Sonic hedgehogexpressing and responding cells generate neuronal diversity in the medial amygdala
© Carney et al; licensee BioMed Central Ltd. 2010
Received: 8 March 2010
Accepted: 27 May 2010
Published: 27 May 2010
The mammalian amygdala is composed of two primary functional subdivisions, classified according to whether the major output projection of each nucleus is excitatory or inhibitory. The posterior dorsal and ventral subdivisions of the medial amygdala, which primarily contain inhibitory output neurons, modulate specific aspects of innate socio-sexual and aggressive behaviors. However, the development of the neuronal diversity of this complex and important structure remains to be fully elucidated.
Using a combination of genetic fate-mapping and loss-of-function analyses, we examined the contribution and function of Sonic hedgehog (Shh)-expressing and Shh-responsive (Nkx2-1+ and Gli1+) neurons in the medial amygdala. Specifically, we found that Shh- and Nkx2-1-lineage cells contribute differentially to the dorsal and ventral subdivisions of the postnatal medial amygdala. These Shh- and Nkx2-1-lineage neurons express overlapping and non-overlapping inhibitory neuronal markers, such as Calbindin, FoxP2, nNOS and Somatostatin, revealing diverse fate contributions in discrete medial amygdala nuclear subdivisions. Electrophysiological analysis of the Shh-derived neurons additionally reveals an important functional diversity within this lineage in the medial amygdala. Moreover, inducible Gli1 CreER(T2) temporal fate mapping shows that early-generated progenitors that respond to Shh signaling also contribute to medial amygdala neuronal diversity. Lastly, analysis of Nkx2-1 mutant mice demonstrates a genetic requirement for Nkx2-1 in inhibitory neuronal specification in the medial amygdala distinct from the requirement for Nkx2-1 in cerebral cortical development.
Taken together, these data reveal a differential contribution of Shh-expressing and Shh-responding cells to medial amygdala neuronal diversity as well as the function of Nkx2-1 in the development of this important limbic system structure.
The mammalian amygdala is an aggregation of 11 to 15 nuclei, which as components of the limbic system mediate distinct aspects of emotional and behavioral processing as well as socio-sexual behaviors (reviewed in ). The amygdala is considered in terms of two functional subdivisions, classified according to whether the major output projection of each nucleus is excitatory or inhibitory. Based on this classification, the nuclei of the basolateral complex and cortical amygdalar nuclei, which have an excitatory output, have been hypothesized to be of a pallial origin, whereas the cortical and medial nuclei are broadly considered similar to the striatum as their output projections are primarily inhibitory. Several studies have revealed that, during embryogenesis, the emerging amygdala is generated from several forebrain embryonic domains, including the pallium, pallial-subpallial boundary, medial ganglionic eminence (MGE), preoptic area (POA) and perhaps the diencephalon [2–10].
The medial amygdala (MeA) subnuclei process pheromonal information, and regulate neuroendocrine function and socio-sexual behaviors. Anatomically, the posterior portion of the MeA is divided into dorsal (medial posterodorsal nucleus (MePD)) and ventral (medial posteroventral nucleus (MePV)) subdivisions, which via their projections to distinct hypothalamic nuclei regulate reproductive and defensive behaviors, respectively (reviewed in [1, 11]). Although not well-characterized, recent studies have revealed insight into the development of the MeA. In terms of circuit formation, the anatomical segregation of efferent projections that regulate reproductive or defensive behaviors is differentially marked by the LIM-containing homeodomain genes Lhx6 and Lhx9 [12, 13]. In the embryonic subpallium, Lhx6 is expressed in tangentially migrating cortical interneurons [14–16], and is a direct transcriptional target of Nkx2-1, which is expressed in the embryonic MGE and POA [17, 18]. In addition, our recent work has revealed that the embryonic telencephalic POA is a major novel source of MeA neurons . Moreover, Nkx2-1-lineage cells have been shown to contribute to the anterior MeA, although their neurochemical fate was not determined . Despite this developmental information, the lineages that contribute to the vast inhibitory neuronal diversity in the MeA remain largely unknown.
Based on the expression patterns of the morphogen Sonic hedgehog (Shh) as well as the Shh-responsive genes Nkx2-1 and Gli1 in the embryonic MGE and POA [19, 20] and, at later developmental stages (by embryonic day 14.5 (E14.5)), expression of Shh in the MePV , we hypothesized that progenitor populations marked by these genes contribute to inhibitory neuronal diversity in the postnatal MeA. In this study, we find that Shh-, Nkx2-1- and Gli1-lineage cells generate inhibitory neuronal diversity in the MePD and MePV in a largely complementary manner. We further reveal a differential functional requirement for Nkx2-1 in the development of the MePD compared to the MePV, and find that Shh-lineage cells generate three functionally distinct classes of neurons in the posterior MeA as defined by electrophysiological criteria. Thus, these data provide novel insights into the lineage and genetic mechanisms that generate the amygdala nuclei that mediate socio-sexual behaviors.
