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Keywords:

  • neural crest;
  • epibranchial placode;
  • parasympathetic ganglion;
  • visceral sensory ganglion;
  • autonomic nervous system

Abstract

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

Background: The neural crest (NC) and placode are transient neurogenic cell populations that give rise to cranial ganglia of the vertebrate head. The formation of the anterior NC- and placode-derived ganglia has been shown to depend on the single activity of either Neurog1 or Neurog2. The requirement of the more posterior cranial ganglia on Neurog1 and Neurog2 is unknown. Results: Here we show that the formation of the NC-derived parasympathetic otic ganglia and placode-derived visceral sensory petrosal and nodose ganglia are dependent on the redundant activities of Neurog1 and Neurog2. Tamoxifen-inducible Cre lineage labeling of Neurog1 and Neurog2 show a dynamic spatiotemporal expression profile in both NC and epibranchial placode that correlates with the phenotypes of the Neurog-mutant embryos. Conclusion: Our data, together with previous studies, suggest that the formation of cranial ganglia along the anterior-posterior axis is dependent on the dynamic spatiotemporal activities of Neurog1 and/or Neurog2 in both NC and epibranchial placode. Developmental Dynamics 241:229–241, 2012. © 2011 Wiley Periodicals, Inc.


INTRODUCTION

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

The cranial placode and neural crest (NC) are essential for generating the complex autonomic circuitry of the vertebrate head and body (Le Douarin et al., 1981; D'Amico-Martel and Noden, 1983; Baker and Bronner-Fraser, 1997, 2001; Schlosser, 2010). In particular, these two transient neurogenic cell populations give rise to cranial sensory and parasympathetic ganglia. The cranial placodes are specialized focal thickenings of the ectoderm located at the lateral surfaces of the face and branchial arches (bas). They give rise to cranial sensory ganglia that detect information related to temperature, touch, balance, hearing, and visceral or autonomic functions (Fode et al., 1998; Ma et al., 1998). The order of these ganglia along the anterior-posterior (AP) axis is as follows: trigeminal, geniculate, vestibulocochlear, petrosal, and nodose ganglia, which contribute to cranial nerves V, VII, VIII, IX, and X, respectively. These ganglia also receive contributions from NC cells that originate from the dorsal midbrain and specific rhombomeres (r) of the hindbrain.

The NC also gives rise to postganglionic (PG) neurons of parasympathetic ganglia that are functional complements of epibranchial placode-derived visceral sensory ganglia associated with cranial nerves VII, IX, and X (D'Amico-Martel and Noden, 1983; Enomoto et al., 2000; Yang et al., 2008; Coppola et al., 2010; Takano-Maruyama et al., 2010; Chen et al., 2011). (Note that the ciliary ganglion associated with cranial nerve III is not included in this category because it is not associated with a complementary epibranchial placode-derived visceral sensory ganglion.) Like the NC-derived precursors of the proximal ganglia, the NC-derived precursors of the parasympathetic ganglia are aligned with specific placodes along the AP axis (Takano-Maruyama et al., 2010). The positional relationship between placode and NC cells is reciprocal, and is critical for their respective migration, differentiation, and gangliogenesis (Stark et al., 1997; Begbie and Graham, 2001; Shiau et al., 2008; Coppola et al., 2010; Takano-Maruyama et al., 2010). It appears that the alignment and subsequent interaction between placode and NC cells are critical for establishing the functional organization of the peripheral circuit corresponding to the various cranial nerves along the AP axis. This appears to be a general feature of craniofacial morphogenesis (Lumsden and Keynes, 1989; Lumsden et al., 1991; Lumsden and Krumlauf, 1996).

The formation of NC- and placode-derived ganglia is controlled by both cell-autonomous and non-cell-autonomous mechanisms. The bHLH transcription factors Neurog1 and Neurog2 are principle cell-autonomous determinants of cranial sensory ganglia (Fode et al., 1998; Ma et al., 1998). Neurog1 is required for the formation of the NC-derived trigeminal and placode-derived vestibulocochlear ganglia, and Neurog2 is required for the formation of placode-derived geniculate ganglia. These observations support a model in which cranial ganglia derived from complementary embryonic tissues (NC and placode) depend on the activity of either Neurog1 or Neurog2. The trigeminal ganglion has also been shown to be under non-cell-autonomous control (Stark et al., 1997; Schwarz et al., 2008; Shiau et al., 2008). Trigeminal gangliogenesis requires the reciprocal interaction between midbrain-derived NC and ophthalmic placode-derived cells. At the molecular level, this interaction appears to be mediated between the receptor Robo2 expressed by placodal cells, and the its cognate ligand Slit1 expressed by NC cells (Shiau et al., 2008).

Like the trigeminal ganglion, the reciprocal interaction between r4-derived NC cells and adjacent cells of the placode-derived geniculate ganglion in ba2 has been shown to be critical for parasympathetic and visceral sensory gangliogenesis, respectively (Begbie and Graham, 2001; Coppola et al., 2010; Takano-Maruyama et al., 2010). In the absence of r4-derived NC cells, the adjacent placode-derived geniculate ganglion fails to migrate inwards and make proper connections with the hindbrain (Begbie and Graham, 2001). Conversely, the loss of the placode-derived geniculate ganglion results in apoptosis of r4-derived NC fated for the parasympathetic sphenopalatine and submandibular ganglia (Coppola et al., 2010; Takano-Maruyama et al., 2010). The reciprocal interaction between complementary placode- and NC-derived cells, therefore, contributes to the formation of a common ganglion (i.e., trigeminal), or complementary-functional ganglia (i.e., visceral sensory geniculate and parasympathetic sphenopalatine/submandibular ganglia) that contribute to a common autonomic neural circuit.

The aforementioned studies focused on the regulation of the anterior cranial ganglia, the trigeminal, geniculate, sphenopalatine, and submandibular (Fode et al., 1998; Ma et al., 1998; Coppola et al., 2010; Takano-Maruyama et al., 2010). However, it remains to be determined whether the molecular and cellular requirements that are essential for the anterior cranial ganglia also apply to the more posterior cranial ganglia. A comprehensive analysis of the molecular and cellular requirements for cranial gangliogenesis along the AP axis may reveal both distinct and common regulatory principles that govern this critical developmental process.

