Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Aug 1.
Published in final edited form as: Dev Dyn. 2011 Jun 14;240(8):1880–1888. doi: 10.1002/dvdy.22679

Plasticity of neural crest-placode interaction in the developing visceral nervous system

Yiju Chen 1, Masumi Takano-Maruyama 1, Gary O Gaufo 1,2
PMCID: PMC3285277  NIHMSID: NIHMS352680  PMID: 21674689

Abstract

The reciprocal relationship between rhombomere (r)-derived cranial neural crest (NC) and epibranchial placodal cells derived from the adjacent branchial arch is critical for visceral motor and sensory gangliogenesis, respectively. However, it is unknown whether the positional match between these neurogenic precursors is hard-wired along the anterior-posterior (A/P) axis. Here we use the interaction between r4-derived NC and epibranchial placode-derived geniculate ganglion as a model to address this issue. In Hoxa1−/−b1−/− embryos, r2 NC compensates for the loss of r4 NC. Specifically, a population of r2 NC cells is redirected towards the geniculate ganglion, where they differentiate into postganglionic (motor) neurons. Reciprocally, the inward migration of the geniculate ganglion is associated with r2 NC. The ability of NC and placodal cells to respectively differentiate and migrate despite a positional mismatch along the A/P axis reflects the plasticity in the relationship between the two neurogenic precursors of the vertebrate head.

Keywords: Neural crest, epibranchial placode, migration, differentiation, Hox, Neurog

INTRODUCTION

The cellular diversification and identity of the animal body depends on the nested expression of the Hox genes and alignment of precursor cells along the anterior-posterior (A/P) axis (McGinnis and Krumlauf, 1992; Lumsden and Krumlauf, 1996; Trainor and Krumlauf, 2000b; Kiecker and Lumsden, 2005). In the developing vertebrate head, the interaction between pluripotent cranial neural crest (NC) cells and adjacent tissues of the developing head and neck areas is critical for the formation of musculoskeletal, connective, cardiovascular, respiratory lymphoid, endocrine, and neuronal tissues that constitute the head, neck, and thoracic-abdominal compartments of the body (Fontaine-Perus et al., 1988; Kirby, 1988; Manley and Capecchi, 1995; Gavalas et al., 1998; Gavalas et al., 2001; Trainor et al., 2002; Macatee et al., 2003; Arenkiel et al., 2004; Matsuoka et al., 2005; Oury et al., 2006; Park et al., 2006; Shiau et al., 2008; Coppola et al., 2010; Takano-Maruyama et al., 2010; Bertrand et al., 2011). The cranial NC is the central player that coordinates the cellular organization in these various tissue compartments. In the head, the cranial NC provides the source of many cell types, and through its interactions with cells in the adjacent branchial arches (ba) coordinates craniofacial morphogenesis. The nested expression of the Hox genes are superimposed on this NC-branchial arch interaction, which ensures that aligned cells are endowed with the positional information that are critical for their integration along the A/P axis (Lumsden et al., 1991; Krumlauf et al., 1993; Bell et al., 1999; Begbie and Graham, 2001; Oury et al., 2006).

In the developing cranial nervous system, the migration of NC cells originating from even-numbered rhombomeres (r) into the ba not only provides the source for the myriad of cell types that constitute the head, but they also act as a cellular conduit to integrate the periphery with the central nervous system (Lumsden and Krumlauf, 1996; Trainor and Krumlauf, 2000b; Trainor and Krumlauf, 2001; Coppola et al., 2010). For example, r4 NC cells migrate into the adjacent ba2 to form myelin forming Schwann cells that ensheath the axons of r4-derived facial motor neurons, which in turn innervate ba2-derived facial muscles (Arenkiel et al., 2003; Arenkiel et al., 2004). In the afferent pathway, r4 NC cells facilitate the inward migration and central connections of the ba2 placode-derived geniculate visceral ganglion (Begbie and Graham, 2001). The specification and formation of these motor and sensory pathways are dependent on the combinatorial activities of Hoxa1 and Hoxb1 (Goddard et al., 1996; Studer et al., 1996; Gavalas et al., 1998; Studer et al., 1998; Rossel and Capecchi, 1999; Gavalas et al., 2001; Gavalas et al., 2003; Arenkiel et al., 2004). The Hox-dependent positional integration of motor and sensory pathways appears to be a fundamental mechanism that is repeated along the A/P axis from the hindbrain to the spinal cord (Goddard et al., 1996; Studer et al., 1996; Carpenter et al., 1997; Gavalas et al., 1997; Tiret et al., 1998; Bell et al., 1999; Lance-Jones et al., 2001; McClintock et al., 2002; Dasen et al., 2003; Gaufo et al., 2003; Arenkiel et al., 2004; Gaufo et al., 2004; Dasen et al., 2005; Oury et al., 2006; Holstege et al., 2008; Chen et al., 2010).

It is established that the cranial NC is critical for coordinating many aspects of craniofacial development, but equally important is the influence of the environment on determining many of its possible fates, thereby reflecting the plasticity of the cranial NC (Fontaine-Perus et al., 1988; Kirby, 1988; Saldivar et al., 1996; Trainor and Krumlauf, 2000a; Trainor et al., 2002; Le Douarin et al., 2004; Bogni et al., 2008; Coppola et al., 2010; Takano-Maruyama et al., 2010). Others and we have shown that the ba2 placode-derived geniculate visceral ganglion is essential for the survival and differentiation of r4 NC-derived visceral postganglionic (PG) precursors of the submandibular and sphenopalatine ganglia (Coppola et al., 2010; Takano-Maruyama et al., 2010). Together with the observation that the inward migration of the geniculate ganglion is dependent on r4-derived NC cells (Begbie and Graham, 2001; Barlow, 2002; Coppola et al., 2010; Takano-Maruyama et al., 2010), we suggested that the reciprocal interaction between positionally aligned cells is critical for the integration of precursors into a common visceral neuronal circuit. However, it is unknown whether this reciprocal interaction is restricted to this A/P position or whether cranial NC and placodal cells show plasticity in their ability to differentiate and migrate, respectively, under different positional conditions along the A/P axis.

Evidence from quail/chick studies have shown that non-neuronal cells, presumably of NC origin, contained within the placode-derived visceral sensory ganglion differentiate into PG neurons when ectopically grafted outside of its normal A/P position (Fontaine-Perus et al., 1988; Kirby, 1988; Le Douarin et al., 2004). These findings provide evidence for the plasticity of the cranial NC, showing that it can differentiate into PG neurons under different positional conditions. In contrast, the geniculate ganglion fails to migrate inwards to the hindbrain when r4 is specifically ablated in the chick embryo (Begbie and Graham, 2001). This suggests that the dependence of the inward migration of the geniculate ganglion is specific to r4 NC cells because neighboring NC cells, which are presumably left intact with this specific surgical ablation, are unable to compensate for the loss of r4 NC cells.

