ARTICLE https://doi.org/10.1038/s42003-020-01616-7 OPEN Gene regulatory networks controlling differentiation, survival, and diversification of hypothalamic Lhx6-expressing GABAergic neurons Dong Won Kim1, Kai Liu1,10, Zoe Qianyi Wang1, Yi Stephanie Zhang1, Abhijith Bathini1, Matthew P. Brown 1, 1234567890():,; Sonia Hao Lin1, Parris Whitney Washington1, Changyu Sun1, Susan Lindtner2, Bora Lee 3, Hong Wang1, ✉ Tomomi Shimogori 4, John L. R. Rubenstein2 & Seth Blackshaw1,5,6,7,8,9 GABAergic neurons of the hypothalamus regulate many innate behaviors, but little is known about the mechanisms that control their development. We previously identified hypothalamic neurons that express the LIM homeodomain transcription factor Lhx6, a master regulator of cortical interneuron development, as sleep-promoting. In contrast to telencephalic inter- neurons, hypothalamic Lhx6 neurons do not undergo long-distance tangential migration and do not express cortical interneuronal markers such as Pvalb. Here, we show that Lhx6 is necessary for the survival of hypothalamic neurons. Dlx1/2, Nkx2-2, and Nkx2-1 are each required for specification of spatially distinct subsets of hypothalamic Lhx6 neurons, and that Nkx2-2+/Lhx6+ neurons of the zona incerta are responsive to sleep pressure. We further identify multiple neuropeptides that are enriched in spatially segregated subsets of hypo- thalamic Lhx6 neurons, and that are distinct from those seen in cortical neurons. These findings identify common and divergent molecular mechanisms by which Lhx6 controls the development of GABAergic neurons in the hypothalamus. 1 Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 2 Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, UCSF Weill Institute for Neurosciences, University of California, San Francisco, CA 94158, USA. 3 Center for Neuroscience, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea. 4 RIKEN Center for Brain Science, Laboratory for Molecular Mechanisms of Brain Development, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. 5 Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 6 Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 7 Center for Human Systems Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 8 Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 9 Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, MD ✉ 21205, USA. 10Present address: Genentech, South San Francisco, CA 94080, USA. email: [email protected] COMMUNICATIONS BIOLOGY | (2021) 4:95 | https://doi.org/10.1038/s42003-020-01616-7 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-020-01616-7 lthough much is now known about both the diversity continuous yet distinct domains of the developing hypothala- and development of GABAergic neurons of the mus: the intrahypothalamic diagonal (ID) and the more pos- A 1,2 4,5 telencephalon , far less is known about their counter- terior tuberomammillary terminal (TT) .Wenextsoughtto parts in the hypothalamus, where over 20% of neurons are more carefully determine the expression pattern of Lhx6 and its GABAergic3. Previous work shows that hypothalamic GABAergic putative regulators during early hypothalamic development. neuronal precursors first appear in a domain that separates the High-quality chromogenic in situ hybridization (ISH) detects anterodorsal and posteroventral halves of the developing hypo- both the ID and TT domain of Lhx6 expression at E11.5, E12.5, thalamus, and is delineated by expression of transcription factors and E14.5 (Fig. 1A–E). By E16.5, hypothalamic Lhx6-expressing that regulate the development of telencephalic GABAergic neurons are observed in the ZI and dorsomedial hypothalamus neurons, including Dlx1/2 and Arx4–8. Within this structure, (DMH), in a pattern that broadly corresponds to the earlier ID which has been termed the intrahypothalamic diagonal/tuber- domain, while expression in the posterior hypothalamus (PH) omammillary terminal (ID/TT), nested expression domains of in turn broadly corresponds to the TT domain (Fig. 1F, G). This LIM homeodomain family genes are observed, in which expres- closely matches the pattern of hypothalamic Lhx6 expression sion of Lhx1, Lhx8, and Lhx6 delineates the anterior–posterior previously reported in adults12. Lhx6-expressing neurons are axis of the ID/TT4. Lhx1 is essential for the terminal differ- only a small minority of hypothalamic GABAergic neurons12, entiation and function of neurons in the master circadian oscil- with single-cell RNA-sequencing (scRNA-Seq) revealing that lator in the suprachiasmatic nucleus9–11. Lhx6-expressing only ~2% of all hypothalamic GABAergic