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Keywords:

  • cellular migration;
  • autonomic;
  • sympathetic;
  • migration;
  • embryo;
  • development;
  • axon

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Prader-Willi syndrome is a neurodevelopmental disorder marked by abnormalities in feeding, drinking, thermoregulation, intestinal motility, and reproduction, suggesting disruption of the autonomic nervous system. Necdin, one of several proteins genetically inactivated in individuals with Prader-Willi syndrome, is important for the differentiation of central and sensory neurons. We now show that formation, migration, and survival of sympathetic superior cervical ganglion neurons are impaired in Ndn-null embryos. We observed reduced innervation of superior cervical ganglion target organs, including the submandibular gland, parotid gland, and nasal mucosa. While the formation of other sympathetic chain ganglia is unaffected, axonal extension is impaired throughout the sympathetic nervous system. These results demonstrate a novel role for necdin in cellular migration, in addition to its roles in survival and axon outgrowth. Furthermore, reduced sympathetic function provides a plausible explanation for deficiencies of salivary gland function in individuals with congenital necdin deficiency consequent to Prader-Willi syndrome. Developmental Dynamics 237:1935–1943, 2008. © 2008 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Many clinically relevant aspects of Prader-Willi syndrome (PWS) suggest dysfunction of the autonomic nervous system (Wharton and Bresnan,1989; DiMario et al.,1994,1996; Goldstone,2004; Hart,1998; Stevenson et al.,2004; Choe et al.,2005). Ndn, encoding the protein necdin, is one of several genes deleted in individuals with PWS, and is highly expressed throughout the nervous system, including peripheral autonomic neurons (Aizawa et al.,1992; MacDonald and Wevrick,1997). A role for necdin in terminal differentiation of neurons is supported by studies showing that PC12 cells transfected with necdin have increased differentiation and accelerated neurite outgrowth (Tcherpakov et al.,2002), that repression of necdin in embryonic dorsal root ganglia suppresses their differentiation (Takazaki et al.,2002), and that expression of necdin induces neurite outgrowth in neuroblastoma cells (Kobayashi et al.,2002). The development and survival of autonomic neurons is dependent on neurotrophin signaling pathways, as demonstrated by defects in transgenic mice harboring mutations in neurotrophic factors and their cognate receptors (Ernfors et al.,1994a,b; Kuruvilla et al.,2004). Necdin participates in neurotrophin signaling through its interaction with both the low affinity neurotrophin receptor p75NTR and tropomyosin-regulated kinase TrkA in multiple systems, including transiently transfected cells, P19 embryonal carcinoma cells, and dorsal root ganglia (Tcherpakov et al.,2002; Andrieu et al.,2003; Kuwako et al.,2005).

Two strains of mice with gene-targeted deletions of necdin exhibit a high degree of neonatal lethality due to a defect in central respiratory rhythm generation (Gerard et al.,1999; Muscatelli et al.,2000; Ren et al.,2003). In the most severe cases, Ndn-null mice have commissural defects in the forebrain and axonal extension, bundling, and branching defects in central nervous system neurons (Lee et al.,2005). An increase in embryonic apoptosis was identified in embryonic sensory neurons of Ndn-null mice, accompanied by loss of sensory innervation in the adult mice (Takazaki et al.,2002; Kuwako et al.,2005; Andrieu et al.,2006). A reduced number of GABAergic but normal numbers of glutamatergic neurons were noted in the Ndn-null developing forebrain, suggesting that survival varies among neuronal populations with necdin-deficiency (Kuwajima et al.,2004). One additional strain of Ndn-null mice has no phenotype (Tsai et al.,1999).