Embryonic expression of Nkx2-1, Shh and Gli1and recombined cells
Progenitor pools in the embryonic telencephalon are marked by spatio-temporal diversity in gene expression patterns. Expression of these genes is typically down-regulated as cells become post-mitotic. To permanently label these transiently marked populations, we used transgenic mouse lines that express Cre recombinase under transcriptional regulation of Shh, Nkx2-1 and Gli1 [5, 21, 22]. The developmental expression of these genes has been well-characterized in previous studies. At E11.5 and E13.5, Nkx2-1 is expressed throughout the MGE progenitor domains (pMGE 1-5) and the progenitor domains of the dorsal (pPOA1) and ventral (pPOA2) subdivisions of the embryonic POA . In addition, previous studies  revealed that Nkx2-1-derived cells, using the same Nkx2-1-Cre mouse line as in this study, migrate toward the developing amygdala. At E11.5, Shh is expressed in the MGE mantle zone and in the progenitor domains of the POA (pPOA1 and pPOA2) and in a small region of the septum [19, 23]. Gli1 is a transcriptional target of Shh and its expression, therefore, is observed near Shh-expressing domains [24–26]. Consequently, at early developmental time points Gli1 mRNA expression is observed in the ventral POA (pPOA2) and the sulcus between the lateral ganglionic eminence (LGE) and the MGE (pLGE4 and pMGE1) [20, 27].
In summary, based on our analyses and previous studies, Shh, Nkx2-1 and Gli1 are spatially and temporally present in progenitor domains known to contribute to the MeA. Therefore, we next focused our analyses on the postnatal characterization of these genetically labeled cell populations.
Nkx2-1- and Shh-lineage cells preferentially accumulate in different subdivisions of the adult posterior MeA
Taken together, these results show that the dorsal (MePD) and ventral (MePV) subdivisions of the adult posterior MeA can be preferentially populated from Nkx2-1- and Shh-lineages, respectively. Nkx2-1-lineage cells are predominantly observed in the MePD whereas Shh-lineage cells are mainly observed in the MePV. In addition, we show that Gli1-derived cells contribute to both subdivisions of the posterior MeA.
Characterization of inhibitory neuronal cell types in the posterior MeA
Neuronal cell diversity in the MeA has not been thoroughly explored. Therefore, we first wanted to characterize the molecular/neurochemical profiles of specific cell types in both the MePD and MePV. We reasoned that this analysis was a necessary prerequisite for subsequent analysis of the fate of Nkx2-1-, Shh- and Gli1-derived populations. To this end, we immunostained P22 wild-type mice with a battery of antibodies against proteins whose expression patterns are well-characterized in other forebrain structures but have not been directly compared to or between the subdivisions of the posterior MeA. As the MeA is largely composed of GABAergic neurons (reviewed in ), expression of Tbr1, which is a marker of excitatory neurons [30, 31], was only observed in the ventral-most portion of the MePV, adjacent to the superficial layer of the MeA (Additional file 1A, B). Conversely both the MePD and MePV were highly immunoreactive for the GABA synthesizing enzymes GAD65/67 (Additional file 1C, D). Surprisingly, we found that expression of Parvalbumin and Neuropeptide Y, which are markers of inhibitory neuronal subtypes in the cerebral cortex and striatum, were nearly absent from the posterior MeA (Additional file 1E-H).