neuronal precursors neurons in the zona incerta (ZI) of the hypothalamus are sleep- (defined by Gad1/2 and Dlx1/2 expression) express Lhx6 promoting and activated by elevated sleep pressure, and betweenE11andE13(Fig.1H)24. hypothalamic-specific loss of function of Lhx6 disrupts sleep This regional pattern of hypothalamic Lhx6 expression is broadly homeostasis12. similar to that reported for Lhx6Cre/+;Ai9 mice (Fig. 1J–D′)12,with Lhx6 has been extensively studied in the developing tele- ~65–70% of tdTomato-expressing neurons in the ZI, DMH, and ncephalon. It is essential for the specification, migration, and PH of Lhx6Cre/+;Ai9 postnatal mice also continuing to express Lhx6 maturation of GABAergic neurons of the telencephalon—particu- (Fig. 1J–U, D′). Notably, we see a few tdTomato-expressing neurons larly the cortex and hippocampus13,14. Lhx6 is expressed in the in other hypothalamic regions, with the largest numbers found in medial ganglionic eminence (MGE) of the embryonic tele- adjacent structures such as the ventromedial hypothalamus (VMH) ncephalon, where it is co-expressed with both Nkx2-1 and Dlx1/ and lateral hypothalamus (LH), although only ~5% of these 215–17. Shh induces expression of Nkx2-118, which in turn directly tdTomato-expressing neurons still express Lhx6 (Fig. 1V–D′). This activates Lhx6 expression16,19. Nkx2-1, in turn, cooperates with shows that, in contrast to telencephalic interneuron precursors, Lhx6 to directly activate the expression of multiple other genes that hypothalamic Lhx6 cells do not appear to undergo long-distance control cortical interneuron specification and differentiation, tangential migration, and that hypothalamic Lhx6-expressing cells including Sox6 and Gsx220,21.Furthermore,Lhx6 is both necessary that do undergo short-range tangential dispersal during early and sufficient for the tangential migration of the great majority of development generally repress Lhx6 expression as they mature. interneuron precursors from the MGE to their final destinations in the cortex and hippocampus17,22,23. Finally, Lhx6 expression per- sists in mature interneurons that express parvalbumin (Pvalb) and Lhx6 is necessary for the survival of hypothalamic neurons. somatostatin (Sst), and is necessary for their expression22. These findings led us to investigate other potential differences The functional role of Lhx6 in hypothalamic development has in the Lhx6 function in hypothalamic neurons relative to the not been previously investigated. However, previous studies imply telencephalon. While Lhx6 does not regulate the survival of this may differ in certain key ways from its function in the cortical interneuron precursors22, hypothalamic-specific loss of developing telencephalon. Notably, the hypothalamic domain of function of Lhx6 leads to substantial changes in sleep patterns12, Lhx6 expression only partially overlaps with that of Nkx2-14. raising the possibility that Lhx6 may be necessary for the viability Furthermore, in sharp contrast to cortical interneurons, Lhx6 is or proper functions of these neurons. not co-expressed with either Pvalb or Sst in the ZI12. In this To investigate this possibility, we tested P8 Lhx6CreER/CreER study, we sought to determine the extent to which gene regulatory mice, in which a CreER cassette has been inserted in frame with networks controlling the development of hypothalamic Lhx6 the start codon to generate a null mutant of Lhx6, to determine if neurons diverge from those that control the development of tel- read-through transcription of endogenous Lhx6 could be detected encephalic Lhx6 neurons. We find that hypothalamic Lhx6 reg- in the hypothalamus (Fig. 2A). Chromogenic ISH of telencephalic ulates neuronal differentiation and survival. Further, we observe structures such as the amygdala and cortex reveals that Lhx6- extensive molecular heterogeneity among mature hypothalamic expressing cells are still detected in both regions, although the Lhx6 neurons and a lack of overlap with annotated subtypes of number of Lhx6-expressing cells in the cortex is substantially Lhx6-expressing cortical interneurons. Combinatorial patterns of reduced in Lhx6CreER/CreER mice relative to Lhx6CreER/+ hetero- transcription factor expression delineate spatial subdomains of zygous controls (Fig. 2B–M). This is consistent with the severe Lhx6 expression within the ID/TT, and we find that Nkx2-1, reduction in tangential migration of cortical interneurons seen in Nkx2-2, and Dlx1/2 each regulate expression of Lhx6 in largely Lhx6-deficient mice22,23. In the hypothalamus, however, no read- nonoverlapping domains. Finally, Lhx6