We now demonstrate that necdin is essential for the normal development of the autonomic nervous system in vivo, by examining the embryonic development of the autonomic nervous system in Ndn-null mice. The neurons that form the superior cervical ganglia (SCG) are most profoundly affected by necdin deficiency, displaying reduced axonal outgrowth, reduced innervation of the salivary glands, and increased cell death. Surprisingly, we found impaired rostral migration of a subset of SCG neurons, demonstrating a previously unknown function for necdin in development. Other sympathetic ganglia in the thoracic and lumbar regions appear be formed normally, although a deficit in axonal outgrowth is observed. These data extend the range of neuronal subtypes affected by necdin-deficiency to include the autonomic nervous system.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Superior Cervical Ganglia Are Smaller and Are Abnormally Located in the Ndn-Null Mouse

The progenitors of sympathetic neurons are neural crest cells that migrate from the dorsal region of the developing neural tube toward the aorta to form the sympathetic trunk. One sub-lineage of these neural crest cells then migrates rostrally to the upper regions of the cervical vertebrae in a stereotypical manner to form the SCG (Nishino et al.,1999). Additional neural crest cells from the vagal region colonize the enteric nervous system (ENS) in the mid- and hindgut. SCG precursors can first be identified as a cell grouping at embryonic day (E) 10.5 in mice and by E11.5, they are aligned along the cervical vertebral column between cervical vertebra 1 (C1) and C7 (Nishino et al.,1999). At E12.5, SCG cells proliferate and begin to migrate away from the stellate ganglia, the closest caudally located ganglia in the sympathetic chain. By E14.5, the SCG neurons have reached their final destination at the bifurcation of the carotid artery. To better understand the role of necdin in the development of the sympathetic nervous system, we examined the SCG using thionin staining for general morphology and tyrosine hydroxylase (TH) immunohistochemistry to detect sympathetic neurons. At both E16.5 and E18.5, we detected TH-positive neurons in the sympathetic chain ganglia of both Ndn-null and control littermate embryos, which suggests that sympathetic noradrenergic neurons are normally specified in the absence of necdin. The most rostral of these neurons form the SCG, which migrate to the bifurcation of the carotid artery between cervical vertebral levels C1–C4 (Fig. 1A). While some SCG neurons have migrated to the C1–C4 region in Ndn-null embryos, the majority of SCG neurons are consistently located more caudally and are significantly reduced in number compared with control littermates (Fig. 1B,C). We also noted increased variability in the rostrocaudal location of the SCG between the left and right sides in Ndn-null embryos. These data suggest that, although noradrenergic specification proceeds normally, necdin is required for the final localization of the superior cervical ganglia.

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Figure 1. Necdin is required for rostral neuronal migration and survival of superior cervical ganglia (SCG) neurons. A: Thionin staining of parasagittal sections of embryonic day (E) 18.5 embryos oriented with the dorsal aspect (d) at the top and rostral (r) to the left. Arrowheads indicate the bifurcation of the carotid artery, arrows mark the SCG. C, cochlea. A′: The SCG in Ndn-null embryos are located in a more caudal position than in the control littermates. B: Whole-mount anti-tyrosine hydroxylase (TH) immunohistochemistry of E16.5 embryos, with the dorsal aspect toward the top and rostral to the left. Arrowheads indicate stellate ganglion. The dashed lines approximately outline the SCG. B′: The SCG in this typical Ndn-null embryo is identified by its expression of TH but extends abnormally caudally. C: Relationship of cell number of the SCG at E18.5 to its rostrocaudal location, expressed as the closest vertebral level to the middle of the ganglion. The two measurements indicated for each of four Ndn-null (NN) and control littermate (WT) embryos (eight measurements total) are from the right and left SCG. The SCG are 45% smaller in Ndn-null embryos than the control littermates (P < 0.0001), are located more caudally, and have greater variability in their final position between the left to right ganglia.