To begin to address this issue, we examined the Neurog requirement for the more posterior placode-derived visceral sensory nodose and NC-derived parasympathetic otic ganglia. We show that unlike the anterior cranial ganglia (Fode et al., 1998; Ma et al., 1998; Coppola et al., 2010; Takano-Maruyama et al., 2010; Chen et al., 2011), the more posterior cranial ganglia require both Neurog1 and Neurog2. In addition to its known expression in the placode and placode-derived visceral sensory ganglia (Fode et al., 1998; Ma et al., 1998), we found through temporal fate mapping that Neurog1 and Neurog2 are expressed in NC cells fated for the parasympathetic otic ganglion. Specifically, Neurog2 has a relatively protracted activity in NC cells fated for the PG lineage, whereas the activity of Neurog1 is transient. This indicates that the synergistic activities of Neurog1 and Neurog2 are both temporally and spatially coordinated, occurring during a narrow time window among a small population of NC cells. Our observations suggest that the formation of cranial ganglia along the AP axis is dependent on the dynamic and differential expression of the Neurog genes in both the placode and the NC.

RESULTS

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

Redundant Functions of Neurog1 and Neurog2 in Placode-Derived Nodose Gangliogenesis

To determine whether the formation of the epibranchial placode-derived geniculate (VIIg), petrosal (IXg), and nodose (Xg) ganglia are sensitive to doses of Neurog1 and Neurog2, we analyzed mouse embryos harboring different combinations of Neurog1 and Neurog2 mutations. The expression of the Phox2b protein in whole-mount e10.5–11 littermate embryos was used to identify the presence of placode-derived ganglia (Pattyn et al., 1997, 1999, 2000; Fode et al., 1998; Ma et al., 1998). We confirmed that the formation of the geniculate ganglion is independent of Neurog1 (Fig. 1A, B). Furthermore, we observed that the geniculate ganglion formed despite the loss of a single allele of Neurog2 in the background of the Neurog1−/− embryo (Neurog1−/−2+/−) (Fig. 1C). The formation of the more posterior petrosal and nodose ganglia also occurred in this mutant background.

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Figure 1. One allele of either Neurog1 or Neurog2 is required for the formation of the placode-derived nodose ganglia. A–F: Lateral view of e10.5–11 whole-mount embryos immunolabeled for Phox2b: control (A, D), Neurog1−/− (B), Neurog1−/−2+/− (C), Neurog2−/− (E), and Neurog1+/−2−/−. Phox2b labels the epibranchial placode-derived geniculate (VIIg), petrosal (IXg), and nodose (Xg) ganglia (boxed area). At this level of analysis, it appears that the geniculate and petrosal ganglia are entirely dependent on Neurog2 (E). In contrast, the more posterior nodose ganglia form despite the absence of 3 alleles of Neurog1 and Neurog2 (C, F), suggesting that Neurog1 and Neurog2 are redundant. Scale bars = 500 μm.

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We next tested whether the requirement of the placode-derived nodose ganglion is dependent on different combinations of the Neurog genes. Previous work has shown that the formation of the geniculate and petrosal ganglia is solely dependent on Neurog2 (Fig. 1D, E) (Fode et al., 1998). We reasoned that a loss of one allele of Neurog1 in the background of Neurog2−/− could alter the formation of the nodose ganglion. Despite the loss of these three alleles, the formation of the nodose ganglion occurred in Neurog1+/−2−/− embryos (Fig. 1D, F). However, we cannot exclude the possibility that the number of Phox2b-expressing neurons were reduced in these mutants. This leaves the possibility that the formation of the posterior-most placode-derived nodose ganglion is dependent on the redundant function of Neurog1 and Neurog2.

To address this, we generated compound mutant Neurog1−/−2−/− embryos. In whole-mount preparations of e9.5 Neurog1−/−2−/− embryos, we observed no remnants of the nodose ganglion, as indicated by the absence of Phox2b protein expression compared to control littermate embryos (Fig. 2A, B). However, the expression of Isl1 protein in the placode was intact in Neurog1−/−2−/− embryos (Fig. 2C, D). This suggests that cells fail to delaminate to form the sensory neurons of the nodose ganglion. This phenotype is consistent with the defect observed in the geniculate placode of Neurog2−/− embryos (Fode et al., 1998).

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Figure 2. Formation of the placode-derived nodose ganglia requires both Neurog1 and Neurog2. A–D: Lateral view of e9.5 whole-mount embryos double-immunolabeled for Phox2b and Isl1: control (A, C) and Neurog1−/−2−/− (B, D). The channels were separated into different panels for clarity. The presence of delaminated Phox2b-expressing neurons of the placode-derived geniculate (VIIg), petrosal (IXg), and nodose (Xg) ganglia is completely absent in the Neurog1−/−2−/− embryo (B), whereas the expression of Isl1 in the geniculate (VIIp), petrosal (IXp), and nodose (Xp) placodes is intact (D). E, F: Sagittal sections of e10.5 embryos double-immunolabeled for Phox2b (red) and Isl1 (green): control (E) and Neurog1−/−2−/− (F). The Phox2b/Isl1-expressing neurons of the placode-derived petrosal and nodose ganglia are completely missing in Neurog1−/−2−/− embryo. However, the Isl1-expressing placodes remain intact. G, H: Sagittal sections of e12.5 embryos double-immunolabeled for Phox2b (red) and Isl1 (green): control (G) and Neurog1−/−2−/− (H). The Phox2b/Isl1-expressing neurons of the placode-derived petrosal and nodose ganglia remain missing in Neurog1−/−2−/− embryo, eliminating the possibility of a delay in delamination and differentiation of placodal cells. R, rostral; D, dorsal. Scale bars = 100 μm.

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We next addressed a possible delay in the differentiation of the nodose ganglion in Neurog1−/−2−/− embryos, as observed for the geniculate ganglion of Neurog2−/− embryos (Fode et al., 1998). In sagittal sections of e10.5 and e12.5 Neurog1−/−2−/− embryos double-immunolabeled for Phox2b (red) and Isl1 (green), we observed no delaminating or differentiating Phox2b+ placode-derived neurons at the clefts of branchial arch 3 (ba3) and 4 (ba4), the origin and areas normally occupied by the petrosal and nodose ganglia, respectively (Fig. 2E–H). This indicates that the loss of the nodose ganglion in Neurog1−/−2−/− embryos is a consequence of a failure of Isl1-expressing placodal cells to delaminate and differentiate into Phox2b-expressing visceral sensory neurons of the nodose ganglion. However, we cannot exclude the possibility that placode-derived cells delaminate, and are rapidly eliminated by apoptosis (Ma et al., 1998; Takano-Maruyama et al., 2010). In contrast to the trigeminal, geniculate, and petrosal ganglia, the formation of the nodose ganglion is, therefore, dependent on the redundant activities of Neurog1 and Neurog2.

