Research Articles: Development/Plasticity/Repair Interneuron origins in the embryonic porcine medial ganglionic eminence https://doi.org/10.1523/JNEUROSCI.2738-20.2021 Cite as: J. Neurosci 2021; 10.1523/JNEUROSCI.2738-20.2021 Received: 27 October 2020 Revised: 18 December 2020 Accepted: 8 January 2021 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 the authors 1 2 Interneuron origins in the embryonic porcine medial ganglionic eminence 3 4 Mariana L. Casalia1, Tina Li1, Harrison Ramsay1, Pablo J. Ross2, Mercedes F. Paredes3,4 5 & Scott C. Baraban3-5 6 7 1Department of Neurological Surgery, University of California, San Francisco, San 8 Francisco, CA 9 2Department of Animal Science, University of California, Davis, Davis, CA 10 3Department of Neurology, University of California, San Francisco, San Francisco, CA 11 4Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, 12 CA 13 5Helen Wills Institute for Neuroscience, University of California, Berkeley, Berkeley, CA 14 15 Abbreviated title: Porcine Medial Ganglionic Eminence 16 17 Correspondence should be addressed to S.C.B. (email: [email protected]) 1 18 19 Pallial (cortical and hippocampal) Interneurons contribute to the complexity of 20 neural circuits and maintenance of normal brain function. Rodent interneurons 21 originate in embryonic ganglionic eminences, but developmental origins in other 22 species are less understood. Here, we show that transcription factor expression 23 patterns in porcine embryonic subpallium are similar to rodents, delineating a 24 distinct medial ganglionic eminence (MGE) progenitor domain. On the basis of 25 Nkx2.1, Lhx6 and Dlx2 expression, in vitro differentiation into neurons expressing 26 GABA and robust migratory capacity in explant assays, we propose that cortical 27 and hippocampal interneurons originate from a porcine MGE region. Following 28 xenotransplantation into adult male and female rat hippocampus, we further 29 demonstrate that porcine MGE progenitors, like those from rodents, migrate and 30 differentiate into morphologically distinct interneurons expressing GABA. Our 31 findings reveal that basic rules for interneuron development are conserved across 32 species, and that porcine embryonic MGE progenitors could serve as a valuable 33 source for interneuron-based xenotransplantation therapies. 34 35 Significance Statement 36 Here we demonstrate that porcine MGE, like rodents, exhibit a distinct transcriptional and 37 interneuron-specific antibody profile, in vitro migratory capacity and are amenable to 38 xenotransplantation. This is the first comprehensive examination of embryonic 39 interneuron origins in the pig, and because a rich neurodevelopmental literature on 40 embryonic mouse MGE exists (with some additional characterizations in other species 2 41 like monkey and human) our work allows direct neurodevelopmental comparisons with 42 this literature. 43 44 Introduction 45 Excitatory glutamatergic neurons and inhibitory GABAergic interneurons represent the 46 two primary neuronal populations in mammalian brain. Cortical and hippocampal 47 inhibitory interneurons are a diverse subset of the total neuronal population, mediate 48 many critical brain functions, and arise from embryonic subpallium regions (Fishell, 2007; 49 Gelman et al., 2012; Kessaris et al., 2014). A wide spectrum of neurological pathologies 50 is associated with loss, or dysfunction, of interneurons including, but not limited to, 51 epilepsy, autism spectrum disorder, Alzheimer’s disease and schizophrenia (Marin, 2012; 52 Inan et al., 2013; Paterno et al., 2020). Studies over the past decade demonstrated that 53 transplantation of interneurons offer great promise for treatment of these disorders 54 (Baraban et al., 2009; Alvarez-Buylla et al., 2000; Bráz et al., 2012; Anderson and 55 Baraban, 2012; Hunt et al., 2013; Tong et al., 2014; Donegan et al., 2017; Juarez-Salinas 56 et al., 2019; Zhu et al., 2019). Interneuron transplantation studies are largely based on 57 harvesting progenitor cells from a medial ganglionic eminence (MGE) subregion. MGE 58 progenitors give rise to cortical GABAergic interneurons subtypes expressing 59 parvalbumin (PV)- and somatostatin (SOM) (Gelman et al., 2012; Hu et al., 2017; Pelkey 60 et al., 2017) along with hippocampal neurogliaform and Ivy cells expressing nitric oxide 61 synthase (nNOS) (Tricoire et al., 2010). These interneurons exhibit highly migratory 62 capabilities and unique functions. To date, the majority of successful MGE progenitor cell 63 transplantations showing efficacy in preclinical animal models