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We then examined the earliest stages of development of sympathetic neurons, to determine the timing of SCG precursor migration abnormalities in Ndn-null mice. Phox2B is a transcription factor required for the differentiation and expression of TH in noradrenergic sympathetic neurons (Goridis and Rohrer,2002). Using RNA in situ hybridization with an antisense probe to Phox2B, we detected SCG precursors in the correct position in all E12.5 embryos, indicating that the initial specification and ventral migration of these cells is not compromised (Fig. 2A). We then performed TH immunohistochemistry on cryosections and whole embryos between E11.5 and E16.5, to follow the progression of the SCG neurons as they migrate rostrally and extend axons toward their target tissues. At E11.5, the most rostral column of cells in the sympathetic chain, the SCG precursors, appears similar in Ndn-null embryos compared with control littermate embryos (Fig. 2B), although we did note a shortened column of SCG precursors in some Ndn-null embryos. At E12.5, there was reduced migration of a subset of SCG neurons but no difference in size of the SCG in Ndn-null embryos (Fig. 2C). By E14.5, the SCG neurons in control littermate embryos have reached their final location between vertebral levels C1 and C4. In contrast, the Ndn-null SCG have an elongated shape and extend from C1 to C7, directly above the stellate ganglia (Fig. 2D). In Ndn-null embryos, the SCG are also smaller at E14.5 compared with control littermates (83 ± 7% of control, P=0.02). In contrast to the continued rostral migration of SCG neurons in the wild-type embryos, no further migration of SCG neurons in Ndn-null embryos was observed from E14.5 to E18.5 (Fig. 1A,B). This suggests that necdin is dispensable for dorsal–ventral migration of neural crest sympathetic precursors but is required for proper caudal–rostral migration of a subset of SCG neurons during embryonic development.

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Figure 2. Normal dorsoventral but abnormal rostral migration of sympathetic precursors during embryogenesis. A: RNA in situ hybridization with a digoxigenin-labeled antisense probe to Phox2b was performed on transverse sections at embryonic day (E) 12.5 and detects neurons of the sympathetic trunk (ST). E, esophagus; Ijv, Internal jugular vein; d, dorsal; v, ventral. A′: The sympathetic trunk expressing Phox2b is comparable in placement and size in the Ndn-null embryo, which indicates that the specification and ventral migration of neural crest sympathetic precursor cells proceeds normally. B: Whole-mount tyrosine hydroxylase (TH) immunohistochemistry at E11.5. Dorsal is toward the top and rostral to the left in B–D. Note that the embryos in B and C are cleared to visualize both the left and right sympathetic trunk, with one side out of focus in the image. Arrows indicate the C7 vertebral level. B′: Formation of sympathetic trunk is comparable between Ndn-null and control littermate embryos at E11.5. C: Whole-mount TH immunohistochemistry (brown) at E12.5. Arrows indicate the nascent superior thoracic ganglia (STG). C′: The distance between the migrating superior cervical ganglia (SCG) and the stellate ganglion is now smaller in the Ndn-null mouse, and a group of cells between the SCG and stellate (ST) ganglion are already delayed in their rostral migration. D: Parasagittal sections at E14.5 immunolabeled for TH (green). The SCG is located at the bifurcation of the carotid artery in the control embryo and has a characteristic ganglion shape. The white lines depict the interval between the SCG and the stellate (ST) ganglion. The vertebral nerve (Ve) that projects from the stellate ganglion is shorter in the absence of necdin. H, heart. D′: The SCG in Ndn-null embryos typically extend to the C7 vertebral level and have an elongated shape.

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Ndn-Null SCG Have Reduced Innervation of Target Tissues and Display Increased Apoptosis