Differential Requirements for Neurog1 and Neurog2 in Parasympathetic Otic Gangliogenesis

We next addressed whether Neurog1 and Neurog2 are also required for the formation of NC-derived postganglionic (PG) neurons of the parasympathetic otic ganglion, or whether they act in a single gene-dependent manner. Analysis of sagittal sections of e12.5 Neurog1−/− embryos show that the formation of Phox2b-expressing PG neurons (red) of the otic ganglion (Og) and Phox2b/Isl1-expressing neurons (yellow) of the geniculate ganglion (VIIg) is unaffected by this mutation compared to control littermate embryos (Fig. 3A, B). In contrast, the Isl1-expressing neurons of the trigeminal ganglion were completely absent as previously described (Fig. 3A, B) (Ma et al., 1998). However, we detected the presence of Phox2b/Isl1-expressing neurons, which may represent placode-derived sensory neurons that contribute to the trigeminal ganglion (Fig. 3B, arrow). In contrast, the PG neurons of the otic ganglion were reduced in e12.5 Neurog2−/− embryos compared to control littermates (Fig. 3C, D). This confirmed our previous report (Chen et al., 2011). The remaining population of PG neurons in Neurog2−/− embryos may, therefore, be dependent on both Neurog1 and Neurog2. To address this, we analyzed Neurog1−/−2−/− embryos. In sagittal sections of e12.5 Neurog1−/−2−/− embryos, we observed a complete loss of PG neurons of the otic ganglion, as well as the putative placode-derived Phox2b/Isl1-expressing neurons of the trigeminal ganglion (Fig. 3E, F; data not shown). Our findings suggest that the formation of NC-derived PG neurons of the otic ganglion is largely dependent on Neurog2, with a requirement for both Neurog1 and Neurog2 in a small subset of PG neurons. In summary, the complete formation of the placode-derived nodose and NC-derived otic ganglia is dependent on the redundant activities of Neurog1 and Neurog2.

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Figure 3. Formation of the NC-derived otic ganglia requires both Neurog1 and Neurog2. A–F: Sagittal sections of e12.5 embryos double-immunolabeled for Phox2b (red) and Isl1 (green): control (A, C, E), Neurog1−/− (B), Neurog2−/− (D), and Neurog1−/−2−/− (F). In the Neurog1−/− embryo, the Isl1-expressing trigeminal ganglion (Vg) is missing, whereas the Phox2b-expressing otic (Og) and Phox2b/Isl1-expressing geniculate (VIIg) ganglia remain intact (A, B). A cluster of Phox2b/Isl1-expressing cells remains intact in the Neurog1−/− embryo (arrow, B). This may represent placode-derived neurons that contribute to the trigeminal ganglion. In the Neurog2−/− embryo, the Phox2b-expressing otic ganglion is significantly reduced, and Phox2b/Isl1-expressing geniculate ganglion is missing. The Isl1-expressing trigeminal ganglion is intact in the Neurog2−/− embryo. In the Neurog1−/−2−/− embryo, all neural crest (Phox2b) and placodal (Phox2b/Isl1) ganglia are absent. Note that unlike the placode-derived geniculate and trigeminal ganglia (arrow in A, B), the placode-derived vestibulocochlear ganglion (VIIIg) expresses Isl1, but not Phox2b. Ov, otic vesicle. Scale bars = 100 μm.

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Interaction of r6 Neural Crest and Placode-Derived Petrosal Ganglion

We next wanted to understand the mechanism by which the NC-derived PG neurons of the otic ganglion were lost in Neurog2−/− or Neurog1−/−2−/− embryos. Previously, we showed that population of r4-derived NC cells fated for the PG lineage of the sphenopalatine and submandibular ganglia differentiated in the adjacent placode-derived geniculate ganglion en route to their final destination (Takano-Maruyama et al., 2010). Moreover, conditional ablation of Phox2b in the placode-derived geniculate ganglion or a null mutation of Neurog2 resulted in a loss of the sphenopalatine and submandibular ganglia (Coppola et al., 2010). We reasoned that a similar cellular relationship occurs in the more posterior population of NC and placodal cells fated for the otic and petrosal ganglia, respectively.

To address this, we first performed genetic lineage labeling to monitor the fate of postotic-derived NC cells. This broad hindbrain origin has been shown in the quail/chick chimera model to give rise to the parasympathetic otic ganglion (D'Amico-Martel and Noden, 1983). To do a more rhombomere-restricted genetic cell lineage analysis, we used the Cre/loxP system together with immunolabeling for the transcription factors Sox10 and Phox2b, which together identify NC cell–derived PG neurons (Pattyn et al., 1999; Enomoto et al., 2000; Britsch et al., 2001; Kim et al., 2003). We used Hoxa3-Cre to express Cre recombinase in NC cells that emerged from r5–r7, and Pax7-Cre to label NC cells from r1/r3/r5 (Mansouri et al., 1996; Macatee et al., 2003; Keller et al., 2004). These mice were crossed with a Rosa-EYFP reporter mouse strain (Srinivas et al., 2001) to trace the fate of Hoxa3- or Pax7-expressing NC cells. Hoxa3 lineage-labeled e12.5 embryos revealed that the majority of Phox2b-expressing PG neurons of the otic ganglion expressed EYFP, indicating that these cells originated from NC cells posterior to r4 (Fig. 4A). Pax7 lineage-labeled e12.5 embryos showed few, if any, Phox2b-expressing PG neurons labeled with EFYP, indicating that r1, r3, and r5 do not contribute appreciable PG neurons to the otic ganglion (Fig. 4B). We also cannot exclude the possibility that r7/r8-derived NC cells contribute to the otic ganglion. However, previous studies have shown that this region of the hindbrain contributes to the caudal migratory vagal NC population that gives rise to enteric neurons (Kulesa et al., 2000; Bogni et al., 2008). Nevertheless, our findings, which are consistent with a previous quail-chick chimera study (D'Amico-Martel and Noden, 1983), suggest that the majority of NC-derived PG neurons of the otic ganglion originate from r6.

