNA extracted from the indicated tissues of C57BL/6 mice. Gapdh RNA was detected as a loading control.

View larger version (123K): [in this window] [in a new window]   Fig. 3. Expression of the Hrk gene in the developing nervous system. (A-G) Para-sagittal paraffin sections of E13.5 (A-E) and transverse sections of E14.5 (F,G) wild-type embryos were hybridised with an Hrk anti-sense cRNA probe (A,B,D-E) or a sense control probe (C). The bright-field images in A,D,F identify the regions of the dark-field images (B,E,G), in which silver grains appear above areas of Hrk mRNA expression. D and E depict insert shown in A at a higher magnification. The Hrk gene shows highly restricted expression. Expression of Hrk was highest in dorsal root ganglia (DRG) at E13.5 (arrow in B; E), the spinal cord at E13.5 (arrowhead in B) and the ventral grey horn of the spinal cord (VH) at E14.5 (short arrow in G) in the area of the motor neurons. DRG at E14.5 (asterisk in G) exhibit a moderate Hrk mRNA signal, as do cells in the lateral grey horn (LH) ventral of the sulcus limitans (long arrow in G) in the area of V0 interneurons. Weak expression of Hrk mRNA is present in other areas of the spinal cord, e.g. the dorsal horn (arrowhead in G). Ce, cerebral cortex; CC, central canal; CG IX, inferior ganglion of glossopharyngeal nerve (IX); CG X, inferior ganglion of vagal nerve (X); DH dorsal grey horn; DRG, dorsal root ganglia; HB, hindbrain; He, heart; LH lateral grey horn; Li, liver; Lu, lung; LVB, cartilage primordium of a lumbar vertebral body; SC, spinal cord; TG, trigeminal ganglion; VL, ventricular layer. Bars, 1.2 mm (A-C) and 93 µm (D-G).

View larger version (12K): [in this window] [in a new window]   Fig. 4. HRK contributes to NGF-deprivation-induced apoptosis in DRG neurons. (A) DRG neurons were isolated from control (wt and Hrk+/–, n=4) or Hrk–/– (n=5) littermates, cultured for 24 hours in NGF, then transferred to medium with or without NGF (supplemented with anti-NGF antibodies) for 72 hours. Cell survival was assessed by counting the number of viable neurons in an assigned field before NGF withdrawal (0 hours), then counting the same field again at 48 and 72 hours after NGF withdrawal. Percent survival was determined relative to the viable neuron number at 0 hours. Asterisks indicate statistically significant survival difference between control and Hrk–/– neurons in the absence of NGF (P<0.05, Student's t-test). (B) SCG neurons were isolated from WT, Hrk–/– or Bim–/– mice and cultured with NGF for 5 to 6 days, then washed and cultured in medium with or without NGF (supplemented with anti-NGF antibodies) for up to 48 hours. Cell survival was assessed from the number of viable neurons remaining in each well at each time point relative to the number present in the same well before NGF withdrawal and were expressed in percent. Data represent the mean ± s.d. Asterisks indicate statistically significant difference between survival of wild-type (WT) and Bim–/– neurons in the absence of NGF (P<0.05, Student's t-test). (C) Q-PCR analysis of Hrk mRNA expression in wt SCG neurons cultured with or without NGF for 16 hours. Expression of neurofilament 3 was used for normalisation. Data are the mean ± s.d. of nine cultures of neurons from three mice (triplicates).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Loss of HRK does not protect cerebellar granule neurons from K+ deprivation. (A) Cerebellar granule neurons were isolated from wild-type (Hrk+/+, n=3) and Hrk–/– (n=3) littermates, cultivated in high-K+ medium for 7 days, then transferred to low-K+ medium to induce apoptosis. Viability was determined 0, 6, 12 and 24 hours after transfer to low K+ using the Live/dead assay (Molecular Probes) to assess the number of viable and dead neurons in five independent fields at each time point for each animal and expressed in percent. Data are the mean ± s.d. (B) Q-PCR analysis of Hrk mRNA expression in wt cerebellar granule neurons cultured for 4 hours in the presence of high (K25+S) or low levels (K5+S) of K+. Expression of neurofilament 3 was used for normalisation. Data are the mean ± s.d. from nine cultures of neurons from three mice (triplicates).

View larger version (109K): [in this window] [in a new window]   Fig. 6. Loss of HRK does not rescue cerebellar Purkinje cells or granule neurons in lurcher mutant mice. Mid-sagittal cerebellar sections stained with H&E showing that the Hrk genotype (Hrk+/+, Hrk+/– or Hrk–/–) does not affect the size of the cerebellum in lurcher (lc) heterozygous (grid+/lc) animals. Bar, 1 mm.

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