The reduced size of the migrating Ndn-null SCG prompted us to examine if increased cell death is observed in these neurons. We identified apoptotic cells by immunostaining with antibodies to cleaved caspase-3, and identified the ganglia by co-labeling the cryosections with neuron-specific antibodies (anti-βIII-tubulin at E12.5 and anti-neurofilament at E14.5 and E18.5). At E18.5, the percentage of cells in the SCG of Ndn-null embryos labeled with cleaved caspase-3 was higher by 1.5-fold (control 1.1 ± 0.4%, Ndn-null 1.6 ± 0.5%; P = 0.01) (Fig. 3). No significant difference was observed at E14.5 (control 0.16 ± 0.02%, Ndn-null 0.18 ± 0.03%), and cell death was not detected at E12.5 in either genotype. Cell proliferation was measured by immunolabeling with an antibody to Ki67, which is expressed in proliferating cells. No difference was observed in the percentage of proliferating cells at E12.5 (control 35 ± 4%, Ndn-null 32 ± 6%) or at E14.5 (control 21 ± 2%, Ndn-null 23 ± 3%). However, we did observe a small increase in proliferation at E16.5 in the Ndn-null embryos (control 19 ± 0.5%, Ndn-null 22 ± 2%; P = 0.02). We then examined axonal outgrowth and innervation of target tissues by the SCG by performing TH immunohistochemistry on whole mount embryos and cryosections at E18.5 (Fig. 4). The SCG neurons normally innervate the parotid and submandibular glands, nasal mucosa, and pupillary muscle. Outgrowth, branching and bundling of axons from the SCG is altered in Ndn-null embryos (Fig. 4A), which results in a large reduction in innervation of both salivary glands and of the nasal mucosa (Fig. 4B–D). We noted greatly reduced innervation of both salivary glands and of the nasal mucosa, with less innervation of the parotid gland than of the submandibular gland (2.8% and 18% of control littermate, respectively; Fig. 4). The glands themselves were of comparable size in wild-type and Ndn-null embryos, and the submandibular ganglia were of normal position and size.

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Figure 3. Cell death and proliferation in Ndn-null SCG. A,A′: Representative parasagittal sections at embryonic day (E) 18.5 co-labeled with antibodies to neurofilament (NF) and cleaved caspase-3 (detecting apoptotic cells). B,B′: Representative parasagittal sections at E14.5 labeled with an antibody to Ki67 (detecting proliferating cells, green) and stained with Hoescht stain (blue) to identify cell nuclei. The superior cervical ganglia (SCG) is outlined with a white dotted line and is more elongated in the Ndn-null embryo.

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Figure 4. Reduced innervation of superior cervical ganglia (SCG) targets is associated with defects in axonal outgrowth and branching. A: Whole-mount tyrosine hydroxylase (TH) immunohistochemistry of embryonic day (E) 16.5 embryos. Note that the embryo is cleared to visualize both the left and right sympathetic trunk, with one side out of focus in the images. Rostral is toward the top and ventral is toward the left. In the control embryo, the nerve from the SCG extends branches to innervate the sublingual glands as indicated by the arrowhead. A′: In the Ndn-null embryo, there is a lack of branching and reduced extension of neurites as indicated by arrows. B–D: Representative transverse sections of E18.5 embryos were labeled with an antibody to TH. B,B′: Nasal mucosa, rostral toward the top. ns, nasal septum, nc, nasal cavity, sk, skin. Rostral (r) is toward the top. C,C′: Submandibular gland. gl, glandular tissue, sk, skin. Rostral (r) is toward the top. D,D′: Parotid gland. sk, skin. Rostral (r) is to the left. E: Quantification of reduced innervation in Ndn-null salivary glands. Innervation of the parotid gland is reduced 35-fold and innervation of the submandibular gland is reduced 8-fold.

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Examination of the Caudal Sympathetic Chain Ganglia, Parasympathetic and Enteric Nervous Systems

