Supporting Information - PNAS · counted. At P1, P2, P6, P13, and P90, neurons were counted in the...
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Supporting Information Holstege et al. 10.1073/pnas.0802176105 SI Text Scoring of Local Excess Grooming. Mice of the same age and gender were videotaped while alone in their cages for consecutive periods of 9 h. The video tapes were viewed, and the periods of lower back grooming were scored and timed. Behavior of control mice and homozygous mutants was scored at 3.5, 4, 5.5, 10, and 11 weeks. The results were presented in a pie diagram, as shown for the 4-week experiment in Fig. S1. Hot Plate Test. The hot plate test with temperatures of 48°C, 51°C, and 53°C was used to determine the thermal pain threshold. In four separate sessions, the mice were habituated to handling and to the hot plate before starting the experiments. Before each measurement, the mice were placed in the experimental room for 20 min to habituate. Thereafter, the mouse was placed on the hot plate, and the time was measured up to the first flinching, licking of the paw, or jumping occurred, after which the mouse was immediately removed from the hot plate. The mouse was removed after 50 s in case it did not show any reaction. For each temperature, the experiment was repeated three times. The three temperatures were tested on separate days with 1 day in between. Lidocaine Test. The mice were allowed to habituate for 20 min in the experimental room before the experiment. For injection of lidocaine, the mouse was held by an assistant and subsequently 200 l of 0.3% lidocaine was injected s.c. underneath the excessively groomed skin area on the lower back. Saline injection served as control. The mice were placed back in their cages and were not disturbed for 15 min. Thereafter, the grooming behav- ior was assessed for 1 s with intervals of 10 s, for a total observation time of 30 min. The grooming behavior was deter- mined as a ‘‘Yes’’ or ‘‘No’’ grooming of the lower back. Capsaicin Test. The mice were placed in the room for 20 min to habituate. For injection, the mouse was held by an assistant, and 25 l of 0.3% capsaicin was injected s.c. in the paw. Subse- quently, the time spent licking was measured in seconds during 20 min immediately after the injection. At 90 min after the capsaicin application, some of the mice were transcardially perfused with saline, followed by 4% paraformaldehyde. The spinal cords were then removed, posfixed in fixation fluid also containing 30% sucrose (overnight at 4°C). Frozen sections (40 m) were cut and processed for c-fos immunocytochemistry WGA-HRP Transganglionic Labeling. One hundred microliters of a 1% wheat germ agglutinin–horseradish peroxidase (WGA– HRP) solution in saline was injected s.c. in the hindpaw. After 4 days survival, the animals were transcardially perfused with saline, followed by 4% paraformaldehyde. Then the lumbar spinal cord was removed, postfixed for 2 h, and transferred to phosphate buffer (pH 7.4) containing 30% sucrose (overnight at 4°C). Frozen sections (40 m) were cut and reacted with tetramethyl benzidine (1). After mounting, dehydration, and coverslipping, the sections were viewed and photographed in a light microscope using dark-field illumination. BrdU Labeling. Pregnant mice of heterozygote crosses at day 12.25 of gestation were injected i.p. with a solution of 3 mg/ml BrdU (BS002; Sigma), at 50 mg per kilogram of body weight. In some of the experiments, we used 100 mg per kilogram of body weight. Embryos were recovered either after 2 hours, or after 1 or 2 days, depending on the experiment. They were then fixed, genotyped, embedded in paraffin and sectioned at 7 m. BrdU incorpora- tion was revealed after incubation with an antibody against BrdU (monoclonal BU 32; Sigma), followed by incubation with a second antibody coupled to Cy3. Apoptosis Assays. Programmed cell death was assayed by using antiactivated caspase 3 (rabbit polyclonal antibody; Cell Signal- ing Technology) on paraffin sections of E13, E15, and E18.5 embryos. It was measured as well by using