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Figure 4. Origin, migration, and differentiation of NC-derived PG neurons of the otic ganglia. A: Sagittal section of an e12.5 Hoxa3Cre/+;RosaEYFP/+ (Hoxa3-L, L for lineage) embryo double-immunolabeled for GFP (green) and Phox2b (red). Postganglionic (PG) neurons of the otic ganglia (Og) express both GFP and Phox2b, whereas neurons of the geniculate ganglia (VIIg) express Phox2b. This indicates that the PG neurons are largely derived from Hoxa3-expressing neural crest cells. B: Sagittal section of an e12.5 Pax7Cre/+;RosaEYFP/+ (Pax7-L) embryo triple-immunolabeled for GFP (green), Phox2b (red), and Sox10 (blue). Most, if not all, PG neurons of the otic ganglia were negative for GFP, suggesting that they do not arise from Pax7-expressing cells. C: Sagittal section through an e9.0 control embryo double immunolabeled for Phox2b (green) and Sox10 (red). The neurons of the placode-derived petrosal ganglia are Phox2bHigh-single positive, whereas the PG precursors that appear to emerge from the petrosal ganglia express both Phox2bLow and the NC marker Sox10 (boxed area). D: Transverse section of an e10.5 Hoxa3Cre/+;RosaEYFP/+ embryo triple-immunolabeled for GFP (green), Sox10 (red), and Phox2b (blue). A stream of r6-derived GFP/Sox10-labeled NC cells appears to be in contact with the placode-derived petrosal (IXg) ganglion. E: Sagittal section of an e11.5 Hoxa3Cre/+;RosaEYFP/+ embryo triple-immunolabeled for GFP (green), Phox2b (red), and Isl1 (blue). A stream of GFP/Phox2b-postive cells (boxed area) appears to be migrating rostrally from the petrosal ganglion toward their terminal target area ventral to the geniculate ganglion. F: Sagittal section of an e12.5 Hoxa3Cre/+;RosaEYFP/+ embryo triple-immunolabeled for GFP (green), Phox2b (red), and Sox10 (blue). A stream of GFP/Phox2b-postive cells (arrow) appears to be migrating rostrally from the petrosal ganglion to the area just ventral to the geniculate ganglion (VIIg), the location of the otic ganglion (Og). R, rostral; V, ventral. Scale bars = (A–C) 50 μm; (D–F) 100 μm.

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We next followed the migratory route of NC cells from r6 to the terminal location of the otic ganglion, just ventral region to the placode-derived geniculate ganglion in ba2 (Enomoto et al., 2000; Chen et al., 2011). At e9.0–9.5, a stream of r6-derived NC cells appeared to be migrating through the adjacent placode-derived petrosal ganglion (Fig. 4C). In contrast to the Phox2bHigh-expressing neurons of the placode-derived petrosal ganglion, the r6-derived NC cells expressed Sox10 and relatively low levels of Phox2b (Fig. 4C, boxed area; and data not shown). In transverse sections of e10.5 Hoxa3 lineage-labeled embryos, a stream of EYFP/Sox10-expressing NC cells persisted from the dorsal edge of the r6 to the adjacent Phox2b+ placode-derived petrosal ganglion (Fig. 4D). We distinguished between the r6 and r7 NC-derived streams by a gap of NC-free zone that spans ∼100 μm along the rostrocaudal axis at e10.5 (data not shown). Between e11.5 and e12.5, we observed appreciable numbers of Hoxa3 lineage-labeled NC cells expressing both Sox10 and Phox2b that appeared to be emerging from the placode-derived petrosal ganglion (Fig. 4E, F; sagittal sections). These findings suggest that PG neurons of the otic ganglion originated from r6, and differentiated into PG neurons within the placode-derived petrosal ganglion, and migrated toward their terminal site for a protracted period (∼e9.0 to e12.5). However, it is unclear weather the NC fated for the PG neurons during this protracted period reside within the petrosal ganglion and/or arise from the NC stream located between r6 and the petrosal ganglion.

The snapshot of Hoxa3 lineage-labeled NC cells spanning from r6 to the petrosal ganglion, and the area just ventral to the geniculate ganglion in ba2, suggests strategic areas that can influence the migration or differentiation of PG precursors. This putative migratory/differentiation pathway is consistent with what we observed for the r4 NC-derived population that differentiated into the more anterior parasympathetic sphenopalatine and submandibular ganglia (Takano-Maruyama et al., 2010). Importantly, it suggests that the loss of the placode-derived petrosal ganglion in Neurog2−/− and Neurog1−/−2−/− embryos may affect the fate of the NC-derived PG precursors of the otic ganglion.

Survival of r6 Neural Crest Associated With the Placode-Derived Petrosal Ganglion

To better understand the fate of r6 NC-derived PG precursors of the otic ganglion in Neurog2−/− embryos, the Hoxa3 lineage reporter was incorporated into this mutant background. The Neurog2−/− background allowed us to follow the fate of PG precursors that give rise to the otic ganglion, which we cannot do in Neurog1−/−2−/− embryos (Fig. 3C, D). We examined the distribution of Hoxa3 lineage–labeled NC cells from the dorsal edge of r6 to the petrosal ganglion at e10.5, and when they appeared to emerge from the petrosal ganglion at later stages between e11.5 and e12.5. In control embryos harboring the Hoxa3 lineage reporter, the EYFP-expressing NC cells delaminated from the dorsal edge of r6 and migrated as a prominent stream to the adjacent Phox2b/Isl1-expressing placode-derived petrosal ganglion (Fig. 5A, arrow). In contrast, the EYFP-expressing NC cells delaminated normally, but the NC stream that is normally present between r6 and the petrosal ganglion was not evident in Neurog2−/− embryos (Fig. 5B).

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Figure 5. Loss of placode-derived petrosal ganglia associated with a decrease in r6 NC-derived migratory PG neurons. A,B: Transverse sections through r6 and ba3 of control (Neurog2+/−;Hoxa3cre;RosaEYFP/+) and Neurog2−/−;Hoxa3cre;RosaEYFP/+ embryos immunolabeled for GFP (green), Phox2b (red), and Isl1 (blue). Arrow shows stream of Hoxa3 lineage–labeled NC cells in the control embryo that is interrupted in the Neurog2−/− embryo. C,D: Transverse sections through r6 and ba3 of control (Neurog2+/−;Hoxa3cre;RosaEYFP/+) and Neurog2−/−;Hoxa3cre;RosaEYFP/+ embryos immunolabeled for GFP (green) and Sox10 (blue), and assayed for TUNEL (red). There is an increase in TUNEL-positive grains in the area proximal to r6 in the Neurog2−/− embryo. Note that the light blue–stained cells represent neural crest cells that co-express EYFP (green) and Sox10 (blue). The TUNEL-positive grains (red) appear orange because each panel represents multiple confocal images that have been stacked. This gives the illusion that the TUNEL-positive grains are co-localized with the EYFP/Sox10-positive neural crest cells. By inspecting individual images, the TUNEL-positive grains are located outside of the cells (data not shown). E,F: Transverse sections through r7 and ba4 of control (Neurog2+/−;Hoxa3cre;RosaEYFP/+) and Neurog2−/−;Hoxa3cre;RosaEYFP/+ embryos immunolabeled for GFP (green) and Sox10 (blue), and assayed for TUNEL (red). Note that the r7-derived neural crest stream between r7 and the placode-derived nodose ganglion is intact in the Neurog2−/− embryo. G: TUNEL-positive grains were counted associated with r6 and r7 NC cells in e10.5 control and Neurog2−/− Hoxa3-lineage-labeled littermate embryos (n=4). Error bar indicates standard deviation. Significant apoptosis was observed for both r6- and r7-derived NC cells in Neurog2−/− embryos, but the effect was more dramatic at the level of r6 (see text). *P<0.001, **P<0.01, bar = standard deviation. Scale bars = 100 μm.