We identified a marked impairment in the migration and axonal outgrowth of SCG neurons in Ndn-null embryos, which prompted us to investigate the remaining sympathetic ganglia located in the thoracic and lumbar regions of the embryo. The sympathetic ganglia located caudal to the SCG are formed by the dorsal–ventral migration of trunk neural crest cells, which are a different group of neural crest cells from those that form the SCG. In contrast to SCG neurons, the neurons of the more caudal sympathetic chain ganglia do not migrate in a caudal–rostral direction. We performed whole-mount TH immunohistochemistry on Ndn-null and control littermate embryos at E16.5 and E18.5 to examine the intact sympathetic nervous system in situ. We observed no difference in the size of the stellate ganglion, which is the first ganglion in the thoracic region of the sympathetic chain (Fig. 5A). Of interest, the vertebral nerve from the stellate ganglion projects in the correct direction but is consistently shorter in Ndn-null embryos (Fig. 5B). The more caudal thoracic and lumbar sympathetic ganglia are normal in size and position (Fig. 5A). The celiac ganglion and the mesenteric ganglia complex are correctly located in the lumbar region of the body, although axonal extensions are shorter in many Ndn-null embryos (Fig. 5C). We noted reduced innervation of intestinal loops and punctate TH-positive staining in the gut (Fig. 5D). This punctate TH staining is similar to what we observed to be associated with neurofilament positive dystrophic spheroidal structures in the brainstem of Ndn-null mice (Ren et al.,2003; Pagliardini et al.,2005). TH-positive axons innervating the heart of Ndn-null embryos also contained punctate structures and were less arborized compared with wild-type (Fig. 5E). Finally, we used Ret tyrosine kinase RNA in situ hybridization on parasagittal sections of E12.5 embryos and detected normal expression in the sympathetic chain ganglia, kidneys, and enteric nervous system in Ndn-null embryos, suggesting that neural crest progenitors can adequately populate the gastrointestinal tract (Fig. 6). Three cranial parasympathetic ganglia (sphenopalatine ciliary, and otic) were also examined but no gross defects were identified in the number of neurons present or the expected location of the ganglia. The adrenal glands were the same size in both genotypes (Ndn-null 96 ± 12% of control) and had similar numbers of cells expressing TH (control 7 ± 2%, Ndn-null 6 ± 1%). In summary, many sympathetic neurons examined had some degree of defective axonal extension. Nonetheless, the neuronal precursors derived from the vagal region of the spinal cord and that undergo rostral migration are preferentially affected by the loss of necdin, compared with those derived from the trunk neural crest.

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Figure 5. Loss of necdin does not affect the specification or localization of the remaining sympathetic chain ganglia but impairs neurite outgrowth. Whole-mount tyrosine hydroxylase (TH) immunohistochemistry of late-stage embryos. Note that the embryos are cleared to visualize both the left and right sympathetic trunk, with one side out of focus in the images. Dorsal (d) is toward the top and rostral (r) is toward the left. Arrowheads mark punctate TH-positive varicosities in the Ndn-null embryos. A,A′: The stellate ganglion (asterisk) and more caudal sympathetic ganglia in the thoracic region are comparable in size and location in Ndn-null and control embryos at embryonic day (E) 18.5. The heads have been removed from these embryos. B: Whole-mount TH-immunohistochemistry of E16.5 embryos. Arrow indicates the terminus of the vertebral nerve projecting from the superior thoracic ganglia (STG). B′: The vertebral nerve that projects from the stellate ganglion is shorter in the absence of necdin. C,C′: Whole-mount anti-TH immunohistochemistry of E18.5 embryos. The prevertebral sympathetic ganglion complex, which includes the inferior and superior mesenteric ganglia, and the celiac ganglion are similarly formed in Ndn-null and control littermates. However, sympathetic innervation of the intestinal tract (i) is visibly reduced in the Ndn-null embryo, with shorter and less branched axons (inset, see D,D′). L, liver. Arrow indicates the celiac ganglion complex. D,D′: Inset from C demonstrating reduced innervation of intestinal loops (arrows). E,E′: Innervation of the heart (H) by the thoracic ganglia is also compromised in Ndn-null embryos and punctate varicosities are seen. L, liver.