the standard TUNEL assay (Apoptosis Detection kit; Sigma) on vibratome sections of agarose-embedded tissues and on paraffin sections of E15.5, E16.5, E17.5, and E18.5 embryos and fetuses and P1, P2, and P13 mice. Dissection of Spinal Cords and Dorsal Root Ganglia (DRGs) for in Situ Hybridization and Immunodetection. Embryos, fetuses, and new- born mice was immersed into 4% paraformaldehyde (PFA) overnight. Spinal cords from older mice were dissected after transcardiac perfusion with PBS and buffered 4% paraformal- dehyde (PFA) under terminal anesthesia. To prepare spinal cord segments, the dorsal roof of the vertebral column was removed before further fixation in buffered PFA for 2 h for immunode- tection experiments and overnight for in situ hybridization. Careful dissection was then performed by using the ribs as axial landmarks to idendify the lumbar DRGs. Following the dorsal roots of the selected DRGs allowed the localization of the corresponding axial levels of the spinal cord. The segment corresponding to levels L4 and L5 of the lumbar spinal cord was processed. To prepare DRGs L4and L5, they were dissected out once identified by means of the vertebral axial landmarks. Agarose-embedded tissues were vibratome sectioned at 40 m and the sections individually collected in PBS-containing 48-well plates. Adjacent sections were then distributed onto different glass slides to be processed for X-Gal and antibody staining, or for in situ hybridization. Tissues embedded in paraffin were sectioned at 10 m and the sections deposited serially onto glass slides to be processed for antibody staining, for in situ hybrid- ization, or for TUNEL assays. Immunocytochemistry and Immunofluorescence. Antibodies used were as follows: Anti-NeuN (Chemicon) was used on either frozen, vibratome, or paraffin sections. The other antibodies were used on both vibratome and frozen sections: Anti-CGRP (Calbiochem), anti-Parvalbumin (Swant), anti-PKC (monoclo- nal C-19; Santa Cruz Biotechnology), anti-Galanin and anti SubP (kindly provided by R. Buijs, Netherlands Brain Institute, Amsterdam), anti-calretinin (Sigma), anti-calbinding (Swant), anti-ChAT (Chemicon), anti-NF200 (Calbiochem), anti-c-fos (monoclonal ab5; Calbiochem), anti Islet 1 (2) (monoclonal antibody 39.4; Developmental Sudies Hybridoma Bank). IB4 (from Sigma) and ExtrAvidin-Cy3 (Cat N; Sigma) were used on vibratome sections. In Situ Hybridization and X-Gal Staining. The expression of Hoxb8, Hoxb6, and Hoxb9 was assayed in whole-mount embryos as described (refs. 2–4, respectively). In situ detection of transcripts of Hoxb8, lacZ (2) and Drg11 (5) was on paraffin sections of the spinal cord. Drg11 probe was kindly provided by Z. 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Transcript of Supporting Information - PNAS · counted. At P1, P2, P6, P13, and P90, neurons were counted in the...
Supporting InformationHolstege et al. 10.1073/pnas.0802176105
SI Text
Scoring of Local Excess Grooming. Mice of the same age and genderwere videotaped while alone in their cages for consecutiveperiods of 9 h. The video tapes were viewed, and the periods oflower back grooming were scored and timed. Behavior of controlmice and homozygous mutants was scored at 3.5, 4, 5.5, 10, and11 weeks. The results were presented in a pie diagram, as shownfor the 4-week experiment in Fig. S1.
Hot Plate Test. The hot plate test with temperatures of 48°C, 51°C,and 53°C was used to determine the thermal pain threshold. Infour separate sessions, the mice were habituated to handling andto the hot plate before starting the experiments. Before eachmeasurement, the mice were placed in the experimental roomfor 20 min to habituate. Thereafter, the mouse was placed on thehot plate, and the time was measured up to the first f linching,licking of the paw, or jumping occurred, after which the mousewas immediately removed from the hot plate. The mouse wasremoved after 50 s in case it did not show any reaction. For eachtemperature, the experiment was repeated three times. Thethree temperatures were tested on separate days with 1 day inbetween.