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To determine how the r6 NC stream was lost in Neurog2−/− embryos, we examined programmed cell death or apoptosis, as reflected by the presence of TUNEL-positive grains or cellular debris at e10.5. We found a significant increase in TUNEL-positive grains among the Hoxa3 lineage–labeled r6 NC stream in Neurog2−/− embryos compared to control littermates (198.5 ± 75 vs. 827.75 ± 225 TUNEL-positive grains/r6 NC stream, P<0.001; n=4 embryos) (Fig. 5C, D, G). Although not as dramatic, we also observed a significant elevation of apoptosis among Hoxa3 lineage–labeled r7 NC stream (100.5 ± 37 vs. 148.25 ± 17 TUNEL-positive grains, P<0.019; n=4) (Fig. 5E, F, G). With the loss of both Neurog1 and Neurog2, we again found a significant elevation of apoptosis associated with the r6 NC stream. However, the apoptosis associated with the r7 NC stream was further enhanced (228.75 ± 37.9 vs. 919.75 ± 232.71 TUNEL-positive grains/r6 NC stream, 190 ± 37.4 vs. 885 ± 161.76 TUNEL-positive grains/r7 NC stream; P<0.001, n=4; pictorial data not shown). At this level of analysis and with limited molecular markers, we cannot distinguish among the various cell lineages that arise from NC cells. Nevertheless, these results suggest that the reduction or complete loss of PG neurons in the otic ganglion of Neurog2−/− or Neurog1−/−2−/− embryos is correlated with apoptosis of the r6-derived NC stream associated with the adjacent petrosal ganglion.

Loss of Neurog2 Affects Specific r6 Neural Crest Population

Despite the reduction of the r6 NC stream in Neurog2−/− embryos, we observed a few Sox10/EYFP-expressing lineage-labeled NC cells in the region normally occupied by the petrosal ganglion at e10.5 (Fig. 6A, B). These Hoxa3 lineage–labeled r6 NC cells were associated with the residual Isl1-expressing placode-derived sensory neurons (Fig. 6A, B, boxed area). This allowed us to follow the putative migratory path of the Hoxa3 lineage–labeled PG precursors from the petrosal ganglion in ba3 to their terminal target in ba2. In e11.5 Neurog2−/− embryos, the Hoxa3 lineage–labeled PG precursors marked by Phox2b and EYFP were detected (albeit reduced) between the region normally occupied by the petrosal ganglion and their target destination in ba2 (Fig. 6C, D, boxed area; and data not shown). At e12.5, no migrating PG precursors were detected in Neurog2−/− embryos, whereas a large number of otic precursors continued to migrate in control littermates (Fig. 6E, F, boxed area). These results suggest that the migration of PG precursors is terminated prior to e12.5 in Neurog2−/− embryos, suggesting a depletion of the NC fated for the PG lineage. Although a defect in r6 NC cell-derived PG precursors was observed in Neurog2−/− embryos, r6 NC-derived neurons of the proximal petrosal ganglion (pIXg) were unaffected (Fig. 6G, H). This suggests that the loss of Neurog2 is associated with a defect in a specific subset of r6-derived NC cells (i.e., those fated for the PG lineage).

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Figure 6. Defect in the placode-derived petrosal ganglia affects a specific population of r6-derived NC cells. A,B: Transverse sections of e10.5 control (Neurog2+/−;Hoxa3Cre/+;RosaEYFP/+) and Neurog2−/−; Hoxa3Cre/+;RosaEYFP embryos immunolabeled for GFP (green), Isl1 (red), and Sox10 (blue). The stream of Hoxa3 lineage-labeled NC (light blue) is interrupted in the Neurog2−/− embryo. In the Neurog2−/− embryo, 1–2 cells can be seen associated with residual placode-derived neurons of the petrosal ganglion (IXg, yellow cells in boxed area). C,D: Sagittal sections of e11.5 control (Neurog2+/−;Hoxa3Cre/+;RosaEYFP/+) and Neurog2−/−; Hoxa3Cre/+;RosaEYFP embryos immunolabeled for GFP (green), Phox2b (red), and Isl1 (blue). A stream of GFP/Phox2b-labeled cells (yellow cells in boxed area) appears to be migrating away from the triple-labeled petrosal ganglion (pink) in the control embryo. The number of GFP/Phox2b-labeled cells in the Neurog2−/− embryo is dramatically reduced (boxed area in D). One residual triple-labeled placodal cell can be identified in the Neurog2−/− embryo (arrow in D). E,F: Sagittal sections of e12.5 control (Neurog2+/−;Hoxa3Cre/+;RosaEYFP/+) and Neurog2−/−; Hoxa3Cre/+;RosaEYFP embryos immunolabeled for GFP (green), Phox2b (red), and Isl1 (blue). At this stage, the putative GFP/Phox2b-expressing PG precursors (yellow) of the otic ganglion continue to emerge from the petrosal ganglion in the control embryo (boxed area in E), but are missing in the Neurog2−/− embryo (boxed area in F). The GFP labels both migrating PG neurons (yellow) and axons (green) derived from neighboring ganglia. G,H: Sagittal sections of e12.5 control (Neurog2+/−;Hoxa3Cre/+;RosaEYFP/+) and Neurog2−/−; Hoxa3Cre/+;RosaEYFP embryos immunolabeled for GFP (green), Sox10 (red), and Phox2b (blue). The proximal petrosal ganglion (pIXg) remains intact in the Neurog2−/− embryo (H). The arrow in H indicates residual placode-derived neurons in the Neurog2−/− embryo. Scale bar = 50 μm (A,B,G,H); 100 μm (C–F).

