Gray de Cristoforis et al. Molecular Brain (2020) 13:85 https://doi.org/10.1186/s13041-020-00623-3 RESEARCH Open Access Differentiation and localization of interneurons in the developing spinal cord depends on DOT1L expression Angelica Gray de Cristoforis1,2,3, Francesco Ferrari3,4, Frédéric Clotman5 and Tanja Vogel1,2,6* Abstract Genetic and epigenetic factors contribute to the development of the spinal cord. Failure in correct exertion of the developmental programs, including neurulation, neural tube closure and neurogenesis of the diverse spinal cord neuronal subtypes results in defects of variable severity. We here report on the histone methyltransferase Disruptor of Telomeric 1 Like (DOT1L), which mediates histone H3 lysine 79 (H3K79) methylation. Conditional inactivation of DOT1L using Wnt1-cre as driver (Dot1l-cKO) showed that DOT1L expression is essential for spinal cord neurogenesis and localization of diverse neuronal subtypes, similar to its function in the development of the cerebral cortex and cerebellum. Transcriptome analysis revealed that DOT1L deficiency favored differentiation over progenitor proliferation. Dot1l-cKO mainly decreased the numbers of dI1 interneurons expressing Lhx2. In contrast, Lhx9 expressing dI1 interneurons did not change in numbers but localized differently upon Dot1l-cKO. Similarly, loss of DOT1L affected localization but not generation of dI2, dI3, dI5, V0 and V1 interneurons. The resulting derailed interneuron patterns might be responsible for increased cell death, occurrence of which was restricted to the late developmental stage E18.5. Together our data indicate that DOT1L is essential for subtype-specific neurogenesis, migration and localization of dorsal and ventral interneurons in the developing spinal cord, in part by regulating transcriptional activation of Lhx2. Keywords: Spinal cord, Interneuron, Specification, Localization, Methyltransferase, DOT1L, Epigenetics Introduction types. Many factors take part in the precise processes that The central nervous system (CNS), composed of spinal initiate and maintain cell-specific transcriptional profiles, cord and brain, forms through invagination of the neural which are necessary for both neurulation and the subse- ectoderm and fusion of the neural folds to generate the quent specification of the diverse neuronal subpopulations neural tube, in a process called neurulation. Fusion of the during neural tube development. neural folds and closure of the neural tube is completed Opposite gradients of signaling pathways, for example between E8.75 and E10 in the mouse [1]. A delicate mo- those of Bone Morphogenetic Protein (BMP) and lecular network tightly orchestrates the development of Wingless-related integration site (WNT) from the roof- the spinal cord in its entire complexity of different cell plate, or Sonic Hedgehog (SHH) from the floorplate, tightly regulate the establishment of diverse progenitor * Correspondence: [email protected] classes along the dorsoventral axis [2]. According to differ- 1Department of Molecular Embryology, Institute of Anatomy and Cell ent marker gene expression patterns and response to Biology, Faculty of Medicine, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany signaling molecules, the developing spinal cord is subdi- 2Spemann Graduate School of Biology and Medicine (SGBM), vided into six dorsal progenitor classes (dp1–6 inter- Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany neuron progenitors) and five ventral progenitor classes Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gray de Cristoforis et al. Molecular Brain (2020) 13:85 Page 2 of 20 (p0–3 interneuron progenitors and pMN motor neurons cord development are still unresolved. Histone H3 lysine progenitors). Two waves of neurogenesis lead to the methylation occurs at different positions including K4, differentiation of the progenitor classes into mature inter- K9 or K27. Recently, modifications of K79 were linked neurons or motor neurons (MN) [3]. In the first wave, with NTD. Specifically, attenuation of H3K79 dimethyla- which takes place between E9.0-E12.5, dorsal interneuron tion (H3K79me2) correlated with NTD occurrence in classes (dI1–6) [4–9] and ventral interneurons (V0–3) brain samples of human embryos presenting with spina differentiate along with MN [10–15], each from the re- bifida [43]. Further, high levels of H3K79 homocysteiny- spective progenitor class. A smaller subset of dorsal inter- lation (Hcy), in place of methylation at this position, neurons, dILA and dILB, is generated in the second wave correlated with decreased expression of specific genes, between E11.5 and E13.5 [6, 9, 16, 17]. Maturing interneu- e.g. Smarca4, Cecr2 and Dnmt3b, which are known to rons migrate from the ventricular zone into the mantle cause NTD upon disbalanced transcription [44]. H3K79 zone to their final position to build and to integrate into mono-, di- and trimethylation (H3K79 me1, me2 or functional neuronal circuits. The dorsal populations dI1- me3) is mainly conferred by the Disruptor of Telomeric dI4 and dILA integrate into circuits involved in proprio- Silencing 1 Like (DOT1L) in mammals [45]. DOT1L ception or touch-related motor control [4, 18, 19], dI4/ function has been linked to a range of cellular processes dILA and dI5/dILB populations into networks transmitting including transcriptional activation, resume of transcrip- information for pain, thermal sensation, itch and touch tion after DNA damage, or cell cycle progression [45]. [20–23]. Mature dI6 interneurons control alternating DOT1L participates in different protein complexes, hindlimb movement and coordination [24, 25]. The ven- which might explain its diverse functionalities [46] and trally generated interneuron classes integrate into loco- it is involved in specific forms of leukemia [47, 48]. It is motor networks, largely projecting on MNs [3]: different also indispensable for development [49], as loss of subsets of V0 participate in locomotion [26] and coordin- DOT1L is lethal at very early embryonic stages in the ation of left-right alternation [27], V1 and V2 contribute mouse [50]. DOT1L is fundamental for development of to limb articulation [28], and V3 to modulation of the diverse organs, as shown for example for cardiomyocytes locomotion rhythm [29]. The MNs, arising from a single differentiation [51] and functionality [52], erythropoiesis progenitor class, differentiate into a wide range of func- [53], and chondrogenesis [54]. Previous studies from our tionally diversified mature classes. MNs located in the laboratory highlighted that DOT1L activity plays import- spinal cord and brainstem are cholinergic and target a ant functional roles for CNS development. In the cere- variety of muscles [30]. bral cortex and cerebellum, but also in other somatic Both impaired neurulation and neurogenesis during organ systems, DOT1L balances progenitor proliferation spinal cord development lead to diverse pathologies. and differentiation [55–58]. Defects in neural tube fusion result in NTDs, which are We here report on our study aiming to explore the among the most common birth defects in human preg- role of DOT1L in neural tube development. Using nancies. The range of severity spans from lethal (anen- Wnt1-cre mediated conditional deletion of DOT1L in cephaly, craniorachischisis) to neurological handicaps the developing mouse neural tube, we here show that (open spina bifida) to asymptomatic conditions (closed in this model system DOT1L is not affecting neural spina bifida) [31]. Further, defective neurogenesis and tube closure, but that it is involved in correct differenti- patterning can lead to neuropathies [2]duetomal- ation of specific subsets of interneurons. Specifically, formed circuitry regarding touch [20], itch [32]and DOT1L contributes to the molecular specification of pain [33]. LHX2-expressing dI1 interneurons and migration of Both genetic and epigenetic factors have been studied the LHX9-expressing dI1 subset together with dI2, dI3, to identify the etiology of NTDs, especially since folate V0 and V1. deficiency has been indicated as a cofactor of NTD oc- currence [34]. Recent studies suggested that histone Materials and methods modifications such as acetylation or methylation corre- Mice lated with the occurrence of NTD in mouse or chick Wnt1-cre animals (Wnt1cre2 [59]) were mated with embryos [35–39]. Further, chromatin modifications have floxed