Lidocaine Test. The mice were allowed to habituate for 20 min inthe experimental room before the experiment. For injection oflidocaine, the mouse was held by an assistant and subsequently200 �l of 0.3% lidocaine was injected s.c. underneath theexcessively groomed skin area on the lower back. Saline injectionserved as control. The mice were placed back in their cages andwere not disturbed for 15 min. Thereafter, the grooming behav-ior was assessed for 1 s with intervals of 10 s, for a totalobservation time of 30 min. The grooming behavior was deter-mined as a ‘‘Yes’’ or ‘‘No’’ grooming of the lower back.
Capsaicin Test. The mice were placed in the room for 20 min tohabituate. For injection, the mouse was held by an assistant, and25 �l of 0.3% capsaicin was injected s.c. in the paw. Subse-quently, the time spent licking was measured in seconds during20 min immediately after the injection. At 90 min after thecapsaicin application, some of the mice were transcardiallyperfused with saline, followed by 4% paraformaldehyde. Thespinal cords were then removed, posfixed in fixation fluid alsocontaining 30% sucrose (overnight at 4°C). Frozen sections (40�m) were cut and processed for c-fos immunocytochemistry
WGA-HRP Transganglionic Labeling. One hundred microliters of a1% wheat germ agglutinin–horseradish peroxidase (WGA–HRP) solution in saline was injected s.c. in the hindpaw. After4 days survival, the animals were transcardially perfused withsaline, followed by 4% paraformaldehyde. Then the lumbarspinal cord was removed, postfixed for 2 h, and transferred tophosphate buffer (pH 7.4) containing 30% sucrose (overnight at4°C). Frozen sections (40 �m) were cut and reacted withtetramethyl benzidine (1). After mounting, dehydration, andcoverslipping, the sections were viewed and photographed in alight microscope using dark-field illumination.
BrdU Labeling. Pregnant mice of heterozygote crosses at day 12.25of gestation were injected i.p. with a solution of 3 mg/ml BrdU(BS002; Sigma), at 50 mg per kilogram of body weight. In someof the experiments, we used 100 mg per kilogram of body weight.
Embryos were recovered either after 2 hours, or after 1 or 2 days,depending on the experiment. They were then fixed, genotyped,embedded in paraffin and sectioned at 7 �m. BrdU incorpora-tion was revealed after incubation with an antibody against BrdU(monoclonal BU 32; Sigma), followed by incubation with asecond antibody coupled to Cy3.
Apoptosis Assays. Programmed cell death was assayed by usingantiactivated caspase 3 (rabbit polyclonal antibody; Cell Signal-ing Technology) on paraffin sections of E13, E15, and E18.5embryos. It was measured as well by using the standard TUNELassay (Apoptosis Detection kit; Sigma) on vibratome sections ofagarose-embedded tissues and on paraffin sections of E15.5,E16.5, E17.5, and E18.5 embryos and fetuses and P1, P2, and P13mice.
Dissection of Spinal Cords and Dorsal Root Ganglia (DRGs) for in SituHybridization and Immunodetection. Embryos, fetuses, and new-born mice was immersed into 4% paraformaldehyde (PFA)overnight. Spinal cords from older mice were dissected aftertranscardiac perfusion with PBS and buffered 4% paraformal-dehyde (PFA) under terminal anesthesia. To prepare spinal cordsegments, the dorsal roof of the vertebral column was removedbefore further fixation in buffered PFA for 2 h for immunode-tection experiments and overnight for in situ hybridization.Careful dissection was then performed by using the ribs as axiallandmarks to idendify the lumbar DRGs. Following the dorsalroots of the selected DRGs allowed the localization of thecorresponding axial levels of the spinal cord. The segmentcorresponding to levels L4 and L5 of the lumbar spinal cord wasprocessed. To prepare DRGs L4and L5, they were dissected outonce identified by means of the vertebral axial landmarks.Agarose-embedded tissues were vibratome sectioned at 40 �mand the sections individually collected in PBS-containing 48-wellplates. Adjacent sections were then distributed onto differentglass slides to be processed for X-Gal and antibody staining, orfor in situ hybridization. Tissues embedded in paraffin weresectioned at 10 �m and the sections deposited serially onto glassslides to be processed for antibody staining, for in situ hybrid-ization, or for TUNEL assays.