Here we use the mouse embryo harboring mutations for Hoxa1 and Hoxb1 – genes that are required for the formation of r4 NC cells, but not the ba2-derived geniculate ganglion (Gavalas et al., 1998; Studer et al., 1998; Rossel and Capecchi, 1999; Gavalas et al., 2001; Gavalas et al., 2003) – as a model to test whether PG neurons differentiate and geniculate ganglion migrate in the absence of the r4 NC population that normally facilitates these processes. In Hoxa1−/−b1−/− embryos, we found that r2-derived NC cells compensate for the loss of r4 NC cells. In this context, r2 NC cells are able to migrate into the geniculate ganglion and differentiate into PG neurons, and associate with the inward migration of the geniculate ganglion. Our study provides evidence for the plasticity of the cranial NC fated for the PG neuronal lineage, and reveals an unanticipated plasticity of the geniculate visceral sensory ganglion in its migratory behavior in the absence of a normal, positionally matched NC population.

RESULTS

Differentiation of postganglionic neurons in the absence of their normal rhombomeric source and migratory environment

Fate mapping of cranial NC in mouse and quail/chick embryos have shown that parasympathetic PG neurons of the submandibular and sphenopalatine ganglia originate from r4, part of the preotic region of the hindbrain (D'Amico-Martel and Noden, 1983; Arenkiel et al., 2003; Yang et al., 2008; Takano-Maruyama et al., 2010). Based on these findings, we predicted that the submandibular and sphenopalatine ganglia would fail to form in mouse embryos harboring compound mutations of Hoxa1 and Hoxb1 due to the absence of r4 NC cells in these mutant embryos (Gavalas et al., 1998; Studer et al., 1998; Rossel and Capecchi, 1999; Gavalas et al., 2001; Gavalas et al., 2003). We unexpectedly found the presence of Phox2b+ PG neurons of the submandibular and sphenopalatine ganglia at e12.5, albeit at significantly reduced numbers and expression levels (Fig. 1A, D; n=4). Postganglionic neurons of the otic ganglion were also observed in reduced numbers in Hoxa1−/−b1−/− embryos, and were situated in their normal position just ventral to the geniculate ganglion (Fig. 1G) (D'Amico-Martel and Noden, 1983; Enomoto et al., 2000). These findings suggest that NC precursors of the submandibular, sphenopalatine, and otic ganglia are able to migrate rostrally through an abnormal branchial arch environment to reach their target areas. Furthermore, the presence of the placode-derived geniculate ganglion in its normal position close to the hindbrain indicates that that the inward migration of this ganglion occurs in the absence of r4-derived NC cells (Fig. 1H, I). This is in contrast to a study in the chick embryo in which r4 was specifically ablated, and the placode-derived geniculate ganglion failed to migrate inwards to the hindbrain (Begbie and Graham, 2001).

Fig. 1. Differentiation of postganglionic neurons in Hoxa1−/−b1−/− mutant embryos in the absence of a normal rhombomere source.

Fig. 1

Open in a new tab

(A, D, G) Statistical analysis of Phox2b-expressing postganglionic (PG) neurons in the submandibular, sphenopalatine, and otic ganglia in e12.5 control (green bar) and Hoxa1−/−b1−/− mutant (red bar, n=4) embryos. The values for Hoxa1−/−b1−/− mutant embryos are expressed as a percentage relative to control embryos, which was set at 100%. The submandibular (23.6% ± 13.4 S.D, p<0.001), sphenopalatine (30.0% ± 14.1 S.D, p<0.002), and otic (3.8% ± 1.0 S.D, p<5×10−7), ganglia of Hoxa1−/−b1−/− mutant embryos (n=4) were significantly reduced compared to control embryos (n=4). The majority of PG neurons in the submandibular, sphenopalatine, and otic ganglia of e12.5 Hoxa1−/−b1−/− mutant embryos expressed Phox2b at low levels, indicative of early differentiating PG precursors (Takano-Maruyama et al., 2010).

(B, C, E, F, H, I) Sagittal sections of e13.5 control (n=5) and Hoxa1−/−b1−/− mutant (n=5) embryos immunolabeled for Phox2b. The control embryo shows that the majority of PG neurons in the submandibular, sphenopalatine, and otic ganglia maintain express high levels of Phox2b. In the Hoxa1−/−b1−/− mutant embryo, the majority of PG neurons express low levels of Phox2b, suggesting that PG neurons are at an early stage of differentiation.

Abbreviations: Gg, geniculate ganglion; Og, otic ganglion.

Scale Bar: Panels B, C, E, F, H, I – 50 µm

Analysis of Hoxa1−/−b1−/− embryos at a later stage (e13.5) showed that the Phox2b+ PG neurons in the submandibular, sphenopalatine, and otic ganglia remained reduced both in number and expression levels compared to control littermates (Fig. 1B, C, E, F, H, I). The persistence of Phox2b at low expression levels suggests these PG precursors may not fully mature into PG neurons, which normally expresses high levels of Phox2b at this stage. Nevertheless, despite the absence of r4 NC and abnormal branchial arch tissues, NC and placodal cells have the capacity to differentiate and migrate to their normal target areas. This suggests a compensatory mechanism to support the early differentiation of PG neurons and facilitate the inward migration of the geniculate ganglion.

Migration of the geniculate ganglion from an A/P mismatch of neural crest and placodal cells

To explore a possible compensatory mechanism, we reexamined hindbrain and NC patterning in Hoxa1−/−b1−/− embryos. Previous reports have shown that the combined loss of Hoxa1 and Hoxb1 resulted in a residual r4 territory, complete loss of NC cells from r4 to the caudal hindbrain and associated NC-derived ganglia, loss of ba2, and fusion of placode-derived geniculate and trigeminal sensory ganglia (Gavalas et al., 1998; Studer et al., 1998; Rossel and Capecchi, 1999; Gavalas et al., 2001). The latter phenotype suggests that the ba2 cleft, the source of the geniculate ganglion, is intact in Hoxa1−/−b1−/− embryos. Indeed, in e10.5 Hoxa1−/−b1−/− embryos, we observed the ba2 cleft, from which Isl1+Phox2b+ neurons of the geniculate ganglion emerged (Fig. 2A, B; asterisk). In whole-mount preparations, we observed fusion of the Isl1+Phox2b+ geniculate and Isl1+ trigeminal ganglia, confirming a previous study (Gavalas et al., 2001). At this level of analysis it is difficult to assess whether the geniculate ganglion has migrated inwards to the hindbrain or remained in a sub-ectodermal position. The latter scenario is possible since the inward migration of the geniculate ganglion is dependent on r4 NC cells (Begbie et al., 1999; Begbie and Graham, 2001). Alternatively, since r3 and r4 are eliminated in Hoxa1−/−b1−/− embryos, the geniculate ganglion may adapt to a new environment that facilitates its inward migration to a more rostral rhombomere. This would suggest that a more rostral NC population associates with the inward migration of the geniculate ganglion.

