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• W3036633926 abstract "•PRISM simplifies lineage studies by anchoring recombination specifically to progenitors•Midbrain but not hypothalamic dopamine neurons originate from Shh+ progenitors•Most globus pallidus and medial amygdala neurons are not derived from Shh+ progenitors•Upper layer cortical neurons are only partly derived from Cux2+ progenitors Lineage tracing aims to identify the progeny of a defined population of dividing progenitor cells, a daunting task in the developing central nervous system where thousands of cell types are generated. In mice, lineage analysis has been accomplished using Cre recombinase to indelibly label a defined progenitor population and its progeny. However, the interpretation of historical recombination events is hampered by the fact that driver genes are often expressed in both progenitors and postmitotic cells. Genetically inducible approaches provide temporal specificity but are afflicted by mosaicism and toxicity. Here, we present PRISM, a progenitor-restricted intersectional fate mapping approach in which Flp recombinase expression is both dependent on Cre and restricted to neural progenitors, thus circumventing the aforementioned confounds. This tool can be used in conjunction with existing Cre lines making it broadly applicable. We applied PRISM to resolve two developmentally important, but contentious, lineages—Shh and Cux2. Lineage tracing aims to identify the progeny of a defined population of dividing progenitor cells, a daunting task in the developing central nervous system where thousands of cell types are generated. In mice, lineage analysis has been accomplished using Cre recombinase to indelibly label a defined progenitor population and its progeny. However, the interpretation of historical recombination events is hampered by the fact that driver genes are often expressed in both progenitors and postmitotic cells. Genetically inducible approaches provide temporal specificity but are afflicted by mosaicism and toxicity. Here, we present PRISM, a progenitor-restricted intersectional fate mapping approach in which Flp recombinase expression is both dependent on Cre and restricted to neural progenitors, thus circumventing the aforementioned confounds. This tool can be used in conjunction with existing Cre lines making it broadly applicable. We applied PRISM to resolve two developmentally important, but contentious, lineages—Shh and Cux2. Anthropomorphically speaking, lineage refers to descent from a common ancestor. In developmental biology, lineage analysis consists of establishing the relationship between progenitors and their cellular progeny (Arendt et al., 2016Arendt D. Musser J.M. Baker C.V.H. Bergman A. Cepko C. Erwin D.H. Pavlicev M. Schlosser G. Widder S. Laubichler M.D. Wagner G.P. The origin and evolution of cell types.Nat. Rev. Genet. 2016; 17: 744-757Crossref PubMed Scopus (216) Google Scholar). In the central nervous system (CNS), these analyses have begun to illuminate how embryonic progenitors, through a series of molecularly guided decisions, generate the enormous diversity of neural cell types. Lineage analyses of neuronal populations are both necessary and challenging, partly because of the extraordinary displacement of neurons from their birth sites, due to complex morphogenetic movements and extensive migration during development. Most current methods of lineage tracing rely on indelibly labeling a progenitor, or a subset of progenitors, and tracking their progeny throughout development. This is now routinely achieved using site-specific recombinases like Cre or Flp (Dymecki and Tomasiewicz, 1998Dymecki S.M. Tomasiewicz H. Using Flp-recombinase to characterize expansion of Wnt1-expressing neural progenitors in the mouse.Dev. Biol. 1998; 201: 57-65Crossref PubMed Scopus (83) Google Scholar, Zinyk et al., 1998Zinyk D.L. Mercer E.H. Harris E. Anderson D.J. Joyner A.L. Fate mapping of the mouse midbrain-hindbrain constriction using a site-specific recombination system.Curr. Biol. 1998; 8: 665-668Abstract Full Text Full Text PDF PubMed Google Scholar), whose expression is driven by gene-specific promoters in discrete regions of the developing neural tube. In these progenitors, the recombinase activates a reporter in a permanent manner, thereby permitting visualization of those cells and their descendants through subsequent developmental stages. Thus, this recombinase-mediated reporter activation results in the indelible labeling of cells that have transiently expressed, or are continuously expressing, a given gene. The standard recombinase-based approach to lineage tracing is subject to one critical limitation—the method is wholly reliant on the specificity of the promoter and transcriptional regulatory elements that are used to drive recombinase expression. To improve specificity, more recently, the development and utilization of inducible and intersectional approaches (Awatramani et al., 2003Awatramani R. Soriano P. Rodriguez C. Mai J.J. Dymecki S.M. Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation.Nat. Genet. 2003; 35: 70-75Crossref PubMed Scopus (154) Google Scholar, Feil et al., 1997Feil R. Wagner J. Metzger D. Chambon P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.Biochem. Biophys. Res. Commun. 1997; 237: 752-757Crossref PubMed Scopus (682) Google Scholar, Hunter et al., 2005Hunter N.L. Awatramani R.B. Farley F.W. Dymecki S.M. Ligand-activated Flpe for temporally regulated gene modifications.Genesis. 