Immunohistochemical Detection of Islet-1 and Neuronal Nitric Oxide Synthase in the Dorsal Root Ganglia (DRG) of Sheep Fetuses During Gestation
Department of Animal Physiology, College of Biological Sciences (HL,SC,DC,JL,ZL) and Faculty of Veterinary Medicine (SC), China Agricultural University, Beijing, People's Republic of China
Correspondence to: Dr. S. Cui, Dept. of Animal Physiology, College of Biological Sciences, China Agricultural University, Beijing 100094, People's Republic of China. E-mail: cuisheng{at}cau.edu.cn
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Summary |
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(J Histochem Cytochem 52:797803, 2004)
Key Words: Islet-1 nNOS DRG immunohistochemistry sheep fetuses
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Introduction |
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It has been well documented that nNOS, acting as an essential synthase for the regulation of nitric oxide (NO), has a variety of functions in the development of the nervous system. nNOS is a calcium/calmodulin-dependent enzyme stimulated by activation of NMDA-type glutamate receptors (Garthwaite et al. 1988; Bredt and Snyder 1990
). NO signaling occurs primarily through activation of soluble isoforms of guanylyl cyclase, and the cellular effects of NO are the results of an NO-induced increase in the level of cGMP (Jaffrey and Snyder 1995
). In the peripheral nervous system, NO plays an important role in nociceptive signaling (Moulton 1996
; Robbins and Grisham 1997
; Wallace and Chin 1997
). nNOS is mainly expressed in small and medium-sized nociceptive DRG neurons in rat and monkey (Zhang et al. 1993
; Qian et al. 1996
). During fetal development, many DRG neurons express nNOS in the early stage, while most of these become nNOS-negative in the late stage in rat (Wetts and Vaughn 1993
) and chick (Ward et al. 1994
), especially when the axons extend peripherally and start to take up nerve growth factor (Thippeswamy and Morris 2002
).
Both Islet-1 and nNOS are expressed and play important roles in developing DRG, but to date there are no data describing the ontogenic patterns of Islet-1 and nNOS expression and their relationships in DRG of sheep fetuses. The aim of this study was to determine the ontogeny of Islet-1 and nNOS expression by IHC in developing DRG of sheep fetuses during gestation.
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Materials and Methods |
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Immunohistochemistry
Immunocytochemical localization of Islet-1 was performed using a mouse monoclonal antibody (Mab) against Islet-1(40.2D6, Developmental Studies Hybridoma Bank), which had been used to detect Islet-1 in chicken (Avivi and Goldstein 1999; Cui and Goldstein 2000
), mouse (Kubota et al. 1999
), and rat (Yamamoto and Henderson 1999
). Comparison of the sequence data of Islet-1 between sheep (Chen and Cui, unpublished data) and mouse showed 98% homology. Immunocytochemistry for Islet-1 detection was performed as described previously (Avivi and Goldstein 1999
; Cui and Goldstein 2000
). Briefly, the sections were dewaxed and rehydrated, and antigen retrieval was performed by microwaving the sections for 20 min (four times for 5 min) at full power in 0.01 M sodium citrate buffer (pH 6.0). Endogenous peroxidase was blocked by incubating the sections in 3% H2O2 in methanol for 30 min. After nonspecific binding was blocked with 10% normal sheep serum in PBS, the sections were then incubated with the Islet-1 antibody (diluted 1:50) overnight at 4C. After further washing in PBS, the sections were incubated with sheep anti-mouse IgG (Serotec, Poole, UK; 1:50) for 2 hr at room temperature (RT). Mouse peroxidaseanti-peroxidase (Scottish Antibody Product Unit, Scotland, UK; 1:50) was then added for 2 hr, and the peroxidase activity was detected with diaminobenzidine (Sigma; St Louis, MO). Finally, the sections were counterstained with hematoxylin and mounted using conventional methods.
nNOS was detected with a rabbit anti-nNOS polyclonal antibody (Upstate Biotechnology, Lake Placid, NY; 1:40) which had been used to detect nNOS in sheep (Peterson et al. 2001). nNOS detection procedures were similar to that for Islet-1, with the exception that the primary, secondary, and tertiary antibodies were rabbit anti-nNOS polyclonal antibody (Upstate Biotechnology; 1:40), biotinylated swine anti-rabbit IgG (DAKO, Glostrup, Denmark; 1:300), and avidinbiotin complex (Vector Laboratories; Burlingame, CA).