To test this possibility, we analyzed Hoxa1−/−b1−/− embryos at e9.5, the time when precursors of ba2 placode-derived geniculate ganglion are initially engaged with r4 NC cells (Begbie and Graham, 2001; Takano-Maruyama et al., 2010). In sagittal sections of control embryos, Phox2b+Isl1+ precursors of the geniculate ganglion have delaminated from the ba2 cleft and aggregated as a sphere directed towards r4 (Fig. 2C, asterisk). A clear boundary is seen between the geniculate ganglion and the more rostral trigeminal ganglion (Fig. 2C, double arrow). In Hoxa1−/−b1−/− embryos, the Phox2b+Isl1+ geniculate ganglion also delaminates from the ba2 cleft and aggregates as a sphere directed towards the hindbrain (Fig. 2D, asterisk). However, the separation between the geniculate and trigeminal ganglia is missing. Instead, these ganglia are fused, with the geniculate ganglion directed towards r2, the entry point of the central processes of the trigeminal ganglion. Furthermore, we verified that the migratory path of the Phox2b+ geniculate precursors, from the ba2 cleft to r4, is completely circumscribed by Sox10+ NC cells in control embryos (Gavalas et al., 1998; Begbie and Graham, 2001; Takano-Maruyama et al., 2010). In contrast, the inward migratory path of the Phox2b+ geniculate precursors in Hoxa1−/−b1−/− embryos were associated with Sox10+ NC cells that stream from r2 to the ba2 cleft (Fig. 2F, asterisk). These findings suggest that in the absence of r3 and r4, the inward migration of the geniculate ganglion appears to be redirected towards r2 by a more rostral r2 NC population normally associated with the trigeminal ganglion.

Fig. 2. Positional mismatch of cranial neural crest and epibranchial placode is conducive to the inward migration of the geniculate ganglion.

Fig. 2

Open in a new tab

(A, B) Whole-mount e10.5 control (n=3) and Hoxa1−/−b1−/− mutant (n=3) embryos immunolabeled for Isl (green) and Phox2b (red). The juxtaposed Isl1+Phox2b+ geniculate and Isl1+ vestibulocochlear ganglia are separated from the more rostral Isl1+ trigeminal ganglion in the control embryo, whereas these ganglia are fused in the Hoxa1−/−b1−/− mutant embryo.

(C, D) Sagittal section through e9.5 control (n=9) and Hoxa1−/−b1−/− mutant (n=9) embryos immunolabeled for Isl1 (green) and Phox2b (red). The Isl1+Phox2b+ geniculate ganglion is separated from the more rostral Isl1+ trigeminal ganglion in the control embryo (double arrow). The area between the geniculate and trigeminal ganglia is missing in the Hoxa1−/−b1−/− mutant embryo, resulting in the juxtaposition of the geniculate and trigeminal ganglia.

(E, F) Sagittal section through e9.5 control (n=9) and Hoxa1−/−b1−/− mutant (n=9) embryos immunolabeled for Sox10 (green) and Phox2b (red). In the control embryo, the Phox2b+ geniculate ganglion is associated with the stream of Sox10+ neural crest cells from r4 to the second branchial cleft (asterisk), the origin of the geniculate ganglion. In the Hoxa1−/−b1−/− mutant embryo, the Phox2b+ geniculate ganglion is directed rostrally, and is associated with the stream of Sox10+ neural crest cells from r2 to the second branchial cleft.

Abbreviations: ov, otic vesicle; 5g, trigeminal ganglion; 7g, geniculate ganglion; r, rhombomere.

Scale Bar: Panels A, B – 200 µm; Panels C–F – 50 µm.

Differentiation of postganglionic neurons from an A/P mismatch of neural crest and placodal cells

The differentiation of PG neurons and inward migration of the geniculate ganglion have been shown to depend on the reciprocal interaction between r4-derived NC and ba2 placode-derived geniculate ganglion, respectively (Begbie and Graham, 2001; Barlow, 2002; Coppola et al., 2010; Takano-Maruyama et al., 2010). Since the inward migration of the geniculate ganglion occurs in the presence of the more rostral r2 NC population in Hoxa1−/−b1−/− embryos, we reasoned that by reciprocity the r2 NC engaged with the geniculate ganglion could differentiate into PG neurons. To explore this possibility, we performed immunolabeling for Sox10 and Phox2b to identify whether r2 NC cells differentiate into PG precursors within the geniculate ganglion (Takano-Maruyama et al., 2010). In control embryos, the Phox2b+ geniculate ganglion is clearly demarcated from the more ventral Sox10+Phox2b+ postotic NC-derived otic ganglion (Fig. 3A, C, E; the orientation of the panels show the otic ganglion to the right of the geniculate ganglion). In Hoxa1−/−b1−/− embryos, we observed a population of Sox10HighPhox2bLow precursors that appeared to stream from the geniculate ganglion into the area normally occupied by the otic ganglion (Fig. 3B, D, F). This expression profile of Sox10 and Phox2b is indicative of early differentiating r4 NC-derived PG precursors of the sphenopalatine and submandibular ganglia as they migrate and emerge from the geniculate ganglion (Takano-Maruyama et al., 2010). However, the position of the PG precursors and the abnormal morphology of branchial arches in Hoxa1−/−b1−/− embryos make it unclear whether these precursors contribute to the rostral sphenopalatine ganglion or the more ventral otic ganglion.

Fig. 3. Positional mismatch of cranial neural crest and the geniculate ganglion is conducive to the differentiation of postganglionic neurons.

Fig. 3

Open in a new tab

(A–F) Sagittal sections of e12.5 control and Hoxa1−/−b1−/− embryos immunolabeled for Sox10 and Phox2b.

(G–L) Transverse sections of e11.5 control and Hoxa1−/−b1−/− embryos immunolabeled for Sox10 and Phox2b.