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Figure 6. Normal expression of Ret tyrosine kinase mRNA in the sympathetic chain ganglia, enteric nervous system and kidneys of Ndn-null embryos. RNA in situ hybridization with a digoxigenin-labeled antisense probe to Ret on parasagittal sections at embryonic day (E) 12.5 in control (A, B) and Ndn-null (A′,B′) embryos. A,A′: Ret expression in the sympathetic chain is marked with an arrow. H, heart. B,B′: Ret expression in the intestine (arrowhead) and kidney (arrow). Dorsal is toward the left, rostral toward the top. L, liver.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Loss of necdin had previously been shown to cause defects in the survival and neurite outgrowth of central and sensory neurons. We now show that loss of necdin in mice also impairs the survival and axonal elongation of sympathetic neurons and greatly reduces innervation of target glands, preferentially affecting the most rostral sympathetic ganglia. Unexpectedly, loss of necdin also greatly impairs the migration of SCG neurons in late embryogenesis, presenting a previously unknown cellular role for necdin. Significant deficits in the formation of other sympathetic chain ganglia were not observed, suggesting that necdin is particularly important for the rostral migration and survival, but not proliferation, of SCG neurons. The neurons that migrate to form the enteric nervous system also migrate a considerable distance into the gastrointestinal tract, but are normally located in the absence of necdin. The ventral migration of sympathetic precursors from the region near the dorsal neural tube to the aorta is also unaffected by the loss of necdin.

The migration of neurons is a universal feature in the development of the nervous system and ultimately requires rearrangement of the cytoskeleton, which is regulated by intracellular signaling pathways that induce cytoskeletal reorganization coordinated by the centrosome/microtubule organizing centre (Badano et al.,2005; de Anda et al.,2005). One important feature of cellular migration is the movement of the centrosome into the region between the nucleus and the leading edge, which determines the direction of migration, and requires an extensive amount of cytoskeletal rearrangement. Notably, axonal outgrowth also requires cytoskeletal rearrangement coordinated with the direction of migration (de Anda et al.,2005). Many proteins have been identified that regulate the cytoskeleton and associated molecular motors during these dynamic processes (Badano et al.,2005). We previously identified interactions between necdin and two proteins important in neuronal cytoskeletal rearrangement, namely fasciculation and elongation protein zeta-1 (Fez-1) and Bardet-Biedl syndrome 4 (BBS4; Lee et al.,2005). Bardet-Biedl syndrome, like PWS, is a genetic syndrome with obesity and developmental delay, but differs from PWS in that it is associated with ciliary dysfunction. Specifically, BBS4 recruits proteins to the pericentriolar region and is required for the anchoring of microtubules and cell cycle progression (Kim et al.,2004). Fez1 is essential for the activation of the microtubule-based molecular motor kinesin-1, but was first described for its role in axon bundling and outgrowth (Bloom and Horvitz,1997; Blasius et al.,2007). We previously observed varicosities in irregularly oriented axons of both serotonergic and motor neurons in Ndn-null embryos, and deficient neurite outgrowth was observed in explants of dorsal root ganglia at E13.5 (Kuwako et al.,2005). We now report similar neurite abnormalities in sympathetic neurons, manifesting as tyrosine hydroxylase-positive varicosities present where the heart and intestinal tract are innervated by sympathetic chain neurons. Thus, accumulating evidence suggests that loss of necdin impairs the cytoskeletal program required for the coordination of neurotrophin signaling and axonal outgrowth, and now additionally for cellular migration.

The survival of specific sets of embryonic neurons is also compromised by necdin-deficiency. In Ndn-null dorsal root ganglia, increased cell death occurs between E12 and E14 (Kuwako et al.,2005; Andrieu et al.,2006), while activity-deprivation related apoptosis is increased in postnatal Ndn-null cerebellar granule cells (Kurita et al.,2006). In this study, we observe increased apoptosis in Ndn-null sympathetic neurons at E18.5. Notably, decreased survival coincides with developmental time points during which target-derived neurotrophic support and intact neurotrophin signaling are essential for survival. The neurotrophic requirements of the SCG neurons have been most finely delineated through gene-targeting in mice. For example, deletion of the gene encoding the neurotrophin NT-3 causes deficits in proximal axon extension that lead to reduced innervation of sympathetic targets and a 50% loss of SCG (Ernfors et al.,1994a; Kuruvilla et al.,2004). Ablation of glial cell line-derived neurotrophic factor signaling through loss of the Ret tyrosine kinase gene causes defective migration and axonal outgrowth of the SCG, other sympathetic chain ganglia, and the ENS (Enomoto et al.,2001). In view of the dual role of neurotrophic factors in promoting neurite outgrowth and supporting neuronal survival, we favor a hypothesis that the limited availability of target-derived neurotrophic growth factors and reduced signaling through neurotrophin receptors combine to impede neurite outgrowth, and reduce the survival of late embryonic Ndn-null neurons. In summary, we propose that necdin plays a critical role in the interrelated processes that link growth factor-responsive intracellular signaling pathways with the cytoskeletal rearrangements required for cellular migration during development.