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Dynamic Activities of Neurog1 and Neurog2 During Neural Crest–Derived Gangliogenesis

The expression pattern of Neurog1 and Neurog2 by RNA in situ hybridization indicates a highly dynamic expression pattern among NC, placode, and cranial ganglia (Ma et al., 1996, 1997, 1998, 2000; Fode et al., 1998; Zirlinger et al., 2002; Kim et al., 2011). To better capture the dynamic spatiotemporal expression pattern of Neurog1/2, and possibly gain insight into the phenotypes associated with Neurog1/2 mutant embryos, we employed the tamoxifen-inducible Cre lineage reporter system to monitor the activity of Neurog1/2 (Zirlinger et al., 2002; Kim et al., 2011). We administered tamoxifen by oral gavage at 6.5, 7.5, and 8.5 days post-coitum (dpc) to pregnant RosaEYFP/EYFP mice that had been crossed to Neurog1CreER or Neurog2CreER male mice. We then harvested embryos at e11.5 and e12.5. Previous studies have shown the dynamic expression of Neurog1 and Neurog2 RNA among placode-derived ganglia (Fode et al., 1998; Ma et al., 1998). We, therefore, focused our analysis on NC-derived PG neurons of the otic ganglion, whose Neurog1/2 expression profiles have yet to be characterized. Neurog1CreER;RosaEYFP/+ embryos treated with tamoxifen at 6.5 dpc showed few if any EYFP labeling among Phox2b-expressing neurons of the otic ganglion at e12.5 (Fig. 7A, A'). In contrast, EYFP labeling was observed in Phox2b/Isl1-expressing neurons of the placode-derived geniculate ganglion, and surrounding mesenchyme. The EYFP labeling in the mesenchyme indicates that Neurog1 may be expressed early in the neural crest progenitor domain (dorsal neural tube), which is independent of its function in the peripheral neuronal lineage. The expression profile of the Neurog1 lineage at this stage resembles that of embryos lineally labeled with the Wnt1-Cre/Rosa-EYFP reporter system (data not shown). However, we cannot exclude the possibility that the expression in the mesenchyme is a consequence of an early transient activity of Neurog1 or leakiness in this BAC-transgenic mouse line. Later tamoxifen administration at 7.5 dpc showed reduced EYFP labeling in the surrounding mesenchyme at e12.5 (Fig. 7B, B'; and data not shown). At this time of tamoxifen-induced activation, EYFP labeling was observed in a small population of Phox2b-expressing neurons of the otic ganglion. In contrast, the majority of Phox2b/Isl1-expressing neurons of the geniculate ganglion were labeled. Tamoxifen administration at 8.5 dpc showed further reduction of EYFP labeling in the mesenchyme, no labeling in the otic ganglion (except for traversing axons), and labeling of most if not all Phox2b/Isl1-expressing neurons of the geniculate ganglion at e12.5 (Fig. 7C, C').

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Figure 7. Dynamic expression of Neurog1 and Neurog2 during placode- and NC-derived cranial gangliogenesis. A, A': Sagittal section of e12.5 Neurog1CreER;RosaEYFP/+ embryo administered with tamoxifen at 6.5 dpc and immunolabeled for GFP (green), Phox2b (red), and Isl1 (blue). B, B': Sagittal section of e12.5 Neurog1CreER RosaEYFP/+ embryo administered with tamoxifen at 7.5 dpc and immunolabeled for GFP (green), Phox2b (red), and Isl1 (blue). C, C': Sagittal section of e12.5 Neurog1CreER;RosaEYFP/+ embryo administered with tamoxifen at 8.5 dpc and immunolabeled for GFP (green), Phox2b (red), and Isl1 (blue). D, D': Sagittal section of e11.5 Neurog2CreER;RosaEYFP/+ embryo administered with tamoxifen at 6.5 dpc and immunolabeled for GFP (green), Phox2b (red), and Isl1 (blue). Note at this stage, e11.5, an initial wave of NC-derived PG neurons (arrow) has arrived to the future site of the otic ganglion (Og). By e12.5, the otic ganglion has formed a large mass (see A–C, E, and F). E, E': Sagittal section of e12.5 Neurog2CreER;RosaEYFP/+ embryo administered with tamoxifen at 7.5 dpc and immunolabeled for GFP (green), Phox2b (red), and Isl1 (blue). F, F': Sagittal section of e12.5 Neurog2CreER;RosaEYFP/+ embryo administered with tamoxifen at 8.5 dpc and immunolabeled for GFP (green), Phox2b (red), and Isl1 (blue). Note that A–C and D–F represent single GFP images that were separated from the triple-labeled images to clarify the expression pattern of Hoxa3-lineage labeled neural crest- and placode-derived cells. Scale bars = 50 μm.

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In contrast to the temporal expression profile of Neurog1, the activity of Neurog2 in NC-derived Phox2b-expressing neurons of the otic ganglion was present at all stages of tamoxifen administration (6.5, 7.5, and 8.5 dpc) (Fig. 7D–F, 7D'–F'). At these stages, EYFP labeling in the geniculate ganglion was predominantly among mesenchymal cells surrounding the Phox2b/Isl1-expressing neurons. Interestingly, the absence of EYFP-labeling among Phox2b/Isl1-expressing neurons suggests that the cell-autonomous requirement of the placode-derived geniculate ganglion for Neurog2 occurs at a later stage (∼e9.5) (see Supp. Fig. S1, which is available online) (Fode et al., 1998; Ma et al., 1998). In summary, the activity of Neurog1 in NC cells fated for the PG lineage of the otic ganglion appears to occur during a narrow time window (∼e7.5), whereas Neurog2 activity appears to be more protracted, spanning between e6.5 and e8.5.

DISCUSSION

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

In this study, we found that the formation of cranial sensory and parasympathetic ganglia requires the differential activity of Neurog1 and Neurog2 along the AP axis. In the anterior region of the head, we confirmed that the formation of the NC-derived trigeminal ganglion requires Neurog1, and the placode-derived geniculate ganglion requires Neurog2. In contrast, the more posterior ganglia require both Neurog1 and Neurog2. Fate mapping reveals that the dynamic spatiotemporal activities of Neurog1 and Neurog2 during NC- and placode-derived gangliogenesis correlates with the phenotypes associated with the Neurog1 and Neurog2 mutations. Lastly, the expression of Neurog1 and Neurog2 in both the placode and the NC suggests that cranial gangliogenesis may be regulated by both Neurog-dependent cell-autonomous and non-cell-autonomous mechanisms.