Immunocytochemistry and Immunofluorescence. Antibodies usedwere as follows: Anti-NeuN (Chemicon) was used on eitherfrozen, vibratome, or paraffin sections. The other antibodieswere used on both vibratome and frozen sections: Anti-CGRP(Calbiochem), anti-Parvalbumin (Swant), anti-PKC� (monoclo-nal C-19; Santa Cruz Biotechnology), anti-Galanin and antiSubP (kindly provided by R. Buijs, Netherlands Brain Institute,Amsterdam), anti-calretinin (Sigma), anti-calbinding (Swant),anti-ChAT (Chemicon), anti-NF200 (Calbiochem), anti-c-fos(monoclonal ab5; Calbiochem), anti Islet 1 (2) (monoclonalantibody 39.4; Developmental Sudies Hybridoma Bank). IB4(from Sigma) and ExtrAvidin-Cy3 (Cat N; Sigma) were used onvibratome sections.
In Situ Hybridization and X-Gal Staining. The expression of Hoxb8,Hoxb6, and Hoxb9 was assayed in whole-mount embryos asdescribed (refs. 2–4, respectively). In situ detection of transcriptsof Hoxb8, lacZ (2) and Drg11 (5) was on paraffin sections of thespinal cord. Drg11 probe was kindly provided by Z. F. Chen.
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Expression of TrkA and ret was assayed on paraffin sections ofthe dorsal root ganglia (DRGs). TrkA and ret probes were kindlyprovided by Q. Ma. Expression of GlyT2 and GAD67 in thespinal dorsal horn was determined in free-floating sectionsgenerated as follows: Animals were transcardially perfused withsaline, followed by 4% paraformaldehyde. The lumbar spinalcord was removed and postfixed in fixation fluid, also containing30% sucrose (overnight at 4°C). Frozen sections (40 �m) werecut and processed for free-floating in situ hybridization asdescribed (6) by using a GlyT2 probe (a generous gift from N.Nelson, Tel Aviv University, Tel Aviv, Israel) or a GAD67 probe(a generous gift from N. J. Tillakaratne, University of Calfornia,Los Angeles) for labeling of glycinergic and GABA-ergic neu-rons respectively.
�-Galactosidase activity was measured by X-Gal staining ofwhole mounts of heterozygote and homozygote mutant embryos
and dissected DRGs, as described (2). lacZ expression in thespinal cords of embryos, fetuses, and adults was determined bymeasuring �-galactosidase activity after X-Gal staining of 40-�mvibratome sections of agarose-embedded spinal cords.
Neuron Counting. At each developmental stage, three mutantsand three controls (wild types or heterozygotes, as indicated inthe text) were used, and a minimum of 5 sections per animal werecounted. At P1, P2, P6, P13, and P90, neurons were counted inthe lamina I and II areas, which can be discerned on thehistological sections and confirmed by overlaying the areaexpressing lacZ at the highest level on adjacent sections. Countswere expressed as the means � SEM. Numerical results in Fig.S2 were generated from the ratios between mutants and controlcounts, with the controls set at 100%. Statistical analysis isdescribed in the legend of Fig. S2.
1. Mesulam MM (1978) Tetramethyl benzidine for horseradish peroxidase neurohisto-chemistry: A non-carcinogenic blue reaction product with superior sensitivity forvisualizing neural afferents and efferents. J Histochem Cytochem 26:106–117.
2. Charite J, de Graaff W, Shen S, Deschamps J (1994) Ectopic expression of Hoxb8 causesduplication of the ZPA in the forelimb and homeotic transformation of axial structures.Cell 78:589–601.
3. Van der Lugt NMT, Alkema M, Berns A, Deschamps J (1996) The Polycomb-group homologBmi-1 is a regulator of murine Hox gene expression. Mech Dev 58:153–164.
4. Van den Akker E, et al. (2002) Cdx1 and Cdx2 have overlapping functions inanteroposterior patterning and posterior axis elongation. Development 129:2181–2193.
5. Chen ZF, et al. (2001) The paired homeodomain protein DRG11 is required for theprojection of cutaneous sensory afferent fibers to the dorsal spinal cord. Neuron31:59–73.
6. Hossaini M, French PJ, Holtege JC (2007) Distribution of glycinergic neuronal somata inthe rat spinal cord. Brain Res 1142:61–69.
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27%13%
B
A
CT MUT
Fig. S1. Localized excess grooming and skin lesions on the lower backs of Hoxb8 null mutant mice. (A) Observed over a period of 9 h, control mice were seento spend 13% of the time grooming their lower backs, whereas mutants spent twice as long doing that. More striking were the long intensive local groomingsessions shown by the homozygous mutants but not by the controls. (B) Photographs of a control and a Hoxb8 null mouse showing the hairless spot on the mutantlower back.
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CT MUT
Fig. S2. Transganglionic labeling of primary afferent fibers in the superficial dorsal horn after WGA–HRP injection in the right paw. WGA–HRP was injectedin the right hind paw, and signal was detected in the superficial laminae of the spinal cord, showing effective transganglionic transport in primary sensoryafferent fibers for both control and homozygous Hoxb8 mutant mice. Some motor neurons were retrogradely labeled as well.
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P90
Galanin
PKCγ
0%25%50%75%
100%
P1 P2 P6 P13 P90
CT MUT
CaBP
Calret
Fig. S3. Detection of dorsal neuron differentiation markers in P90 control and Hoxb8 mutant mice. From top to bottom: Immunostaining of Calretinin (labelingsome neurons of lamina I and II), Calbinding (labeling some neurons of lamina I and II), Galanin (lamina I and IIo), and PKC� (lamina IIi) on sections of mutantand control lumbar spinal cord. The bottom graph is a representation of the lower number of dorsal spinal neuron counts in the Hoxb8 null mutants versuscontrols at different developmental stages. The areas considered for counting were delimited on the basis of visible extent of laminae I and II on the histologicalsections, as shown in Fig. 3. They were confirmed by overlaying the X-Gal-stained area of an adjacent section on the photograph of the NeuN-stained section.In the graph, neuron counts in the mutants are represented as the percentages of counts in the controls. Each point represents the means of counted neuronsin five to six sections of two to three animals. Data for controls and mutants were analyzed by using the Mann–Whitney test. The difference between controland mutants gave the following P values: P1, P � 0.009; P2, P � 0.002; P6, P � 0.004; P13, P � 0.009; P90, P � 0001.
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BrdUMUT
BrdUCT
E13.5 E14.5
Fig. S4. Distribution of BrdU-labeled dorsal neurons. BrdU-labeled cells in lumbar cross-sections of control and mutant embryos labeled between E12.25 (justbefore late born neurons arise from the ventricular zone progenitors) and E13.5 (Left) and between E12.25 and E 14.5 (Right).
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MUTCT CTMUTCERVICAL LUMBAR
B
A
NeuN
LacZ
Fig. S5. Alterations of laminae I and II are specific for lumbar levels of the spinal cord. Serial sections of the spinal cord of control and mutant at P35 takenat lumbar and cervical levels have been stained for NeuN immunoreactivity (A) and for lacZ expression (X-Gal staining) (B).
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