Nkx2-1-, Shh- and Gli1-lineage cells contribute to inhibitory neuronal diversity in the posterior MeA
Next, we analyzed the percentage co-expression of the markers Calbindin, nNOS, FoxP2 and SST in recombined cells in all three Cre lines. Calbindin was expressed by almost half of the recombined cells in the MePD of Nkx2-1-Cre (43 ± 13%, n = 740 β-gal+ cells; Figure 4A, A'), Shh Cre (46 ± 16%, n = 350 β-gal+ cells; Figure 4B, B') and Gli1 CreER(T2) brains (39 ± 4%, n = 476 β-gal+ cells; Figure 4C, C'). As mentioned previously, nNOS is expressed in a relatively low number of cells in the MePD compared to the MePV. Interestingly, for the MePD, the contributions of the Nkx2-1 lineage (20 ± 10%, n = 1,200 β-gal+ cells; Figure 4D, D') and the Shh-lineage (22 ± 3%, n = 432 β-gal+ cells; Figure 4E, E') to the nNOS population were remarkably similar. This likely reflects the contribution of the preoptic domains pPOA1 and pPOA2 in which Nkx2-1 and Shh are co-expressed  and is consistent with previous work from our laboratory showing that Dbx1+ cells from the POA generate nNOS+ cells in the MeA . Furthermore, from early-labeled Gli1 CreER(T2) (TM E9.5) brains we found that MePD nNOS+ cells also derive from this Shh-responding population (15 ± 6%, n = 621 β-gal+ cells; Figure 4F, F'). In addition, FoxP2+ cells were derived from both Nkx2-1 (37 ± 10%, n = 903 β-gal+ cells; Figure 4G, G') and Shh (27 ± 12%, n = 477 β-gal+ cells; Figure 4H, H') lineages in the MePD, although this difference was not statistically significant (P = 0.48). Gli1-derived cells co-expressing FoxP2 were abundant in the MePD (23 ± 6%, n = 450 β-gal+ cells; Figure 4I, I'), showing that progenitor cells that respond to Shh signaling from an early developmental stage generate this inhibitory cell type in the posterior MeA. Interestingly, in the MePD we found a statistically significant (P < 0.01) difference in the generation of SST-positive cells from the Nkx2-1-lineage cells (10 ± 1%, n = 1116 β-gal+ cells; Figure 4J, J') versus the Shh-lineage cells (2 ± 2%, n = 431 β-gal+ cells; Figure 4K, K'). SST+ cells were also significantly generated from Gli1-expressing progenitors (20 ± 10%, n = 590 β-gal+ cells; Figure 4L, L'), which has also been observed for the cerebral cortex .
In summary, these data revealed that: Nkx2-1- and Shh-lineage cells show a complementary distribution between the dorsal and ventral subdivisions in the adult posterior MeA - in contrast, Gli1-derived neurons contribute more predominantly to the MePD but heavily to both subdivisions; nNOS+ cells, which are preferentially localized in the MePV, are primarily derived from Shh-lineage cells; Nkx2-1-lineage cells generated a higher proportion of SST+ cells in the MePD and MePV than Shh-lineage cells; Nkx2-1-derived cells showed a higher co-expression with FoxP2 than Shh-lineage cells, although within both lineages there was no discrimination between the MePV and the MePD; and early-generated Gli1+ progenitor cells generated inhibitory neuronal cells in proportions similar to those derived from the Nkx2-1 lineage.
Nkx2-1 mutant analysis demonstrates a greater functional role for Nkx2-1in the MePD compared to the MePV
Having shown that the Nkx2-1 lineage contributes to neural diversity in the posterior MeA, we next sought to determine whether there is a functional requirement for Nkx2-1 in the generation of these cell types. To this end, we analyzed the expression of genes that mark the above-described inhibitory neurons in Nkx2-1 mutant mice . This analysis was accomplished at E18.5, the latest stage possible due to perinatal lethality. First, we used GAD67 mRNA expression as a pan-inhibitory neuronal marker, which in controls is abundantly expressed throughout the whole telencephalon, including the hippocampus, cortex, striatum and amygdala (Figure 6A). In agreement with prior studies [18, 38, 39], we observed a major reduction in GAD67 mRNA expression throughout the Nkx2-1 mutant telencephalon, including the posterior MeA (Figure 6B). In particular, the MePD displayed intense GAD67 mRNA expression in the control (Figure 6A), which was reduced in the absence of Nkx2-1 (Figure 6B). The mutant MePV also showed a clear reduction in GAD67 mRNA expression compared to control (Figure 6A, B). Next, we examined the expression of Lhx6, which, similar to GAD67, exhibited notably more intense expression in the MePD than the MePV in control brains (Figure 6C). Prior studies have described a complete loss of Lhx6 in the Nkx2-1 mutant telencephalon [18, 40], which was later explained by the demonstration that Nkx2-1 directly regulates Lhx6 transcription . Therefore, we were surprised to find a subset of cells in the posterior MeA that expressed Lhx6 mRNA in the Nkx2-1 mutant (Figure 6D). These 'remnant' Lhx6 mRNA-expressing cells were located in the MePD, whereas the MePV was devoid of Lhx6 expression (Figure 6D). Subsequently, we examined Sst mRNA expression. In control, there were numerous Sst mRNA-expressing cells in the MePD, whereas the MePV contained only a few positive cells (Figure 6E). However, similar to Lhx6, we observed a number of remaining Sst mRNA-expressing cells that were primarily observed in the MePD (Figure 6F). This is in interesting contrast to other areas of the telencephalon, such as the cerebral cortex and hippocampus, that display a complete absence of Sst mRNA-expressing cells. A large number of Sst mRNA-expressing cells in the central nucleus was also maintained in the absence of Nkx2-1 (Figure 6F).
This analysis demonstrates that Nkx2-1 plays an important role in the specification of inhibitory neuronal subtypes in the posterior MeA. Surprisingly, however, we noticed that a population of cells that expressed Lhx6 and Sst mRNA persisted in the MePD in the Nkx2-1 mutant. Therefore, in interesting contrast to the cerebral cortex, a small population of posterior MeA Lhx6+ and Sst+ cells is not dependent on Nkx2-1 function.
In summary, the Nkx2-1 mutant analysis at E18.5 suggests a functional role for this gene in both the MePD and MePV, with primary effects on the MePD. This is consistent with the fate-mapping analysis (Figure 2) in which Nkx2-1-lineage cells show a major contribution to the MePD.
Electrophysiological characterization reveals three distinct functional classes of Shh-lineage neurons in the posterior MeA
When hyperpolarized to -100 mV by injecting a step current (-40 to 120 pA, 1,000 ms), the first class of YFP+ neurons (class I; 9 out of 28 neurons) was characterized by clear depolarizing sag current (5.5 ± 2 mV; n = 9), usually resulting in a single rebound spike upon repolarization (n = 7 out of 9; Figure 8A). A small depolarizing step current at rheobase value (10 to 40 pA, 1,000 ms) typically induced a series of three to four spikes separated by relatively large and slow rising afterhyperpolarization (amplitude = -11.2 ± 2.5 mV; decay half-time = 50.7 ± 14.7 ms). Larger depolarizing current steps (100 to 200 pA, 1,000 ms) were injected to determine their maximal discharge frequency (24 ± 5 Hz) and showed accommodating firing patterns (accommodation ratio = 0.46 ± 0.08). Post hoc immunohistochemical analysis showed that all neurons in this class were nNOS+/FoxP2- (Figure 8B), with the exception of one cell that was immunonegative for both markers. Interestingly, both the electrophysiological and immunohistochemical profile of this first class of YFP+ neurons made them indistinguishable from nNOS+ MeA neurons generated from the Dbx1-derived progenitors previously described by our laboratory .
A second class of YFP+ neurons (class II; 8 out of 28 neurons) could be distinguished by their inability to repetitively fire even when large depolarizing currents were injected (100 to 300 pA, 1,000 ms) (Figure 8C). These neurons exhibited a single or dual spike discharge upon depolarization. At rheobase value (20 to 80 pA), the discharge systematically rode on a slow-depolarizing envelope (amplitude = 18 ± 8 mV), suggesting the presence of a low-threshold Ca++ current (IT). These neurons were exempt of sag currents when hyperpolarized to -100 mV but would exhibit a slow-depolarizing envelope upon repolarization, confirming the likely presence of large IT currents in these cells. Interestingly, all of these neurons were FoxP2+ and most were also nNOS+ (6 out of 8 cells; Figure 8D).
The third class of YFP+ neurons (class III; 11 out of 28 neurons) was characterized by an irregular firing (frequency 5 to 20 Hz) in response to a large depolarization (Figure 8E). At rheobase value (20 to 80 pA), these neurons displayed a single early discharge of one to three spikes riding a small depolarizing envelope (10 ± 5 mV) and exhibited sharp fast-rising afterhyperpolarization (amplitude = -13.2 ± 1.5 mV; decay half-time = 15.4 ± 4.2 ms). In response to larger depolarizing current steps (100 to 200 pA, 1,000 ms), these cells exhibited a very variable maximal discharge frequency (11 ± 5 Hz) ranging from a single initial burst of three to five spikes to a series of two or three bursts of three to five spikes each. When hyperpolarized to -100 mV, these cells exhibited no or a minimal sag current (1.5 ± 0.8 mV) and small rebound IT-like currents were observed (10 ± 5 mV). In most cells (9 out of 11 cells) these currents were not large enough to induce a rebound spike. All cells in this third class of cells were immunonegative for both nNOS and FoxP2 (Figure 8F).
Taken together, the results from the electrophysiological and post hoc immunohistochemical analyses revealed that Shh-lineage neurons generate functionally distinct inhibitory neuronal classes in the posterior MeA. We find that in addition to the class I neurons that are similar to neurons derived from the Dbx1 + lineage , at least two other classes of neurons have distinct electrophysiological and molecular profiles. Based on their expression of nNOS, a marker of MeA inhibitory projection neurons, as well as their morphology, we speculate that the classes I and II are inhibitory projection neurons. Our electrophysiological analysis shows that our Shh-lineage class III neurons that lack expression of both nNOS and FoxP2 exhibit electrophysiological characteristics that partially reflect a glutamatergic phenotype in the posterior MeA, which has been suggested by a prior study . However, the restriction of these neurons to a Tbr1-negative region indicates that they may possess inhibitory character. Interestingly, we found that Shh-lineage cells gave rise to a subset of deep layer neurons in the cerebral cortex with a pyramidal morphology (data not shown), indicating that these progenitors can give rise to a subset of glutamatergic neurons. However, the vast majority of Shh-lineage cells in the amygdala are likely to be inhibitory because the MeA is primarily GABAergic in neuronal output, as shown by intense expression of GAD65 and GAD67 ( and this study) and because nNOS  and Calbindin, which are expressed in most Shh-lineage neurons, are markers of inhibitory neurons.
Shh- and Nkx2-1-lineage cells generate distinct classes of inhibitory neurons in the posterior MeA
Shh is the most studied of the Hedgehog family of secreted glycoproteins and is a potent morphogen that plays a crucial role in ventral patterning of many tissues, including the forebrain, midbrain, cerebellum, spinal cord and the limb (reviewed by [47–52]). Here, we used a Cre line under the transcriptional regulation of Shh  to indelibly label Shh-lineage (Shh Cre +) cells from early embryonic development through to adulthood. Using this genetic fate-mapping tool we were able to identify a predominant contribution of Shh-expressing cells in the generation of inhibitory neural diversity in the MeA. Specifically, we show that Shh-lineage cells preferentially generate the cells in the MePV, which express nNOS (68% of recombined cells), a marker of hypothalamic projecting inhibitory output neurons . Interestingly, a subset of these Shh-lineage, nNOS+ cells (the class I type) display electrophysiological and molecular signatures reminiscent of our previously identified Dbx1-derived MeA neurons . Therefore, this subpopulation likely derives from the ventral subdivision of the POA (pPOA2), which expresses Dbx1 . Thus, similar to the generation of midbrain dopaminergic neurons , Shh-lineage cells appear to give rise to diverse neural subtypes in the telencephalon.
The co-expression of FoxP2 with nNOS in the class II Shh-derived neurons is also quite intriguing. FoxP2 has been identified as essential for development of brain circuitry involved in speech in humans and vocalization in other vertebrates. Mutations in FoxP2 have also been implicated in both language disorders and autism, the latter of which is characterized by both language and amygdala-based impairments (reviewed in [54, 55]). Therefore, it is possible that the electrophysiological and marker diversity of Shh-lineage neurons underlies important differences in both pathways of connectivity and function of MeA output neurons. For example, as the MeA is a central component of circuitry that regulates innate behaviors such as feeding, reproduction, aggression and maternal bonding, perhaps these different Shh-lineage populations differentially modulate these behaviors. Developmentally, it is also possible that transcription factor genes that show discrete and overlapping expression domains within the POA, such Dlx1, Nkx6-2, Nkx5-1 or Lhx2 [19, 46], may selectively or combinatorally generate the class I to III electrophysiological subtypes that we describe here. In addition, as Shh and Dbx1 are expressed in the embryonic hypothalamus [23, 56, 57], which itself has been speculated to generate cells destined for the amygdala [4, 6], ], further complexity or refinement of neural populations may be related to more precise origins within the developing forebrain.
We also find that, in contrast to Shh-lineage neurons, Nkx2-1-derived cells preferentially fate-map to the MePD, a nucleus that regulates reproductive behaviors. A previous study showed that Nkx2-1-lineage cells generate inhibitory cells in the MeA , in addition to the known and recently identified roles for Nkx2-1 in generating inhibitory cells for the cerebral cortex [5, 18, 58] and globus pallidus [29, 59], respectively. We find that analogous to the cerebral cortex, a number of SST+ cells are Nkx2-1-derived, although another subset are derived from Shh-lineage progenitor cells. Nkx2-1-lineage cells show high co-expression with Calbindin and FoxP2 in both the MePD and MePV. Whereas Shh-lineage cells generate the vast majority of nNOS+ cells in the MePV, Nkx2-1-lineage cells generate an equivalent portion of nNOS+ cells within both nuclei. Therefore, this study reveals that inhibitory neural diversity in the posterior MeA is generated from Shh-expressing and Shh-responding (Nkx2-1-Cre + and Gli1 CreER(T2)+ ) components of the Shh signaling pathway (see Figure 9 for schematic).
Function of Nkx2-1in medial amygdala patterning
The transcription factor Nkx2-1 plays a major role in the generation of inhibitory neurons in the forebrain [5, 18, 60] and the expression of Nkx2-1 is largely dependent on Shh . The notable exception to this is at the level of the prospective amygdala, where a small focal expression domain of Shh remains in Nkx2-1 mutants . Interestingly, as previously shown, this remnant expression is coincident with the persistence of markers of the oligodendrocyte lineage . Here, we show that Shh-lineage cells generate inhibitory neurons as shown by the differential expression of Calbindin, nNOS, FoxP2 and SST. Our gene expression analysis in Nkx2-1 mutant embryos revealed that Nkx2-1 has a significant functional role in the development of the MePD, where we observed major decreases in GAD67, Lhx6 and Sst mRNA gene expression. This finding is consistent with previous studies that have described a complete loss of Lhx6 mRNA expression in the Nkx2-1 mutant telencephalon [18, 40]. Surprisingly, we observed a sparing of a subset of these GAD67 + , Lhx6 + and Sst + neurons in the MeA. As Nkx2-1 has more recently been shown to directly regulate Lhx6 transcription , our results suggest that in at least a subpopulation of amygdala neurons, this might not be the case. Indeed, it is likely that in this spared population, the remnant Shh expression domain in the embryonic caudal telencephalon in Nkx2-1-mutant mice is sufficient to directly specify these inhibitory neurons directly.
Fate-mapping of embryonic Gli1 + cells reveal the precocious generation of inhibitory neural diversity in both subdivisions of the posterior MeA compared to other forebrain structures
Shh signaling is mediated through the Gli family of transcription factors, which are homologous to the Drosophila zinc finger transcription factor cubitus interruptus, which mediates all Hedgehog signaling in the fly . Of the three mammalian Gli family members, Gli1 and Gli2 primarily act as activators whereas Gli3 functions as a repressor [63–65]. Analysis of loss of function mutant mice has shown that Shh signaling is required for the initial transcriptional activation of endogenous Gli1, but not Gli2, in the forebrain . Here, by administering a single tamoxifen dose at E9.5, we were able to label Gli1 + Shh-responder cells born from approximately E10.5 in the Gli1 CreER(T2) line, a readout of Shh activity . From this analysis, we show that cells that respond to Shh signaling also contribute to inhibitory neural diversity in the posterior MeA. Co-expression was observed with all inhibitory markers used in this study: Calbindin, nNOS, FoxP2 and SST. Furthermore, numerous LacZ+ recombined cells in the MePD and MePV in Gli1 CreER(T2) ; Tau mGFP (TM E9.5) brains lend credence to prior observations that indicate that neurons of the MeA are born earlier in development than those destined for other amygdala nuclei, such as those of the basolateral complex [3, 4, 7, 8].
In this study, we have shown that Nkx2-1- and Shh-lineage cells preferentially fate-map to the dorsal and ventral subdivisions of the posterior MeA, with differential contributions to both local and projection inhibitory neurons. Previous tracing studies have identified that MePD efferent neurons project to three interconnected nuclei that are involved in reproductive behaviors: the medial preoptic nucleus, the ventrolateral part of the ventromedial hypothalamic nucleus and the ventral premamillary nucleus. In contrast, projections from the MePV involved in defensive behaviors terminate in the anterior hypothalamic nucleus and the dorsomedial portion of the ventromedial hypothalamic nucleus [12, 66]. In a behavioral context, the reproductive and defensive actions are closely interrelated, and upon the appearance of threatening behaviors, a 'gate-control' mechanism ensures the rapid shut-down of reproductive behaviors to aid survival. The use of different Lhx transcription factors that delineate these projections to their hypothalamic targets may potentially serve as a neural substrate to integrate conflicting reproductive and defensive behavioral cues . Here, we show the inhibitory neural diversity of these nuclei is generated from Shh-expressing and Shh-responsive cells, implicating the Shh-pathway component of MeA development. Therefore, the data from the current study provide novel insights into the gene network complexity and genetic mechanisms involved in the development of the MeA.
Materials and methods
Mouse lines used in this study were: Swiss-Webster (SW; Taconic Farms, Albany, NY, USA), Nkx2-1 mutant , Shh Cre , ROSA-YFP , Tau mGFP , Gli1 CreER(T2)  and Nkx2-1-Cre . Mice were maintained according to protocols approved by the Animal Welfare and use committee at Children's National Medical Center, Washington DC, National Institutes of Health, Bethesda, MD, and NYU School of Medicine, NY. Nkx2-1 mutant mice were maintained on a SW background. The Nkx2-1-Cre and Shh Cre lines were maintained on a C57Bl/6 background, the Gli1 CreER(T2) mice were kept on a SW background and the reporter mice on C57Bl/6 × SW mixed backgrounds.
Animal crosses and genotyping
For staging of the embryos, midday of the day of vaginal plug detection was considered as E0.5. The day of birth was considered P0. To avoid potential sexual dimorphic differences in the MeA, only males were used for postnatal analyses. Mice were transcardially perfused with 4% paraformaldehyde (PFA) and brains were post-fixed in 4% PFA for at least 6 hours to overnight. Pregnant females from Gli1 CreERT2 ; Tau mGFP crosses were administered a single dose (100 mg/kg in corn oil) of tamoxifen (Sigma, St Louis, MO, USA) by oral gavage at E9.5, and embryos collected at E12.5 or pups were perfused at P22. For early embryonic analysis (E10.5 to E13.5) pregnant dams were euthanized by CO2 inhalation and the embryos were harvested by Caesarian section and whole heads were fixed for 2 hours (for X-gal staining) or overnight in 4% PFA at 4°C. Pregnant dams of E18.5 litters were anaesthetized with Nembutal and the embryos were transcardially perfused. The isolated brains were post-fixed overnight at 4°C. Genomic DNA for genotyping was isolated by phenol:chloroform extraction. Mice for fate-mapping and electrophysiological analyses were identified by PCR for Cre  and yellow fluorescent protein (YFP)  for transgenic and reporter lines, respectively. Nkx2-1 mutant embryos were identified by PCR using a GC rich kit (Roche, Indianapolis, IN, USA) and previously described primers [18, 23], and morphologically by the absence of lungs .
Tissue preparation and histology
After rinsing in PBS, postnatal brains were embedded in 4% agarose and sectioned coronally at a thickness of 50 μm using a Vibroslicer (Leica VT1000S, Leica, Nussloch, Germany). Embryonic brains were cryoprotected by graded sucrose immersion (10%, 20% then 30% overnight) and embedded in Tissue-Tek OCT Compound (Sakura Finetek USA Inc., Torrance, CA, USA). Coronal sections at a thickness of 20 (E10.5 to E13.5) or 30 μm (E18.5) were collected on glass slides, air-dried and stored at -20°C.
Non-radioactive dioxygenin-labeled RNA in situhybridization
mRNA in situ hybridization was carried out as described previously  with the exception that the reaction product was visualized by NBT/BCIP (Roche) diluted in AP buffer (10 μl/100 mM NaCl, 100 mM Tris pH 9.5, 50 mM MgCl2). The following cDNA probes were used in this study: Cck, Foxg1, GAD67, Gli1, Lhx6, Lhx9, Nkx2-1, Shh and Sst.
Free-floating adult vibratome sections were rinsed in PBS prior to blocking in 10% normal serum (NS) in PBS with Triton 0.03% (PBST; Sigma) for 1 hour at room temperature (RT), then overnight incubation in the primary antibodies diluted in PBST with 1% NS at RT for free-floating or 4°C for mounted tissue. The sections underwent three 10-minute rinses in PBS and were incubated in the appropriate donkey anti-Cy3, Cy5 (1:200; Jackson Immunoresearch, West Grove, PA, USA) or donkey anti-Alexa 488 (Invitrogen, Carlsbad, CA, USA) secondary antibodies, diluted in PBST with 1% normal donkey serum (NDS) for 2 hours at RT. The sections were rinsed twice then incubated in DAPI (1:1,000 in PBS; Sigma) for 10 minutes. The sections were mounted on Superfrost slides and coverslipped using Gel Mount aqueous mounting media (Sigma). The primary antibodies used in distinct combinations were: goat anti-β-gal (1:500; Biogen, Cambridge, MA, USA), goat anti-FoxP2 (1:500; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), goat anti-GFP (1:1,000; Novus Biologicals, Littleton, CO, USA), rabbit anti-β-gal (1:1,000; ICN Pharmaceuticals Inc, Costa Mesa, CA, USA), rabbit anti-Calbindin (1:1,000; Calbiochem, La Jolla, CA, USA), rabbit anti-nNOS (1:1,000; Sigma), rat anti-GFP (1:1,000; Nacalai USA, San Diego, CA, USA) and rat anti-SST (1:250; Millipore, Billerica, MA, USA).
Free-floating adult vibratome sections and E18.5 cryostat sections were rinsed in PBS and incubated in PBS:methanol:30% H2O2 (Sigma) in an 8:1:1 ratio for 20 minutes at RT to quench endogenous peroxidases. The sections underwent further rinses in PBS prior to blocking of non-specific binding sites using 10% NS in PBST. The primary antibodies were incubated overnight at RT. Further rinses in PBS preceded and followed incubations for 1 hour at RT in biotinylated antibodies (1:500; Vector Labs, Burlingame, CA, USA) then extravidin-peroxidase (1:2,000; Sigma), all diluted in 1% NS in PBST. The reaction product was visualized using a DAB kit (Vector Labs) according to the manufacturers' instructions. The reaction was stopped in Tris-buffered saline pH7.5 and the sections were mounted and/or coverslipped using permanent mounting media (Sigma). The primary antibodies used were as follows: goat anti-FoxP2 (1:1,000), goat anti-GFP (1:1,000), mouse anti-Parvalbumin (1:1,000; Sigma), rabbit anti-Calbindin (1:2,000), rabbit anti-nNOS (1:3,000), rabbit anti-Tbr1 (1:1,000; kind gift of R Hevner), rabbit anti-Neuropeptide Y (1:1,000; Immunostar, Hudson, WI, USA), rabbit anti-GAD65/67 (1:1,000; Millipore), and rat anti-SST (1:250).
Electrophysiological analysis of Shh-lineage cells in the posterior MeA was performed on male mice at P17 to P21 from Shh Cre ; ROSA26-YFP crosses. Mice were anesthetized with isoflurane, decapitated, and their brains were removed. Brain hemispheres were rapidly dissected on ice and cut into 300-μm thick coronal sections on a Leica VT100S vibratome in an ice-cold oxygenated solution consisting of (in mM) 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 7 MgCl2, 0.5 CaCl2, 25 NaHCO3, 25 glucose, 75 sucrose (347 mOsmol) at pH 7.4. Slices were stored in the same solution at 35°C for 30 minutes, then transferred into artificial cerebrospinal fluid of the following composition (in mM): 124 NaCl, 3 KCl, 2.5 CaCl2, 1.3 MgSO4, 26 NaHCO3, 1.25 NaHPO4, 15 glucose; saturated with 95% O2/5% CO2 at room temperature (20 to 25°C).
Slices were transferred to the recording chamber and perfused with artificial cerebrospinal fluid at a rate of 1 to 2 ml/s. Cells were visualized using an upright Olympus BX51W microscope equipped with infrared and fluorescent illumination, Normasky optics, and infrared camera (all from Olympus). Cells were only chosen based on their YFP fluorescence and apparent viability (clear membrane, nucleus not visible) without bias in terms of location within the MePD or MePV, cell size or morphology. Patch electrodes had resistances between 3 and 6 MΩ when filled with the intracellular solution of the following composition (in mM): 130 K-gluconate, 10 NaCl, 2 Mg-ATP, 0.3 Na-GTP, 10 HEPES, 0.6 EGTA, biocytin 5 mg/ml, solution adjusted to pH 7.2, 275 mOsm (junction potential = 13 mV). Whole-cell recordings were obtained using a Multiclamp 700B (Molecular Devices, Sunnyvale, CA, USA) and recordings were monitored via a PC running pClamp 9.2 (Molecular Devices). After recording membrane potential, capacitance and resistance values, cells were categorized on the basis of their response to depolarizing and hyperpolarizing current pulses. Off-line analysis was performed using Clampfit 9.2 (Molecular Devices) and Mini Analysis (Synaptosoft Inc., Fort Lee, NJ, USA). In all experiments, data were filtered at 10 kHz during capacitance compensation and 5 kHz during subsequent data recording. The traces were digitized at 10 kHz. All voltage measurements and steps were corrected for a junction potential offset.
Analysis of in situ hybridization experiments was performed using bright-field microscopy (Olympus BX51, Olympus, Center Valley, PA, USA) and high-resolution digital images were captured under a 4× objective using an Olympus D570 camera. For fluorescence, digital photographs were obtained from epifluorescence microscopy (Olympus BX61) and selected for further analysis using a Zeiss LSM 510 META confocal microscope (Thornwood, NY, USA). For confocal analysis, each fluorophore was scanned sequentially and confocal images are presented as individual optical sections. Figures were prepared using Adobe Photoshop CS and Adobe Illustrator CS software (Adobe Systems, San Jose, CA, USA). Adjustments to contrast were applied across each image as a whole and equally to control and mutant brains. For anatomical considerations we included the embryonic expression domain terminology of Flames and colleagues . For the adult anatomy we used the atlas of Franklin and Paxinos . General anatomical consultations were made from prior publications on the posterior MeA [2, 4, 33, 41]. All n values for immunohistochemistry, in situ hybridization and electrophysiology are detailed in each corresponding figure legend.
To assess the co-expression of immunohistochemical markers in recombined cells from fate-mapping analysis in adults from the three Cre lines, we performed cell counts in the MePD and MePV from two rostro-caudal levels of the posterior MeA, corresponding to Bregma levels -1.46 mm and -1.82 mm. Cells were counted in a 400 μm × 400 μm boxed area through 10 adjacent sections (1-μm steps) of each Z-stack using the LSM510 software. For the fate-mapping analysis we used the Tau mGFP reporter mouse, which was designed with a nuclear localization signal for LacZ . Therefore, we used an anti-β-gal antibody for nuclear staining of recombined cells, which, along with DAPI counterstaining, facilitated quantification of cells expressing other makers that were also primarily nuclear. The numerical data from the fate-mapping analysis are presented as the average percentage and standard deviation of recombined β-gal+ cells that co-expressed the second marker. Statistical significance was determined using an unpaired t-test (alpha value was set at 0.05) and was calculated using online GraphPad software (GraphPad Software Inc., La Jolla, CA, USA).
lateral ganglionic eminence
medial posterodorsal nucleus
medial posteroventral nucleus
medial ganglionic eminence
neuronal nitric oxide synthase
PBS with Triton 0.03%
progenitor domain of the preoptic area
- Shh :
yellow fluorescent protein.
The authors would like to thank Dr Robert Hevner for the Tbr1 antibody. The authors thank Ms Berenice Alfonso for technical assistance at the start of this study. The authors also acknowledge members of the Corbin and Haydar labs in the Center for Neuroscience Research for their helpful discussions.
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