Differential Contribution of Neurog1 and Neurog2 in Cranial Gangliogenesis Along the AP Axis

Combined with previous studies, a general principle for how Neurog1 and Neurog2 contribute to NC- and placode-derived gangliogenesis along the AP axis has emerged. It appears that the more anterior cranial ganglia depend on the single activity of the Neurog genes, whereas the more posterior cranial ganglia depend on the combined activities of Neurog1 and Neurog2. For example, the midbrain NC-derived trigeminal and r4 NC-derived sphenopalatine and submandibular ganglia require either Neurog1 or Neurog2, respectively (Ma et al., 1998; Coppola et al., 2010; Takano-Maruyama et al., 2010). The placode-derived geniculate and vestibulocochlear ganglia also depend on the single gene activity of Neurog2 and Neurog1, respectively (Fode et al., 1998; Ma et al., 1998). However, the contribution of Neurog1 and Neurog2 becomes more apparent at posterior levels of the head. We found that although the majority of NC-derived PG neurons of the otic ganglion require Neurog2 (Chen et al., 2011), the formation of a small population of PG neurons is dependent on both Neurog1 and Neurog2. This was also true for the placode-derived petrosal ganglion, where the majority of Phox2b/Isl1-expressing neurons required Neurog2, and a smaller population depended on both Neurog1 and Neurog2. In contrast to the placode-derived otic and petrosal ganglia, the formation of the more posterior placode-derived nodose ganglion was entirely dependent on both Neurog1 and Neurog2.

What is the significance of the differential requirement of the cranial ganglia for the Neurog genes along the AP axis? The requirement for Neurog1 and Neurog2 on the formation of the dorsal root ganglia (DRG) may provide insight into this question. In the absence of both Neurog1 and Neurog2, the DRG fail to form along the entire length of the spinal cord (Ma et al., 1999). However, the loss of either Neurog1 or Neurog2 revealed that each gene was required for distinct waves of neurogenesis that corresponded to the formation of distinct neuronal subtypes in the DRG. The early wave of neurogenesis produces trkB+ and trkC+ sensory neurons that are dependent on Neurog2, whereas the later wave produces trKA+ sensory neurons that are dependent on Neurog1. It is possible that this developmental phenomenon applies to other ganglia along the AP axis, where different neuronal subtypes are patterned by a specific Neurog code. In the present study, for example, we observed that the majority of PG neurons of the otic ganglion required Neurog2, and the remaining population was dependent on both Neurog1 and Neurog2. The differential requirement and relative distribution of Neurog1 - and Neurog2 -lineage labeled PG neurons appear to correlate with the different neuronal subtypes in the mature otic ganglion. The mature otic ganglion contains predominantly secretomotor cholinergic neurons and a small population of vasodilatory non-cholinergic PG neurons (Izumi, 1995; Khosravani et al., 2006; Proctor and Carpenter, 2007). It is possible that the specification of the more numerous cholinergic neurons require Neurog2, while the smaller non-cholinergic population is dependent on both Neurog1 and Neurog2. Studies to investigate long-term fate mapping of Neurog1, from embryogenesis to adulthood, could provide insight into this hypothesis.

As described above the Neurog genes have specific contributions to cranial gangliogenesis, but our data also suggest a broader role. In the case of the DRG and otic ganglion, the Neurog genes appear to control distinct waves of neurogenesis, which is critical for generating different neuronal subtypes. With respect to the trigeminal and geniculate ganglia, it is possible that the Neurog genes control the formation of all neuronal populations. This suggests that the Neurog genes control an earlier stage of neurogenesis, one that impacts subsequent waves of neurogenesis that give rise to the different neuronal subtypes. Together with the temporal fate map of Neurog1 and Neurog2, the requirements of different waves of cranial neurogenesis (early versus late) for the Neurog genes may be a consequence of variations in their dynamic spatiotemporal activities along the AP axis.

Non-Cell- and Cell-Autonomous Requirements of Autonomic Cranial Gangliogenesis

Our findings show snapshot images of r6 lineage–labeled NC cells differentiating into PG precursors as they migrate through the adjacent placode-derived petrosal ganglion. This supports the notion that the differentiation of PG precursors may be regulated by a non-cell-autonomous mechanism through their interaction with placode-derived neurons (Coppola et al., 2010; Takano-Maruyama et al., 2010). Lineage labeling of Neurog1 and Neurog2 supports this idea. We found that both genes are expressed in precursors that give rise to all neurons of the placode-derived petrosal ganglia (data not shown) (Fode et al., 1998; Ma et al., 1998). However, we also found that early expression of Neurog2 results in labeling of NC that gives rise to all PG neurons of the otic ganglion. This invokes a cell-autonomous mechanism by which Neurog2 may act directly or cell-autonomously on the NC fated for the PG lineage.

In an elegant study in which Phox2b was systematically ablated in either Wnt1-expressing NC cells or Isl1-expressing placodal cells, the formation of various parasympathetic ganglia (sphenopalatine and submandibular) were lost in each cell domain–specific knockout (Coppola et al., 2010). This finding not only supports the idea that Phox2b is required cell autonomously by NC-derived PG neurons, but it suggests a non-cell-autonomous mechanism by which the placode-derived ganglia control the formation of NC-derived PG neurons. The function of Phox2b is also required in progenitor and postmitotic precursors of motor neurons (Coppola et al., 2010). Together, these findings support the hypothesis that Phox2b orchestrates the formation of the cranial autonomic reflex circuit (Pattyn et al., 1999; Dauger et al., 2003; Coppola et al., 2010). It is possible that the Neurog genes contribute to this Phox2b-dependent circuit.

To gain insight into this idea, at least two observations in the present study have to be considered: the onset of Neurog activity in the NC fated for the PG lineage, and the stage at which the NC commits to the PG lineage. The earliest activity of Neurog2 among PG precursors of the otic ganglion that we tested occurred ∼e6.5. In contrast, the earliest commitment of NC for the PG lineage (i.e., induction of Phox2b expression) occurs much later at ∼e9.0. This suggests that Neurog2 is far upstream in the genetic cascade leading to Phox2b expression in PG precursors. The ∼e9.0 stage also corresponds to the approximate onset of Neurog2 expression in the petrosal placode (Fode et al., 1998; Ma et al., 1998). Together, this suggests that the early activity of Neurog2 in NC cells may be required cell-autonomously for the early specification of NC fated for the PG lineage. In contrast, the later activity of Neurog2 in the placode may be required non-cell-autonomously for the differentiation of NC into PG neurons. The activities of the Neurog genes during the formation of the peripheral autonomic circuit are, therefore, sequential: first in the NC, and later in the placode. Ablation of the Neurog genes in a spatial- and temporal-dependent manner in the neural tube, NC, placode, and placode-derived ganglia may provide further insight into the genetic hierarchy that regulates the precursors of the cranial autonomic circuit.