Boxed areas in panels A, B, G, and H (10x) are magnified in panels C, D, I, J, E, F, K, and L (40x).

Abbreviations: 5g, trigeminal ganglion; 8g, vestibulocochlear ganglion; 7g, geniculate ganglion; Og, otic ganglion; R, rostral; C, caudal; D, dorsal; V, ventral.

Scale Bar: panels A, B, G, H – 100 µm; panels C, D, E, F, I, J, K, L – 50 µm.

To clarify this issue, we analyzed transverse sections of e11.5 embryos to better visualize the relationship between precursors of the sphenopalatine ganglion that emerge from the geniculate ganglion, and the more ventrally situated otic ganglion. In control embryos, the precursors of the sphenopalatine ganglion emerge from the ventromedial side of the geniculate ganglion and migrate rostrally alongside the sphenopalatine artery (Fig. 3G, I, K; see arrow in I). The more differentiated otic ganglion, as defined by the combination of Sox10HighPhox2bHigh expression, is located directly ventral to the geniculate ganglion. This relationship of geniculate, sphenopalatine, and otic precursors in the transverse plane is also observed in Hoxa1−/−b1−/− embryos (Fig. 3H, J, L). A sagittal section through the geniculate ganglion would therefore capture the otic ganglion in the same plane, but miss the more medially located precursors of the sphenopalatine ganglion. Based on this anatomical criterion, the aforementioned stream of Sox10HighPhox2bLow precursors in Hoxa1−/−b1−/− embryos likely contribute to the otic ganglion (Fig. 3B, D, F). Altogether, these findings indicate that the more rostral r2 NC cells engaged with the geniculate ganglion have the capacity to differentiate into PG precursors of the sphenopalatine and otic ganglia, and likely the submandibular ganglion.

Multi-rhombomeric origin of preganglionic precursors of the otic ganglion

Our finding that r2 NC cells in Hoxa1−/−b1−/− embryos migrate through the geniculate ganglion to give rise to the sphenopalatine, submandibular, and otic ganglia reflects the plasticity of the cranial NC. Previous genetic lineage labeling of Hoxb1 demonstrated that precursors of the sphenopalatine and submandibular ganglia originated from r4 (Yang et al., 2008; Takano-Maruyama et al., 2010), whereas the otic ganglion, as revealed by a chick/quail transplantation study, showed that its precursors originated from postotic levels of the hindbrain (posterior to r4) (D'Amico-Martel and Noden, 1983). The emergence of PG precursors of the otic ganglion from the geniculate ganglion suggests that this may be a consequence of the reorganization of the hindbrain and craniofacial area in Hoxa1−/−b1−/− embryos (Rossel and Capecchi, 1999), or a possible normal contribution from an unappreciated preotic source (i.e., r4).

To address this, we performed genetic lineage analyses with the Hoxa3-Cre and Hoxb1-Cre drivers coupled with the ROSA-YFP reporter to label NC cells from r5 (postotic) and r4 (preotic and postotic) to the caudal hindbrain, respectively (Srinivas et al., 2001; Arenkiel et al., 2003; Macatee et al., 2003). We confirmed that PG neurons of the sphenopalatine and submandibular ganglia originate from r4-derived NC cells in e12.5 Hoxa3Cre/+;ROSAYFP/+ and Hoxb1Cre/+;ROSAYFP/+ embryos (Fig. 4A–D) (D'Amico-Martel and Noden, 1983; Arenkiel et al., 2003; Yang et al., 2008; Takano-Maruyama et al., 2010). In Hoxa3 lineage-labeled embryos, which label postotic NC cells from r5 to the caudal hindbrain, no GFP-labeling was observed among Sox10+Phox2b+ PG neurons of the sphenopalatine and submandibular ganglia (Fig. 4A, C). In Hoxb1 lineage-labeled embryos, which label r4 and postotic NC cells, GFP labeled all Sox10+Phox2b+ PG neurons of the sphenopalatine and submandibular ganglia. These findings suggest that all PG neurons of the sphenopalatine and submandibular ganglia originate from r4 NC. The NC origin of PG neurons of the otic ganglion revealed a more complex picture. In Hoxa3 lineage-labeled embryos, the majority of PG neurons in the otic ganglion were triple-labeled with GFP, Sox10 and Phox2b (Fig. 4E). However, we identified a small population of Sox10+Phox2b+ devoid of GFP expression. The absence of GFP expression in these cells may be due to the efficiency of the Cre recombinase, or the possibility that they arise from a preotic source. To address this, we analyzed the otic ganglion in Hoxb1 lineage-labeled embryos. In these embryos, we observed a greater number of triple-labeled cells in the otic ganglion compared to Hoxa3 lineage-labeled embryos (Fig. 4F). This suggests that the small population of PG neurons that were unlabeled with GFP in Hoxa3 lineage-labeled embryos may originate from r4.

Fig. 4. Contribution of r4 neural crest to postganglionic neurons of the otic ganglion is dependent on the geniculate ganglion.

Fig. 4

Open in a new tab

(A, C, E) Sagittal sections of e12.5 Hoxa3Cre/+;ROSAYFP/+ immunolabeled for Phox2b, Sox10, and GFP. The Phox2b+Sox10+ postganglionic (PG) neurons in the sphenopalatine and submandibular ganglia are devoid of GFP expression, whereas the otic ganglion is mostly triple-labeled with a small population of Phox2b+Sox10+ double-positive PG neurons.

(B, D, F) Sagittal sections of e12.5 Hoxb1Cre/+;ROSAYFP/+ immunolabeled for Phox2b, Sox10, and GFP. The Phox2b+Sox10+ PG neurons in the sphenopalatine and submandibular ganglia express GFP, indicating that they are all derived from r4 NC cells. The otic ganglion has a greater number of triple-labeled PG neurons compared to Hoxa3Cre/+;ROSAYFP/+ embryos, indicating that the double-positive PG neurons in the Hoxa3Cre/+;ROSAYFP/+ embryo arise r4.

(G, H, I) Sagittal sections of e12.5 control (Neurog2+/+;Hoxa3Cre/+;ROSAYFP/+) and Neurog−/−Hoxa3Cre/+;ROSAYFP/+ embryos immunolabeled for Phox2b, Sox10, and GFP. The otic ganglion in the control embryo contains both triple-labeled Phox2b+Sox10+GFP+ and double-labeled Phox2b+Sox10+ PG neurons. The Neurog2−/− embryo contains only triple-labeled Phox2b+Sox10+GFP+ PG neurons. Statistical analysis show an 80.7% reduction in the number of triple-labeled Phox2b+Sox10+GFP+ PG neurons in the otic ganglion of Neurog2−/− embryos compared to control embryos (1146 ± 98 S.D. vs. 221 ± 166 S.D. neurons/ganglion, p<0.001; n=3).

