The hypothalamus is a complex multi-nucleated structure in which individual nuclei function to direct a variety of behaviors essential for survival, adaptation and species propagation [2, 3]. Despite the extensive study of hypothalamic function and anatomy, only recently has there been a greater understanding of the mechanisms of hypothalamic development, predominantly via a combination of gene expression and lineage tracing studies. Our previous study focusing on the function of the embryonic expressed homeodomain encoding transcription factor, Dbx1 revealed Dbx1 to have a restricted function in the specification of hypothalamic neurons required for innate stress responses but not other innate behaviors [19]. Here, we sought to extend these findings in order to provide deeper insight into the Dbx1-lineage contribution to diverse neuronal hypothalamic populations and to determine if Dbx1-derived neurons are activated by specific innate behaviors. We found a large amount of Dbx1-derived neuronal diversity across hypothalamic nuclei and broad activation of the Dbx1-lineage by different innate behaviors. Interestingly, the broad fate and generally non-selective activation of Dbx1-derived neurons to a variety of innate behaviors was not predicted by our previous finding of the restricted function of Dbx1 in specification of neurons solely required for innate stress responses. However, taken together these data are consistent with a model in which there are distinct gene sets expressed during hypothalamic development that, while maybe widespread in their lineage contribution, perform select functional roles in specification of distinct hypothalamic subpopulations.
Previous fate-mapping studies of the hypothalamus have begun to provide a general understanding of the relationship between progenitor domains and mature nuclei [3, 16, 18, 28, 61]. These studies have included examination of the lineage of a variety of developmentally defined subpopulations such as those that express the transcription factors Nkx2.1, Dlx, Nr5a1 [62, 63] and secreted factors such as Shh [48]. Complementing this fate-mapping work are a series of detailed and highly informative hypothalamic developmental gene expression studies [27, 28, 64]. Collectively these studies have revealed that: 1) there are gene sets that give rise to neurons and function across hypothalamic nuclei (e.gs Rax, Nkx2.1, Asc1) and complementary gene sets that appear to be restricted in expression and function in specification of specific nuclei (e.gs Bsx, Nr5a1) [18] and 2) consistent with our findings, embryonic gene expression domains appear to be generally predictive of the location of mature nuclei, suggesting a general lack of widespread migration across domains. This is in contrast to the telencephalon where the ventral embryonic ganglionic eminence developmental domains (MGE and CGE) give rise to immature neurons that migrate to distant areas such as the cerebral cortex and hippocampus [65–67].
Within this framework, similar to progenitors expressing the developmentally regulated genes Shh and Nkx2.1, we found that Dbx1
+
progenitors generate a wide variety of neuronal subtypes across multiple hypothalamic nuclei. Dbx1-derived cells were also present, although to a lesser degree, in the VMH. In contrast, regions of the anterior hypothalamus were devoid of Dbx1-derived neurons. A gradient of Dbx1-derived cells was also observed radiating from the tuberal domain into the anterior domain, a pattern that is more pronounced in medial portions of the ventral diencephalon. This pattern of Dbx1-derived neuronal location was generally shared with the pattern of location of Shh-lineage neurons [48]. This finding perhaps reflects the overlapping embryonic expression domains of Shh and Dbx1 and may further indicate putative positive control of Dbx1 expression by Shh during forebrain development.
The hypothalamus has been thought to develop in an ‘outside-in’ manner, stemming from studies using traceable thymidine analogs indicating the lateral hypothalamic nuclei are typically born prior to medial nuclei [49, 68, 69]. More recent fate-mapping studies and analysis of molecularly defined cell types have provided another layer of complexity of development for select cell populations [48, 70]. Here, using inducible fate-mapping, we demonstrate that medial Dbx1-derived neurons in both LH and Arc are recombined earlier (before E9.5), with lateral populations recombined at later ages (after E9.5). Although our study is limited in that we did not conduct a birth-dating analysis of Dbx1-derived neurons, this observed pattern is consistent with previous Shh fate-mapping studies [48], and is supportive of a more complex pattern of medial-lateral development.
We further found Dbx1-derived neurons contribute to diverse molecularly defined populations in the LH, Arc and VMH. Within the LH, the majority of Pmch+ cells, which also express Nfn+, and Cart+, were Dbx1-derived. This finding is predicted by our loss-of-function studies in which the Pmch+ population was dramatically reduced [19]. In the Arc, our Dbx1 loss-of-function studies demonstrated a ~50 % reduction of Agrp and Cart expression, with no changes in the Pomc+ or TH+ populations [19]. Here we demonstrate that ~50 % of Agrp+ and Cart+ neurons were Dbx1-derived. Collectively these data are consistent with a cell autonomous function of Dbx1 in generation of LH Pmch+ and Arc Agrp+ populations. However, surprisingly, a significant proportion of Arc Pomc+ and TH+ cells were also Dbx1-derived. Thus, while Dbx1
+
progenitors generate diverse populations in the Arc, it appears that Dbx1-independent mechanisms are required for specification of the Pomc+ and TH+ neurons.
At the behavioral level, the number of c-Fos+ cells in response to innate behavioral cues was increased in a predictable manner consistent with previous work [19, 55–57]. Building upon these results, we assessed the Dbx1-lineage contribution to these patterns of activation. We previously demonstrated that at the behavioral level conditional Dbx1 hypothalamic loss-of-function resulted in a specific defect in innate stress responses, but not other innate behaviors such as mating or aggression [19]. Based on these findings, we anticipated that Dbx1-derived neurons would also be engaged (c-Fos+) selectively during innate stress behaviors (predator odor and fasting), but not other social behaviors (mating and aggression). In contrast, we found that across hypothalamic nuclei, Dbx1-derived neurons were active during multiple innate behavior tasks. Most broadly tuned to many behaviors was the LH, in which the percent of Dbx1-derived neurons expressing c-Fos increased after every behavioral paradigm tested. While this was not predicted by our previous loss-of-function studies, as we show here that a large portion the Pmch+ and Hcrt+ populations were Dbx1-derived, it is perhaps not surprising that the Dbx1-derived populations in the LH are responsive to a variety of innate cues.
These activation patterns, while still encompassing multiple behaviors, were more specific in the Arc and VMH. We observed an increase in the proportion of activated Dbx1-derived neurons after fasting, mating and male aggression in the Arc, and an increase after mating and male aggression in the VMH. In the Arc, while less than 50 % of the feeding neurons (Pomc, Agrp, and Cart) were Dbx1-derived, these neurons were c-Fos+ during fasting, likely reflecting their involvement in this major function of the Arc. In contrast, Arc Dbx1-derived neurons were less engaged in responses to predator odor. In the VMH, the Dbx1-derived neurons contributed to large portions of the ER?+ and Arom+ neuronal subpopulations, which are known to influence mating and aggressive behaviors [14, 43–45, 71]. This was reflected in the behavioral activation patterns, where the Dbx1-derived neurons were selectively activated during mating and aggression. While further experiments are needed to define the Dbx1-derived circuits that are required for specific hypothalamic-driven behaviors, these studies present novel insight into the link between developmental lineage and behavioral control.