Necdin is a member of the type II MAGE (melanoma antigen) family of proteins, which have distinct roles in apoptosis and cell cycle progression in nervous system development (Barker and Salehi,2002). Notably, three MAGE proteins (NRAGE/MAGED1, MAGEH1, and MAGEG1) also interact with p75NTR and may have overlapping functions in the development of the nervous system, providing some functional redundancy in specific neuronal subsets (Salehi et al.,2000,2002; Tcherpakov et al.,2002; Kuwako et al.,2005; Kurita et al.,2006). NRAGE is expressed in the superior cervical ganglia and the sympathetic chain (Kendall et al.,2002). We then reviewed the expression profiles presented in an analysis of expressed sequence tag counts, using the National Center for Biotechnology Information EST Profile Viewer. Only necdin, MAGED2, and MAGEE1 are represented among 10,966 transcripts derived from murine sympathetic ganglia cDNA libraries. These three genes and MAGED1 are also more widely expressed in the brain and other parts of the nervous system.

People with Prader-Willi syndrome have a congenital absence of necdin (Jay et al.,1997; MacDonald and Wevrick,1997; Sutcliffe et al.,1997). We previously proposed that defects in axonal extension and arborization in the developing central nervous system could be implicated in cognitive and respiratory compromise in people with PWS (Ren et al.,2003; Pagliardini et al.,2005). We observed reduced innervation of the salivary glands in Ndn-null embryos, which if present in people with PWS, could account for the observed abnormalities in saliva output that generate adverse effects, most notably difficulty with swallowing (Hart,1998). A defect in parasympathetic innervation of the gut has previously been described as a possible reason for gastrointestinal impairments observed in PWS (Goldstone,2004). We did not observe such innervation defects in the gut of Ndn-null embryos, nor did we find defects in the population of the gut by enteric neurons, but sympathetic innervation of the intestinal tract was compromised. Given the complexity of the MAGE protein family, it is likely that functional redundancies in mice may not completely mirror those that are present in humans. Notably, the MAGE protein MAGEL2 is also congenitally absent in people with PWS (Boccaccio et al.,1999; Lee et al.,2000). MAGEL2 is highly expressed in the hypothalamus, which conveys information about autonomic status to the peripheral autonomic system through the lateral medulla. It is possible that the combined loss of necdin and MAGEL2 could have a more severe impact on autonomic function than loss of necdin alone, through combined central and peripheral autonomic dysfunction. Alternatively, the loss of necdin function in the smooth muscle of the intestine could contribute to gastric dysmotility independent of autonomic dysfunction. Further studies that examine whether there is reduced innervation or decreased smooth muscle function in the gastrointestinal tract in people with PWS, and that examine the effect of the concurrent loss of necdin and Magel2 in mice are needed to further test these hypotheses.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Mouse Breeding and Genotyping

All animal studies procedures were conducted in accordance with the Canadian Council on Animal Care Guidelines and Policies with approval from the Health Sciences Animal Policy and Welfare Committee for the University of Alberta. The Ndntm2Stw mice were originally on a mixed genetic background (W9.5 (129S1) / C57Bl/6; Ren et al.,2003) and subsequently back-crossed to C57Bl/6 for at least 14 generations. The Ndntm2Stw mouse colony was maintained by breeding Ndn−/+ female mice carrying a maternally inherited Ndn-lacZ knockin allele with C57Bl/6 male mice to generate heterozygous, functionally wild-type offspring. C57Bl/6 female mice were then bred to Ndn−/+ male mice carrying a maternally inherited Ndn-lacZ knockin allele, to generate Ndn+/− embryos carrying a paternally inherited lacZ knockin allele (referred to as Ndn-null) and Ndn+/+ (control littermate) embryos. Because of imprinting that silences the maternally inherited allele, heterozygous Ndn-null mice with a paternally inherited lacZ knockin allele retain expression only of this mutant allele and have no expression of Ndn. Mice were genotyped from tissue samples or ear notch biopsies as described (Ren et al.,2003).