Since the differentiation of r4 NC-derived PG precursors of the submandibular and sphenopalatine ganglia has been shown to depend on their interaction with the geniculate ganglion (Coppola et al., 2010; Takano-Maruyama et al., 2010), we reasoned that the population of r4 NC cells that contribute to the otic ganglion could also depend on the interaction with the geniculate ganglion. To address this, we generated Neurog2−/−;Hoxa3Cre/+;ROSA YFP/+ embryos to monitor the fate of lineage-labeled postotic NC cells in the absence of the geniculate ganglion. In e12.5 control sagittal sections labeled for Phox2b, Sox10, and GFP, we observed both triple-labeled Phox2b+Sox10+GFP+ and double-labeled Phox2b+Sox10+ precursors in the otic ganglion (Fig. 4G). In contrast, only triple-labeled Phox2b+Sox10+GFP+ precursors were observed in the otic ganglion of Neurog2−/−;Hoxa3Cre/+;ROSAYFP/+ embryos (Fig. 4H). The absence of double-labeled Phox2b+Sox10+ precursors suggests that the r4 NC-derived population that contributes to the otic ganglion may be dependent on the geniculate ganglion for differentiation. However, the 5-fold reduction of PG neurons in the otic ganglion of Neurog2 mutant embryos cannot be simply due to the loss of the r4 NC population (Fig. 4I). This dramatic reduction might be due to a dependence of postotic NC-derived precursors on the placode-derived petrosal ganglion, which is also missing in Neurog2 mutant embryos (Fode et al., 1998). These findings suggest that a small r4 NC population contributes to the otic ganglion, and imply that the ectopic differentiation of PG neurons in the otic ganglion of Hoxa1−/−b1−/− embryos is mediated through the interaction of r2-derived NC cells with the geniculate ganglion. Furthermore, since Hoxa1−/−b1−/− embryos were reported to be deficient in postotic NC cells (Gavalas et al., 1998), it is unlikely that the postotic hindbrain provides the source of PG neurons in the otic ganglion.

DISCUSSION

In this study we provide genetic evidence that the alignment and interaction of r4-derived NC and ba2-derived placodal cells along the A/P axis is not absolutely required for the differentiation of PG neurons of the submandibular and sphenopalatine ganglia and inward migration of the geniculate ganglion, respectively. In the absence of r4 NC cells in Hoxa1−/−b1−/− embryos, the r2 NC compensates, showing that it has the capacity to migrate towards and differentiate within the geniculate ganglion. The ability of the r2 NC to differentiate into PG neurons is consistent with the idea that the cranial NC is not prepatterned, but is dependent on environmental signals (Fontaine-Perus et al., 1988; Kirby, 1988; Saldivar et al., 1996; Trainor et al., 2002; Le Douarin et al., 2004; Bogni et al., 2008). However, it remains to be determined whether this phenomenon is unique to NC cells derived from even-numbered rhombomeres, or whether NC cells from other A/P axial levels have the potential to be induced into PG neurons by the placode-derived visceral sensory ganglion. This idea also puts into question whether other placode- or NC-derived ganglion related to other sensory modalities can induce NC cells to differentiate into PG neurons. Our data suggest that the epibranchial placode-derived visceral sensory ganglion may be unique in this process. For example, although the placode-derived vestibulocochlear (balance and hearing) and placode/NC-derived trigeminal (somatosensory) ganglia are intact in Neurog2−/− embryos, these ganglia fail to provide the compensatory environment to attract or induce the differentiation of PG neuronal precursors (Coppola et al., 2010; Takano-Maruyama et al., 2010). This suggests that the geniculate ganglion has molecular features – perhaps shared by other epibranchial placode-derived visceral sensory ganglia – that are critical for the attraction, differentiation, and/or survival of the NC fated for the PG neuronal lineage (Pattyn et al., 1999; Enomoto et al., 2000; Dauger et al., 2003; Bogni et al., 2008; Shiau et al., 2008; Coppola et al., 2010). This interaction may be specific for the parasympathetic PG neuronal lineage, as NC cells derived from the trunk spinal cord differentiate into sympathetic PG neurons in the absence of the dorsal root ganglia in Neurog1−/−2−/− embryos (Ma et al., 1999). How about PG neurons of the sacral division of the parasympathetic nervous system? Are NC cells from this caudal level of the neural tube dependent on a placode-like cellular environment to differentiate into PG neurons? A detailed analysis of the cellular environment encountered by the migratory NC population at this level of the A/P level axis could provide insight into this question, and possibly to a broader mechanism that regulates the specification of both cranial and sacral parasympathetic PG neurons. At this point, our findings indicate that the interaction between the NC and the epibranchial placode-derived sensory visceral ganglion may be unique to the differentiation of cranial parasympathetic PG neurons.

Our study also reveals an unanticipated plasticity of the placode-derived geniculate ganglion – the ability to associate with an A/P mismatched NC population and migrate inwards to the hindbrain. Previous studies have shown that surgical removal of r4 in the chick embryo resulted in failure of the geniculate ganglion to migrate inwards to the hindbrain, indicating an essential role for r4-derived NC cells in this migratory process (Begbie et al., 1999; Begbie and Graham, 2001). These findings imply that neighboring NC cells that are intact in the chick embryos, presumably those derived from r2, are unable to compensate for the loss of r4. In contrast, we observed that in r4 NC-deficient Hoxa1−/−b1−/− embryos (Gavalas et al., 1998; Studer et al., 1998; Rossel and Capecchi, 1999; Gavalas et al., 2001), r2 NC cells compensate by associating with the inward migration of the geniculate ganglion, but whether this association is functional remains to be tested. Nevertheless, the discrepancy between the reported studies (Begbie et al., 1999; Begbie and Graham, 2001) and our findings may reflect how the hindbrain and surrounding tissues respond to surgical versus genetic manipulation. The inability of r2-derived NC cells to compensate for the surgical loss of r4 may depend on a time window in which r2 NC cells and the geniculate ganglion are able to respond to one another, or simply to the severity of the surgical ablation along the A/P axis (Begbie et al., 1999; Begbie and Graham, 2001; Trainor et al., 2002). With the loss of Hoxa1 and Hoxb1 in the germline, it is possible that the plasticity that we observed for the geniculate ganglion is a consequence of an early coordinated reorganization of the hindbrain and surrounding branchial arch tissues (Rossel and Capecchi, 1999). Alternatively, the loss of r3 and the boundary between the trigeminal and geniculate ganglia in Hoxa1−/−b1−/− embryos (Fig. 2C–F), which may be a manifestation of this reorganization, eliminates a non-permissive migratory boundary (Lumsden and Guthrie, 1991). This in turn may allow r2-derived NC cells to engage the geniculate ganglion, thereby facilitating its inward migration. Short of resolving this issue, future work will focus on identifying the time window and the molecular requirements that control the full repertoire of cell types that can differentiate from the even-numbered rhombomere NC and epibranchial placode-derived cell interaction (Fontaine-Perus et al., 1988; Trainor et al., 2002; Le Douarin et al., 2004).

EXPERIMENTAL PROCEDURES

Mouse Lines

The Hoxa1, Hoxb1, and Neurog2 mutant mice, and Hoxb1-Ires-Cre, Hoxa3-Ires-Cre, and ROSA-YFP reporter mice have been reported (Ma et al., 1999; Rossel and Capecchi, 1999; Srinivas et al., 2001; Arenkiel et al., 2003; Macatee et al., 2003).

Immunohistochemistry and Antibody Generation

Embryos were dissected in cold phosphate-buffered saline (PBS) and fixed in cold 4% paraformaldehyde (Sigma, St. Louis, MO) for 30 min at 4°C and cryoprotected in 30% sucrose at 4°C, followed by embedding in OCT compound (Tissue-Tek). The cryostat sections were blocked in PBS-T (phosphate-buffered saline with 0.02% TritonX-100) with 5% skimmed milk and 5% normal goat sera for 5 min at room temperature, followed by incubation with the appropriate dilution of primary antibody in PBS-T containing 2% skimmed milk and 5% goat sera overnight at 4°C. The following primary antibodies used for this study were mouse anti-Isl1/2 (Developmental Studies Hybridoma Bank, Iowa, IA), mouse anti-Sox10 (a gift from D. Anderson), and sheep anti-GFP (Invitrogen). The rabbit polyclonal antibody was generated against a 15-amino acid mouse Phox2b peptide sequence conjugated to cysteine, CPNGAKAALVKSSMF (Pattyn et al., 1997). The synthesis and purification of the Phox2b peptide, and subsequent immunization and bleeding of rabbits were conducted at the Covance facilities. For visualization of antibody binding, the sections were incubated with Alexa 594 conjugated goat anti-rabbit (Fab)2 fragment (Invitrogen), Alexa 647 conjugated goat anti-mouse (Fab)2 fragment (Invitrogen) and Alexa 488 conjugated goat anti-mouse (Fab)2 (Invitrogen) for 45min at 4°C. Detection of apoptotic cells was performed with In situ cell death detection kit (Roche Applied Science, Indianapolis, IN). The sections were incubated with TdT and secondary antibodies for 1hr at 37°C. The slides were examined with a Zeiss LSM 5 PASCAL confocal microscope. Cell counting was performed using Imaris software (Bitplane AG, Zurich, Switzerland) in combination with manual verification of the automated results.

Statistical Analysis

Results are expressed as means ± SD. Statistical analysis of differences between two groups was performed using the unpaired Student's t-test (two-tailed analysis). Differences with P values of <0.05 were considered statistically significant.

ACKNOWLEDGEMENTS

We would like to thank Drs. M. Capecchi, A.M. Moon, and D.J. Anderson for providing mice and reagents, Dr. R. Krumlauf and members of the NIH-SNRP-SAC for helpful comments, and O. Trevino for technical assistance. This study was supported by grants to G.O.G from the NIH (NS060658) and the Whitehall Foundation.

Grant Support: NIH (NS060658) and Whitehall Foundation

Footnotes

Contributions: Y.C., M.T.-M., and G.O.G. designed research and analyzed data; Y.C. and M.T.-M. performed research; and G.O.G. wrote the paper.

The authors declare no conflict of interest.