For dual labeling of Islet-1 and nNOS, the primary antibodies were same as those for the single staining. The sections were incubated with a mixture of anti-Islet-1 MAb (1:50) and anti-nNOS polyclonal antibody (1:40) overnight at 4C. The slides were then rinsed in PBS three times and incubated in a mixture of sheep anti-mouse IgG (Serotec; 1:50) and biotinylated swine anti-rabbit IgG (DAKO; 1:300) for 2 hr at RT. The mixture of mouse alkaline phosphataseanti-alkaline phosphatase (DAKO; 1:30), and avidinbiotin complex (Vector) was then added for 2 hr. After three further washes in PBS, the alkaline phosphatase was visualized by the addition of NBT/BCIP for 10 min and the color reaction was stopped by washing in water. The peroxidase was visualized by the addition of diaminobenzidine (Sigma) for 2 min.
DRG cell proliferation was detected with the same procedures as that for Islet-1 detection except that the primary antibody was replaced by a mouse anti-proliferating cell nuclear antigen (PCNA) MAb (Zymed, S. San Francisco, CA; 1:200).
The controls included replacement of anti-Islet-1 and anti-PCNA Mabs and of anti-nNOS polyclonal antibody by normal mouse serum and normal rabbit serum, respectively, and by the omission of the primary, secondary, and tertiary antibodies, all of which eliminated staining (data not shown).
Data Analysis
From day 60 of gestation, DRG neuron profiles could be distinguished. In all cases, only the neurons that had distinguishable nuclei were observed and counted under a high-magnification objective with an eyepiece graticule. In each specimen, five fields were selected randomly throughout the DRG and the number of neurons immunopositive for Islet-1 or nNOS were counted and expressed as a percentage of the total number of neurons in that field, while the dual staining neurons were expressed as a percentage accounting for the total number of Islet-1+ or nNOS+ neurons, respectively. Results were presented as mean ± SEM. The differences between means of the repeated measures (n=6) over time at different stages of sheep fetuses were tested for significance using ANOVA Student's t-test.
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Results |
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Discussion |
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Similar to the Islet-1 expression, nNOS immunoreactivity was detected in over 90% of DRG neurons of the sheep fetuses at day 60 of gestation, but this proportion sharply declined to around 30% at day 90 of gestation and persisted at that level until birth. This changing pattern of nNOS expression is in accordance with that in rat (Wetts and Vaughn 1993) and chick embryo (Ward et al. 1994
). Although the relationship of nNOS expression to DRG development and functional regulation remains to be further investigated, the present results support the reports that nNOS expression is important for the differentiation of DRG neurons, especially in determining the developmental direction of DRG neurons by regulating the synthesis of NO in the early stage of gestation (Robbins and Grisham 1997
; Thippeswamy and Morris 2002
). However, from the middle stage of gestation until birth the relative proportion of nNOS-immunopositive neurons was around 30% accounting for the DRG neurons, and sheep sensory innervation of skin and muscle occurred from day 75 of gestation (Rees et al. 1994
,1995
). During this period, many DRG neurons extend peripherally (Rees et al. 1994
,1995
) and begin to take up nerve growth factor (NGF) (Thippeswamy and Morris 2002
), accompanied by depression of nNOS expression (Wetts and Vaughn 1993
; Ward et al. 1994
). Furthermore, most nNOS-immunopositive neurons were small and medium-sized DRG neurons. These observations suggest that nNOS expression may be associated with the functional maintenance of some nociceptive DRG neurons.
It has been documented that Islet-1 functions mainly through interactions with other proteins to regulate the transcription of some genes relative to development (Chen and Chiu 1992; Ericson et al. 1992
; Dawid et al. 1995
; Hobert and Westphal 2000
), whereas nNOS regulates the synthesis of NO, which acts as a molecular signal to induce an increase in intracellular cGMP levels and triggers a series of cellular effects that include enhancement of neurotrophin-induced neurite outgrowth and prevention of neuron apoptosis (Farinelli et al. 1996
; Hindley et al. 1997
; Thippeswamy and Morris 2002
). The dual IHC staining results first demonstrated that Islet-1 and nNOS were co-expressed in more than 60% of DRG neurons at day 60 of gestation. This implies that Islet-1 may have cooperative effects with nNOS or NO in DRG development. In addition, after the middle stage of gestation only a subset of DRG neurons co-expressed Islet-1 and nNOS. These were mainly small and medium-sized neurons that were mainly related to nociceptive properties (Zhang et al. 1993
; Qian et al. 1996
). This suggests that Islet-1 and NO may interact with each other to maintain some functions of a subset of DRG neurons after the middle stage of gestation, supported by reports that the expression of nNOS was upregulated (Verge et al. 1992
; Fiallos-Estrada et al. 1993
; Zhang et al. 1993
) and Islet-1 was downregulated in the DRG during peripheral nerve regeneration (Pfaff et al. 1996
; Hol et al. 1999
). However, further study is needed to determine the functions and the related mechanisms of Islet-1 and nNOS in the development and functional regulation of the nervous system.
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Acknowledgments |
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The Islet-1 monoclonal antibody 40.2D6 was developed by Thomas Jessel and was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA.
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Footnotes |
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Literature Cited |
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