harvest fresh murine 3 64 embryonic tissue. Human “MGE-like” embryonic or induced pluripotent stem cell derived 65 interneurons could offer an alternative, however, these neurons fail to (i) migrate 66 extensively or (ii) differentiate to cortical or hippocampal interneuron subtypes seen with 67 embryonic MGE progenitors, and (iii) exhibit protracted functional maturation (Nicholas et 68 al., 2013; Liu et al., 2013; Maroof et al., 2014). Human stem cells also present a risk of 69 tumorigenesis (Carpentino et al., 2008). Although embryonic allografted fetal human 70 tissue in Parkinson's disease patients ameliorated some symptoms of this disease in the 71 1990s (Björklund et al., 2003), practical and ethical problems using aborted human fetal 72 tissue have since led to an extended exploration for an alternative source(s) of suitable 73 fetal material for transplantation. 74 75 76 Most successfully applied to organs, xenotransplantation from pig-to-nonhuman primate 77 (or even pig-to-human) raise the exciting possibility that embryonic porcine tissue - 78 perhaps coupled with techniques to genetically modify a donor pig using transgenic or 79 CRISPR/Cas9 technologies (Cowan et al., 2019; Hryhorowicz et al., 2020) - could be a 80 viable source of transplantable interneurons. These efforts are supported by data showing 81 similarities at the gene and protein level between humans and pigs (Sjöstedt et al., 2020). 82 It is well established that murine MGE can be defined by transient anatomical landmarks 83 and expression of a unique set of transcription factors e.g., Nkx2.1, Dlx2 and Lhx6 (Corbin 84 et al., 2003; Du et al., 2008; Flames et al., 2007; Gelman et al., 2012). Analysis in human 85 and macaque (nonhuman Old World) monkey demonstrated a recapitulation of these 86 murine MGE transcription factor expression patterns (Hansen et al., 2013; Ma et al., 4 87 2013). Tangential migration of interneurons from MGE-to-cortex or -hippocampus is also 88 believed to be highly conserved across species. Although some features of porcine 89 ganglionic eminences are known, this information is limited to lateral ganglionic eminence 90 (LGE) (Jacoby et al., 1999), a subpallial region that mainly generates olfactory bulb 91 interneurons and lacks tangential migratory properties in vitro or following transplantation 92 into postnatal brain. 93 94 Here we analyzed temporal and regional expression of MGE-specific transcription factors 95 in fetal porcine brain at embryonic days 30 and 35 (E35). Cultured porcine progenitors 96 express MGE-specific transcription factors, differentiate to GABAergic SOM+ 97 interneurons and migrate extensively in vitro. Similar to human, embryonic pig ganglionic 98 eminence organization showed distinct organization of cells into doublecortin-positive 99 clusters, a pattern not seen in murine MGE. Xenotransplantation of E35 MGE progenitors 100 into adult rat hippocampus resulted in migration across all hippocampal sub-fields and 101 differentiation into GABAergic SOM+ interneurons up to 60 days after transplantation 102 (DAT). Overall, patterns of transcription factor expression, differentiation into distinct 103 interneuron subpopulations and tangential migration capacity all appear to be conserved 104 from rodents to pig MGE during evolution. 105 106 Materials and Methods 107 108 Animals. Donor porcine (Sus scrofa domesticus) embryos were procured from 109 Department of Animal Science, University of California, Davis (English Large white) or 5 110 National Swine Resource and Research Center (NSRRC:0016 GFP NT92; Whitworth et 111 al., 2009), University of Missouri. Adult male Sprague Dawley rats (Rattus norvegicus) 112 were purchased from Charles River Laboratories (strain code 400) and housed under a 113 standard 12 hr light/dark cycle with food and water provided ad libitum. A total of 133 rats 114 were used for xenotransplantation studies (79 female; 54 male); 75 rats received 115 intraperitoneal (i.p.) injection of an immunosuppression cocktail containing 116 methylprednisolone acetate (2 mg/kg), cyclosporine (20 mg/kg) and azathioprine (5 117 mg/kg) 3 times per week (Table 1). All protocols and procedures were approved by the 118 Institutional Animal Care and Use Committee (IACUC protocol #AN181524-02) and 119 adhered to University of California, San Francisco (UCSF; San Francisco, CA) Laboratory 120 Animal Resource Center and United States Public Health policy on Humane Care and 121 Use of Laboratory Animals guidelines. 122 123 Tissue collection and processing. Individual embryonic day 30 (E30) or E35 pig 124