REFERENCES

  1. Arenkiel BR, Gaufo GO, Capecchi MR. Hoxb1 neural crest preferentially form glia of the PNS. Dev Dyn. 2003;227:379–386. doi: 10.1002/dvdy.10323. [DOI] [PubMed] [Google Scholar]
  2. Arenkiel BR, Tvrdik P, Gaufo GO, Capecchi MR. Hoxb1 functions in both motoneurons and in tissues of the periphery to establish and maintain the proper neuronal circuitry. Genes Dev. 2004;18:1539–1552. doi: 10.1101/gad.1207204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barlow LA. Cranial nerve development: placodal neurons ride the crest. Curr Biol. 2002;12:R171–R173. doi: 10.1016/s0960-9822(02)00734-0. [DOI] [PubMed] [Google Scholar]
  4. Begbie J, Brunet JF, Rubenstein JL, Graham A. Induction of the epibranchial placodes. Development. 1999;126:895–902. doi: 10.1242/dev.126.5.895. [DOI] [PubMed] [Google Scholar]
  5. Begbie J, Graham A. Integration between the epibranchial placodes and the hindbrain. Science. 2001;294:595–598. doi: 10.1126/science.1062028. [DOI] [PubMed] [Google Scholar]
  6. Bell E, Wingate RJ, Lumsden A. Homeotic transformation of rhombomere identity after localized Hoxb1 misexpression. Science. 1999;284:2168–2171. doi: 10.1126/science.284.5423.2168. [DOI] [PubMed] [Google Scholar]
  7. Bertrand N, Roux M, Ryckebusch L, Niederreither K, Dolle P, Moon A, Capecchi M, Zaffran S. Hox genes define distinct progenitor sub-domains within the second heart field. J Dev Biol. 2011;353:266–274. doi: 10.1016/j.ydbio.2011.02.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bogni S, Trainor P, Natarajan D, Krumlauf R, Pachnis V. Non-cell-autonomous effects of Ret deletion in early enteric neurogenesis. Development. 2008;135:3007–3011. doi: 10.1242/dev.025163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carpenter EM, Goddard JM, Davis AP, Nguyen TP, Capecchi MR. Targeted disruption of Hoxd-10 affects mouse hindlimb development. Development. 1997;124:4505–4514. doi: 10.1242/dev.124.22.4505. [DOI] [PubMed] [Google Scholar]
  10. Chen SK, Tvrdik P, Peden E, Cho S, Wu S, Spangrude G, Capecchi MR. Hematopoietic origin of pathological grooming in Hoxb8 mutant mice. Cell. 2010;141:775–785. doi: 10.1016/j.cell.2010.03.055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Coppola E, Rallu M, Richard J, Dufour S, Riethmacher D, Guillemot F, Goridis C, Brunet JF. Epibranchial ganglia orchestrate the development of the cranial neurogenic crest. PNAS USA. 2010;107:2066–2071. doi: 10.1073/pnas.0910213107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. D'Amico-Martel A, Noden DM. Contributions of placodal and neural crest cells to avian cranial peripheral ganglia. Am J Anat. 1983;166:445–468. doi: 10.1002/aja.1001660406. [DOI] [PubMed] [Google Scholar]
  13. Dasen JS, Liu JP, Jessell TM. Motor neuron columnar fate imposed by sequential phases of Hox-c activity. Nature. 2003;425:926–933. doi: 10.1038/nature02051. [DOI] [PubMed] [Google Scholar]
  14. Dasen JS, Tice BC, Brenner-Morton S, Jessell TM. A Hox regulatory network establishes motor neuron pool identity and target-muscle connectivity. Cell. 2005;123:477–491. doi: 10.1016/j.cell.2005.09.009. [DOI] [PubMed] [Google Scholar]
  15. Dauger S, Pattyn A, Lofaso F, Gaultier C, Goridis C, Gallego J, Brunet JF. Phox2b controls the development of peripheral chemoreceptors and afferent visceral pathways. Development. 2003;130:6635–6642. doi: 10.1242/dev.00866. [DOI] [PubMed] [Google Scholar]
  16. Enomoto H, Heuckeroth RO, Golden JP, Johnson EM, Milbrandt J. Development of cranial parasympathetic ganglia requires sequential actions of GDNF and neurturin. Development. 2000;127:4877–4889. doi: 10.1242/dev.127.22.4877. [DOI] [PubMed] [Google Scholar]
  17. Fode C, Gradwohl G, Morin X, Dierich A, LeMeur M, Goridis C, Guillemot F. The bHLH protein NEUROGENIN 2 is a determination factor for epibranchial placode-derived sensory neurons. Neuron. 1998;20:483–494. doi: 10.1016/s0896-6273(00)80989-7. [DOI] [PubMed] [Google Scholar]
  18. Fontaine-Perus J, Chanconie M, Le Douarin NM. Developmental potentialities in the nonneuronal population of quail sensory ganglia. J Dev Biol. 1988;128:359–375. doi: 10.1016/0012-1606(88)90298-9. [DOI] [PubMed] [Google Scholar]
  19. Gaufo GO, Thomas KR, Capecchi MR. Hox3 genes coordinate mechanisms of genetic suppression and activation in the generation of branchial and somatic motoneurons. Development. 2003;130:5191–5201. doi: 10.1242/dev.00730. [DOI] [PubMed] [Google Scholar]
  20. Gaufo GO, Wu S, Capecchi MR. Contribution of Hox genes to the diversity of the hindbrain sensory system. Development. 2004;131:1259–1266. doi: 10.1242/dev.01029. [DOI] [PubMed] [Google Scholar]
  21. Gavalas A, Davenne M, Lumsden A, Chambon P, Rijli FM. Role of Hoxa-2 in axon pathfinding and rostral hindbrain patterning. Development. 1997;124:3693–3702. doi: 10.1242/dev.124.19.3693. [DOI] [PubMed] [Google Scholar]
  22. Gavalas A, Ruhrberg C, Livet J, Henderson CE, Krumlauf R. Neuronal defects in the hindbrain of Hoxa1, Hoxb1 and Hoxb2 mutants reflect regulatory interactions among these Hox genes. Development. 2003;130:5663–5679. doi: 10.1242/dev.00802. [DOI] [PubMed] [Google Scholar]
  23. Gavalas A, Studer M, Lumsden A, Rijli FM, Krumlauf R, Chambon P. Hoxa1 and Hoxb1 synergize in patterning the hindbrain, cranial nerves and second pharyngeal arch. Development. 1998;125:1123–1136. doi: 10.1242/dev.125.6.1123. [DOI] [PubMed] [Google Scholar]
  24. Gavalas A, Trainor P, Ariza-McNaughton L, Krumlauf R. Synergy between Hoxa1 and Hoxb1: the relationship between arch patterning and the generation of cranial neural crest. Development. 2001;128:3017–3027. doi: 10.1242/dev.128.15.3017. [DOI] [PubMed] [Google Scholar]
  25. Goddard JM, Rossel M, Manley NR, Capecchi MR. Mice with targeted disruption of Hoxb-1 fail to form the motor nucleus of the VIIth nerve. Development. 1996;122:3217–3228. doi: 10.1242/dev.122.10.3217. [DOI] [PubMed] [Google Scholar]
  26. Holstege JC, de Graaff W, Hossaini M, Cano SC, Jaarsma D, van den Akker E, Deschamps J. Loss of Hoxb8 alters spinal dorsal laminae and sensory responses in mice. PNAS USA. 2008;105:6338–6343. doi: 10.1073/pnas.0802176105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kiecker C, Lumsden A. Compartments and their boundaries in vertebrate brain development. Nat Rev Neurosci. 2005;6:553–564. doi: 10.1038/nrn1702. [DOI] [PubMed] [Google Scholar]