EXPERIMENTAL PROCEDURES

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

Mouse Lines

The Neurog1+/−, Neurog2+/−, Neurog1CreER, Neurog2CreER, Hoxa3Cre/+, and RosaEYFP/+ mice have been reported (Ma et al., 1998, 1999; Srinivas et al., 2001; Zirlinger et al., 2002; Macatee et al., 2003; Koundakjian et al., 2007). The Hoxa3-Cre or Rosa-EYFP allele was introduced into the Neurog2 mutant background to generate Neurog2+/−;Hoxa3Cre/+ mice or Neurog2+/−;RosaEYFP/+ mice. These mice were intercrossed to generate embryos harboring a reporter that allow the fate of r6 neural crest to be followed. All animal experiments were approved by the IACUC protocol MU041 at the University of Texas at San Antonio.

Temporal Fate Mapping

Neurog1CreER or Neurog1CreER male and RosaEYFP/+ female mice were bred to generate Neurog1CreE;RosaEYFP/+ and Neurog1CreER;RosaEYFP/+ embryos. Female mice were checked for plugs at 9 am, and mice with visible plugs the morning after were designated as e0.5. Tamoxifen was administered by oral gavage at 9 am on e6.5, e7.5, and e8.5. Tamoxifen (Sigma, St. Louis, MO) was reconstituted in peanut oil at a concentration of 10 mg/ml. Progesterone was also added at 5 mg/ml. Ten μl of this tamoxifen/progesterone solution was administered per 1 g of body weight. The embryos from tamoxifen/progesterone-treated mice were harvested at e12.5, and processed for immunohistochemistry.

Immunohistochemistry

The embryos were dissected and fixed for 30 min in 4% paraformaldehyde in phosphate buffered saline (PBS), washed 3–4 times in PBS, and then cryoprotected in a sequential series of 15% sucrose/PBS (3–4 h), and 30% sucrose/PBS (overnight). All steps were done at 4°C. Embryos were then embedded in OCT compound-filled molds (Tissue-Tek®), and then frozen rapidly. Embryos were sectioned at 12 μm and were mounted on charged glass slides, and washed in PBS containing 0.2% Triton X-100 (PBST) for 5 min. The sections were then incubated overnight with primary antibodies in PBST with 10% normal goat serum and 2% powdered skimmed milk at 4°C. For visualization, the sections were incubated with secondary antibodies in PBST with 10% goat sera and 2% skim milk for 45 min at 4°C, and washed 3 times with PBST.

Antibodies

The primary antibodies used in this study were as follows: mouse anti-Isl1, mouse anti-β-galactosidase (βgal), mouse anti-AP2 (1:50, Developmental Studies Hybridoma Bank, Iowa City, IA), sheep anti-Green Fluorescent Protein (GFP) (1:1,000, Covance, Princeton, NJ), mouse anti-Tuji1 (1:8,000, Covance), and mouse anti-BrdU (1:300, Sigma-Aldrich). Rabbit anti-Phox2a and anti-Phox2b antibodies were generated in our laboratory, and the mouse anti-Sox10 antibody was kindly gifted by Dr. D. Anderson. The secondary antibodies used in this study were as follows: Alexa 488 conjugated goat anti-rabbit (Fab)2 fragment, Alexa 488 conjugated goat anti-mouse (Fab)2 fragment, Alexa 488 conjugated goat anti-sheep IgG, Alexa 594 conjugated goat anti-rabbit (Fab)2 fragment, Alexa 647 conjugated goat anti-mouse (Fab)2 fragment, and Alexa 647 conjugated goat anti-rabbit (Fab)2 fragment (1:1,000, Invitrogen, Carlsbad, CA).

Programmed Cell Death Assay

Detection of apoptotic cell death was performed using In Situ Cell Death Detection Kit, TMR red (Roche Applied Science, Indianapolis, IN). The sections were examined with an LSM 5 PASCAL confocal microscope. Cell counting was performed using Imaris software (Bitplane Scientific Software) in combination with manual verification of the automated results.

Statistical Analysis

Results are expressed as means ± SD. Statistical analyses of differences between two groups were performed using the unpaired Student's t-test (two-tailed analyses). Differences of P<0.01 were considered statistically significant.

Acknowledgements

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

We are grateful to Drs. O. M. Pereira-Smith, P. Mueller, and S. Howard-Byerly for critical review of the manuscript; Drs. A. Moon, D. Anderson, Q. Ma, T. Reh, and J. Brzezinski for providing transgenic and knockout mice; Drs. D. Ko and C. Witt for statistical and imaging assistance; and H.-Y. Kao and O. Trevino for technical assistance. This study was supported by grants to G.O.G from the NIH (NS060658) and the Whitehall Foundation.

REFERENCES

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

Supporting Information

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

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_22785_sm_SuppFig1.tif1857KSupp. Fig. S1. Neurog2 protein expression in the placode and delaminating placode-derived visceral sensory neurons. The image shows a transverse section of an e9.5 Hoxb1 lineage labeled embryo (Hoxb1Cre/+; RosaEYFP/+) immunolabeled for GFP (A, white; D, green), Neurog2 (B, white; D, red), and Phox2a (C, white; D, blue). D: Merged image of A–C. This section was obtained at the level of rhombomere 4 (r4) and the second branchial arch. The Hoxb1 lineage specifically labels r4 and r4-derived neural crest (NC) cells. The Neurog2 protein labels the geniculate placode (P) and the placode-derived delaminating visceral sensory neurons (dP). Note the relative difference in the expression levels of Neurog2 in the placode (high) and delaminating cells (low). At this stage and earlier stages that we studied (<e8.5, data not shown), the expression level of Neurog2 in the placode and NC areas is difficult to detect beyond normal background expression. The Phox2a protein is also expressed in the placode and the placode-derived delaminating visceral sensory neurons (dP). However, the expression levels are similar between the placode (P) and placode-derived delaminating sensory neurons (dP). Nevertheless, the expression of Neurog2 in the placode at e9.5 supports the hypothesis that Neurog2 is required cell autonomously to pattern the placode-derived geniculate ganglia. Scale bar = 50 μm.

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