Histological Staining and Immunohistochemistry

Embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline (PFA) and were cryosectioned at 20 to 30 μm. Sections were stained with thionin and adjacent sections were prepared for immunohistochemistry. Results were confirmed in Ndn-null and control littermates from multiple litters. Antibodies were rabbit anti- TH (Chemicon, used at 1:2,000), mouse anti-neurofilament (2H3, Developmental Studies Hybridoma Bank, used at 1:2,000), neuron-specific βIII-tubulin (TUJ1, Chemicon, used at 1:1,000), rabbit anti-cleaved caspase-3 (Cell Signaling, used at 1:1,000), and rabbit anti-Ki67 (AbCam, used at 1:1,000). Secondary antibodies for immunofluorescence were Alexa 594 goat anti-rabbit and Alexa 488 goat anti-mouse (Molecular Probes) both used at a dilution of 1:1000.

For whole-mount anti-TH immunohistochemistry, embryos were fixed in 4% PFA, dehydrated in a methanol series and incubated overnight in 20% dimethyl sulfoxide (DMSO)/80% methanol with 3% hydrogen peroxide to quench endogenous peroxidase activity. Tissues were rehydrated, blocked overnight in blocking solution (5% skim milk powder/5% DMSO/1% Tween-20 in PBS) and incubated for 48–72 hr at 4°C with a sheep anti-TH antibody (1:500, Chemicon) in blocking solution. Next, tissues were incubated overnight with a peroxidase-conjugated anti-sheep secondary antibody in blocking solution. After washing in PBS with 1% Triton X-100, tissues were incubated with diaminobenzidine for 30–60 min in the dark then the color was developed by adding hydrogen peroxide solution. The reaction was stopped by placing tissues in 4% PFA overnight. Upon dehydration in a methanol series, tissues were cleared in 1:2 benzyl alcohol: benzyl benzoate and stored at 4°C until photography.

RNA In Situ Hybridization on Mouse Embryos

RNA in situ hybridization with digoxigenin-labeled probes on 25-μm cryosections was performed as previously described (Lee et al.,2003). The Phox2b probe was kindly provided by Dr. J.F. Brunet (CNRS, Marseilles). The Ret probe pMCRET7 was kindly provided by Dr. V. Pachnis (National Institute for Medical Research, London, UK). Control experiments with no probe or sense probes gave either no signal or a uniformly low background staining, as expected (data not shown).

Image Analysis

All organs were reconstructed from images of serial sections with Adobe Photoshop and analyzed with ImageJ software. Relative SCG sizes were estimated by thionin staining as the area occupied on the three to five consecutive sections that contained the entire ganglion, in multiple embryos from multiple litters. SCG cell numbers were determined counting nuclei labeled with Hoechst (total cells) or labeled by anti-cleaved caspase-3 (for cell death) or Ki67 (for proliferation). The innervation of SCG target organs was measured as the amount of anti-TH immunofluorescence as a fraction of total area occupied by each organ. The relative size of the adrenal gland was estimated by the area labeled with the anti-TH antibody on sections through the entire gland, measuring every third section. Statistical comparisons between Ndn-null and control littermate embryos (four to eight ganglia from each genotype) were made using the Student t-test with P ≤ 0.05 considered significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We gratefully acknowledge the support of the Foundation for Prader-Willi Research and One Small Step. AAT was supported by a studentship from the Natural Sciences and Engineering Research Council of Canada, the Alberta Heritage Foundation for Medical Research, and the CIHR Training Program in Maternal, Fetal and Newborn Health. C.B.G. was supported by a studentship from the Alberta Heritage Foundation for Medical Research.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES