Molecular and behavioral profiling of Dbx1-derived neurons in the arcuate, lateral and ventromedial hypothalamic nuclei.

BACKGROUND: Neurons in the hypothalamus function to regulate the state of the animal during both learned and innate behaviors, and alterations in hypothalamic development may contribute to pathological conditions such as anxiety, depression or obesity. Despite many studies of hypothalamic development and function, the link between embryonic development and innate behaviors remains unexplored. Here, focusing on the embryonically expressed homeodomain-containing gene Developing Brain Homeobox 1 (Dbx1), we explored the relationship between embryonic lineage, post-natal neuronal identity and lineage-specific responses to innate cues. We found that Dbx1 is widely expressed across multiple developing hypothalamic subdomains. Using standard and inducible fate-mapping to trace the Dbx1-derived neurons, we identified their contribution to specific neuronal subtypes across hypothalamic nuclei and further mapped their activation patterns in response to a series of well-defined innate behaviors. RESULTS: Dbx1-derived neurons occupy multiple postnatal hypothalamic nuclei including the lateral hypothalamus (LH), arcuate nucleus (Arc) and the ventral medial hypothalamus (VMH). Within these nuclei, Dbx1 (+) progenitors generate a large proportion of the Pmch-, Nesfatin-, Cart-, Hcrt-, Agrp- and ERα-expressing neuronal populations, and to a lesser extent the Pomc-, TH- and Aromatase-expressing populations. Inducible fate-mapping reveals distinct temporal windows for development of the Dbx1-derived LH and Arc populations, with Agrp(+) and Cart(+) populations in the Arc arising early (E7.5-E9.5), while Pmch(+) and Hcrt(+) populations in the LH derived from progenitors expressing Dbx1 later (E9.5-E11.5). Moreover, as revealed by c-Fos labeling, Dbx1-derived cells in male and female LH, Arc and VMH are responsive during mating and aggression. In contrast, Dbx1-lineage cells in the Arc and LH have a broader behavioral tuning, which includes responding to fasting and predator odor cues. CONCLUSION: We define a novel fate map of the hypothalamus with respect to Dbx1 expression in hypothalamic progenitor zones. We demonstrate that in a temporally regulated manner, Dbx1-derived neurons contribute to molecularly distinct neuronal populations in the LH, Arc and VMH that have been implicated in a variety of hypothalamic-driven behaviors. Consistent with this, Dbx1-derived neurons in the LH, Arc and VMH are activated during stress and other innate behavioral responses, implicating their involvement in these diverse behaviors

Embryonic Transcription Factor Expression Predicts Neuronal Identity and Innate Behavioral Activation Patterns in the Limbic System

Instinctive behaviors such as mating and aggression are key for the survival and propagation of species. As innate behaviors manifest without prior training, there must be embryonic genetic mechanisms that specify these innate behavioral circuits. Focusing on the MeA and hypothalamus, both major integration centers of olfactory inputs, first, we sought to elucidate the link between embryonic transcription factor expression, neuronal identity and innate behavioral activation patterns in the MeA, and second, the link between embryonic transcription factor expression and instinctive behavioral activation patterns in hypothalamic subnuclei. Using mice as a model organism, we observed that the MeA progenitor niche in the preoptic area (POA) is comprised of distinct progenitor populations differentially marked by the transcription factors Dbx1 and Foxp2. Both embryonically and postnatally, Dbx1-derived and Foxp2+ subpopulations remain spatially segregated. We also observed that Dbx1-derived and Foxp2+ neurons differentially express sets of sex-steroid pathway proteins. Furthermore, both subpopulations differed in their intrinsic and extrinsic electrophysiological properties. Additionally, behavioral activation patterns were investigated in both subpopulations by determining the co-expression of the immediate early gene c-fos, an indirect marker of neuronal activity. During aggressive encounters, both Dbx1-derived and Foxp2+ neurons were activated in male and female mice; however, during mating cues, Dbx1-derived neurons in male and female mice were activated while only Foxp2+ neurons in male mice were activated and not in female mice. This denotes sex-specific differences in behavioral activation patterns in the MeA. Thus, parcellation of MeA neuronal subpopulations based on developmental genetics predicts molecular, electrophysiological, and behavioral specificity. Secondly, we were interested in determining whether embryonic transcription factor expression would be predictive of innate behavioral activation patterns in other limbic system structures implicated in the generation of innate behaviors such as the hypothalamus. Interestingly, we observed the presence of Dbx1-derived neurons in the lateral (LH), arcuate (Arc) and ventromedial (VMH) hypothalamic subnuclei. As Foxp2+ neurons are not present in the hypothalamus, we only analyzed Dbx1-derived neurons in these three hypothalamic regions. We show that Dbx1-derived neurons are activated in these structures during mating and aggression in both male and female mice. Thus, embryonic transcription factor expression in the hypothalamus is also linked to postnatal behavioral activation patterns. Taken together our findings indicate that embryonic transcription factor expression is predictive of behavioral activation patterns in the limbic system. We found that progenitor populations present in the same region but expressing distinct transcription factors, can generate MeA postnatal diversity based on molecular, electrophysiological and behavioral activation patterns. Furthermore, this can be generalized to other limbic system structures such as the hypothalamus, in which embryonic transcription factor expression of Dbx1 is also predictive of activation patterns during instinctive behavioral cues

A Genetically Encoded Fluorescent Sensor for Rapid and Specific In Vivo Detection of Norepinephrine

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Amygdala Neural Development Mechanisms Are Linked to Innate Social and Non-social Behaviors During Adulthood

Innate behaviors for fight, flight and reproduction are essential for species survival and propagation. As these innate behaviors manifest without prior training, there must be embryonic developmental mechanisms that specify these circuits. The medial amygdala (MeA), as a major target for olfactory inputs, has been implicated in the regulation of these innate behaviors. I aim to elucidate how embryonic transcription factors generate distinct neuronal subpopulations that regulate diverse social and non-social innate behaviors. During amygdala development there are at least two neuronal progenitor pools that contribute to MeA neuronal diversity. We focused on two embryonic progenitor populations that are marked by the expression of the transcription factors: Dbx1 and the autism susceptibility gene Foxp2. We found that neuronal progenitors expressing the transcription factors Dbx1 or Foxp2 will become two distinct non-overlapping adult MeA neuronal subpopulations. These two populations express different molecular markers and possess distinct intrinsic electrophysiological properties. Furthermore, Dbx1-derived and Foxp2+ MeA neurons were activated during distinct innate behaviors involved in reproduction, aggression and predator avoidance. Therefore, activation of distinct neuronal subpopulations during key social and non-social behaviors are linked to transcription factor expression during development. Thus, future research should focus on how alterations in embryonic transcription factor expression can lead to social disorders characterized by amygdala based behavioral deficits, such as autism

Additional file 3: Figure S3. of Molecular and behavioral profiling of Dbx1-derived neurons in the arcuate, lateral and ventromedial hypothalamic nuclei

Increased c-Fos in Arc, LH, and VMH after innate behaviors. (A-D.i) Images of the c-Fos expression in the Arc (A-D), LH (E-H), and VMH (I-L) after mice were exposed to mating (A, E, and I), aggression (B, F, and J), fasting (C, G, and K), or predator (D, H, and L) behavioral paradigms. The number of cells expressing c-Fos in the Arc, LH, and VMH are increased after exposure to the four behavioral paradigms. The scale bar represents 500 μm in the LH and VMH (A-D.i, I-L.i) and 250 μm in the Arc (E-H.i). (TIF 172032 kb

Additional file 2: Figure S2. of Molecular and behavioral profiling of Dbx1-derived neurons in the arcuate, lateral and ventromedial hypothalamic nuclei

Dbx1-derived cells in nuclei as defined by specific markers. (A-D) Schematic of medial (top) to lateral (bottom) sagittal views of the embryonic forebrain. (A.i-D.i) As shown by YFP expression in E13.5 Dbx1 Cre ;Rosa26YFP embryos, Dbx1-derived cells are are found in primordial hypothalamic nuclei including in the paraventricular, arcuate, ventral medial and lateral progenitor domains. (A.ii-D.iii) As shown by ISH of serial sections, specific markers of the paraventriclar (Sim1, A.ii; Fezf1, A.iii), arcuate (Pomc, B.ii; Bsx, B.iii), ventral medial (Fezf1, C.ii; Nr5a1, C.iii) and lateral hypothalamic (Pmch, D.ii; Lhx9, D.iii) nuclei overlap with expression of YFP. The scale bar represents 500 μm. (TIF 157696 kb