  28. Kirby ML. Nodose placode contributes autonomic neurons to the heart in the absence of cardiac neural crest. J Neurosci. 1988;8:1089–1095. doi: 10.1523/JNEUROSCI.08-04-01089.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Krumlauf R, Marshall H, Studer M, Nonchev S, Sham MH, Lumsden A. Hox homeobox genes and regionalisation of the nervous system. J Neurobiol. 1993;24:1328–1340. doi: 10.1002/neu.480241006. [DOI] [PubMed] [Google Scholar]
  30. Lance-Jones C, Omelchenko N, Bailis A, Lynch S, Sharma K. Hoxd10 induction and regionalization in the developing lumbosacral spinal cord. Development. 2001;128:2255–2268. doi: 10.1242/dev.128.12.2255. [DOI] [PubMed] [Google Scholar]
  31. Le Douarin NM, Creuzet S, Couly G, Dupin E. Neural crest cell plasticity and its limits. Development. 2004;131:4637–4650. doi: 10.1242/dev.01350. [DOI] [PubMed] [Google Scholar]
  32. Lumsden A, Guthrie S. Alternating patterns of cell surface properties and neural crest cell migration during segmentation of the chick hindbrain. Development Suppl. 1991;2:9–15. [PubMed] [Google Scholar]
  33. Lumsden A, Krumlauf R. Patterning the vertebrate neuraxis. Science. 1996;274:1109–1115. doi: 10.1126/science.274.5290.1109. [DOI] [PubMed] [Google Scholar]
  34. Lumsden A, Sprawson N, Graham A. Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development. 1991;113:1281–1291. doi: 10.1242/dev.113.4.1281. [DOI] [PubMed] [Google Scholar]
  35. Ma Q, Fode C, Guillemot F, Anderson DJ. Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia. Genes Dev. 1999;13:1717–1728. doi: 10.1101/gad.13.13.1717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Macatee TL, Hammond BP, Arenkiel BR, Francis L, Frank DU, Moon AM. Ablation of specific expression domains reveals discrete functions of ectoderm- and endoderm-derived FGF8 during cardiovascular and pharyngeal development. Development. 2003;130:6361–6374. doi: 10.1242/dev.00850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Manley NR, Capecchi MR. The role of Hoxa-3 in mouse thymus and thyroid development. Development. 1995;121:1989–2003. doi: 10.1242/dev.121.7.1989. [DOI] [PubMed] [Google Scholar]
  38. Matsuoka T, Ahlberg PE, Kessaris N, Iannarelli P, Dennehy U, Richardson WD, McMahon AP, Koentges G. Neural crest origins of the neck and shoulder. Nature. 2005;436:347–355. doi: 10.1038/nature03837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. McClintock JM, Kheirbek MA, Prince VE. Knockdown of duplicated zebrafish hoxb1 genes reveals distinct roles in hindbrain patterning and a novel mechanism of duplicate gene retention. Development. 2002;129:2339–2354. doi: 10.1242/dev.129.10.2339. [DOI] [PubMed] [Google Scholar]
  40. McGinnis W, Krumlauf R. Homeobox genes and axial patterning. Cell. 1992;68:283–302. doi: 10.1016/0092-8674(92)90471-n. [DOI] [PubMed] [Google Scholar]
  41. Oury F, Murakami Y, Renaud JS, Pasqualetti M, Charnay P, Ren SY, Rijli FM. Hoxa2- and rhombomere-dependent development of the mouse facial somatosensory map. Science. 2006;313:1408–1413. doi: 10.1126/science.1130042. [DOI] [PubMed] [Google Scholar]
  42. Park EJ, Ogden LA, Talbot A, Evans S, Cai CL, Black BL, Frank DU, Moon AM. Required, tissue-specific roles for Fgf8 in outflow tract formation and remodeling. Development. 2006;133:2419–2433. doi: 10.1242/dev.02367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF. Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis. Development. 1997;124:4065–4075. doi: 10.1242/dev.124.20.4065. [DOI] [PubMed] [Google Scholar]
  44. Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF. The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature. 1999;399:366–370. doi: 10.1038/20700. [DOI] [PubMed] [Google Scholar]
  45. Rossel M, Capecchi MR. Mice mutant for both Hoxa1 and Hoxb1 show extensive remodeling of the hindbrain and defects in craniofacial development. Development. 1999;126:5027–5040. doi: 10.1242/dev.126.22.5027. [DOI] [PubMed] [Google Scholar]
  46. Saldivar JR, Krull CE, Krumlauf R, Ariza-McNaughton L, Bronner-Fraser M. Rhombomere of origin determines autonomous versus environmentally regulated expression of Hoxa-3 in the avian embryo. Development. 1996;122:895–904. doi: 10.1242/dev.122.3.895. [DOI] [PubMed] [Google Scholar]
  47. Shiau CE, Lwigale PY, Das RM, Wilson SA, Bronner-Fraser M. Robo2-Slit1 dependent cell-cell interactions mediate assembly of the trigeminal ganglion. Nat Neurosci. 2008;11:269–276. doi: 10.1038/nn2051. [DOI] [PubMed] [Google Scholar]
  48. Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM, Costantini F. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol. 2001;1:4. doi: 10.1186/1471-213X-1-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Studer M, Gavalas A, Marshall H, Ariza-McNaughton L, Rijli FM, Chambon P, Krumlauf R. Genetic interactions between Hoxa1 and Hoxb1 reveal new roles in regulation of early hindbrain patterning. Development. 1998;125:1025–1036. doi: 10.1242/dev.125.6.1025. [DOI] [PubMed] [Google Scholar]
  50. Studer M, Lumsden A, Ariza-McNaughton L, Bradley A, Krumlauf R. Altered segmental identity and abnormal migration of motor neurons in mice lacking Hoxb-1. Nature. 1996;384:630–634. doi: 10.1038/384630a0. [DOI] [PubMed] [Google Scholar]
  51. Takano-Maruyama M, Chen Y, Gaufo GO. Placodal sensory ganglia coordinate the formation of the cranial visceral motor pathway. Dev Dyn. 2010;239:1155–1161. doi: 10.1002/dvdy.22273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tiret L, Le Mouellic H, Maury M, Brulet P. Increased apoptosis of motoneurons and altered somatotopic maps in the brachial spinal cord of Hoxc-8-deficient mice. Development. 1998;125:279–291. doi: 10.1242/dev.125.2.279. [DOI] [PubMed] [Google Scholar]
  53. Trainor P, Krumlauf R. Plasticity in mouse neural crest cells reveals a new patterning role for cranial mesoderm. Nat Cell Biol. 2000a;2:96–102. doi: 10.1038/35000051. [DOI] [PubMed] [Google Scholar]
  54. Trainor PA, Ariza-McNaughton L, Krumlauf R. Role of the isthmus and FGFs in resolving the paradox of neural crest plasticity and prepatterning. Science. 2002;295:1288–1291. doi: 10.1126/science.1064540. [DOI] [PubMed] [Google Scholar]
  55. Trainor PA, Krumlauf R. Patterning the cranial neural crest: hindbrain segmentation and Hox gene plasticity. Nat Rev Neurosci. 2000b;1:116–124. doi: 10.1038/35039056. [DOI] [PubMed] [Google Scholar]
  56. Trainor PA, Krumlauf R. Hox genes, neural crest cells and branchial arch patterning. Curr Opin Cell Biol. 2001;13:698–705. doi: 10.1016/s0955-0674(00)00273-8. [DOI] [PubMed] [Google Scholar]
  57. Yang X, Zhou Y, Barcarse EA, O'Gorman S. Altered neuronal lineages in the facial ganglia of Hoxa2 mutant mice. J Dev Biol. 2008;314:171–188. doi: 10.1016/j.ydbio.2007.11.032. [DOI] [PubMed] [Google Scholar]

RESOURCES