2005; 41: 99-109Crossref PubMed Scopus (47) Google Scholar) have led to more refined fate maps in various regions of the embryonic CNS (Farago et al., 2006Farago A.F. Awatramani R.B. Dymecki S.M. Assembly of the brainstem cochlear nuclear complex is revealed by intersectional and subtractive genetic fate maps.Neuron. 2006; 50: 205-218Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, Jensen et al., 2008Jensen P. Farago A.F. Awatramani R.B. Scott M.M. Deneris E.S. Dymecki S.M. Redefining the serotonergic system by genetic lineage.Nat. Neurosci. 2008; 11: 417-419Crossref PubMed Scopus (179) Google Scholar, Miyoshi et al., 2010Miyoshi G. Hjerling-Leffler J. Karayannis T. Sousa V.H. Butt S.J.B. Battiste J. Johnson J.E. Machold R.P. Fishell G. Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons.J. Neurosci. 2010; 30: 1582-1594Crossref PubMed Scopus (375) Google Scholar). Despite these advancements, one important caveat remains; most genes that are expressed in progenitors are also expressed in lineally related or unrelated postmitotic neurons. The expression of the recombinase in postmitotic neurons obfuscates lineage analysis, as recombination occurs in cells regardless from which progenitor populations they originated. An overwhelming number of Cre drivers suffer from this limitation, although this is not always recognized. Indiscriminately combining progenitor and postmitotic-derived labeling confounds lineage analysis, which in turn, obscures our understanding of cell fate specification. To overcome these limitations, here, we developed an intersectional platform for lineage analysis, termed progenitor-restricted intersectional fate mapping (PRISM). In this approach, Flp recombinase expression is both dependent on Cre and restricted to neural progenitors, and thus circumvents the aforementioned limitation, in that recombinase activity is anchored specifically in the progenitor domain. PRISM excludes postmitotic Cre recombinase activity from the fate map, thus provides significant advantages over current lineage tracing methods as it allows the unequivocal ascertainment of cell lineage. Using this approach, we uncovered lineage relationships using the Shh-Cre driver, as well as rectified improper lineages attributed to progenitors expressing this gene. We also used this method to resolve a debated topic in the field of cortical development—whether upper layer neurons are entirely derived from Cux2+ progenitors—and reveal that only a minority of upper layer neurons are derived from Cux2+ progenitors. This approach is broadly applicable and can be used to accurately reveal bona fide progenitor-progeny relationships with any Cre or CreERT2 driver of interest. We aimed to develop a strategy that would exclusively label genetically defined neural progenitors and their progeny (Figure 1A). To achieve this, we utilized the specificity of intersectional genetic approaches that use two distinct recombinases to label a population of cells based on the expression of two genes. By utilizing Nestin, a gene encoding an intermediate filament that is a well-established marker of neural stem cells (Kawaguchi et al., 2001Kawaguchi A. Miyata T. Sawamoto K. Takashita N. Murayama A. Akamatsu W. Ogawa M. Okabe M. Tano Y. Goldman S.A. Okano H. Nestin-EGFP transgenic mice: visualization of the self-renewal and multipotency of CNS stem cells.Mol. Cell. Neurosci. 2001; 17: 259-273Crossref PubMed Scopus (260) Google Scholar, Lendahl et al., 1990Lendahl U. Zimmerman L.B. McKay R.D. CNS stem cells express a new class of intermediate filament protein.Cell. 1990; 60: 585-595Abstract Full Text PDF PubMed Scopus (2672) Google Scholar, Zimmerman et al., 1994Zimmerman L. Parr B. Lendahl U. Cunningham M. McKay R. Gavin B. Mann J. Vassileva G. McMahon A. Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors.Neuron. 1994; 12: 11-24Abstract Full Text PDF PubMed Scopus (477) Google Scholar), we aimed to restrict the activity of Flp recombinase to neural progenitors. We initially generated mice using the promoter and second intron sequence of the rat Nestin gene (Mignone et al., 2004Mignone J.L. Kukekov V. Chiang A.S. Steindler D. Enikolopov G. Neural stem and progenitor cells in nestin-GFP transgenic mice.J. Comp. Neurol. 2004; 469: 311-324Crossref PubMed Scopus (512) Google Scholar) to drive Flp recombinase, using traditional transgenesis or site-directed transgenesis approaches at the H11 locus (Tasic et al., 2011Tasic B. Hippenmeyer S. Wang C. Gamboa M. Zong H. Chen-Tsai Y. Luo L. Site-specific integrase-mediated transgenesis in mice via pronuclear injection.Proc. Natl. Acad. Sci. USA. 2011; 108: 7902-7907Crossref PubMed Scopus (136) Google Scholar). However, these strains were hampered by mosaicism and/or positional effects. Thus, we generated a knockin mouse by modifying the coding region of the Nestin locus to introduce destabilized GFP (d4GFP) followed by a stop cassette, both flanked by loxP sites, and located upstream of the codon-optimized Flp recombinase (Flpo; Figures 1B and S1 for detailed map). By this design, Flpo can only activate a reporter in a Nestin-expressing cell where the stop cassette has been removed by Cre (Figure 1C). Although the d4GFP cassette was originally included to visualize Nestin expression, the undetectable fluorescence prevented the usability of this feature. This d4GFP cassette, being flanked by rox sites, can easily be removed using a Dre deleter mouse (Figure S1). In summary, we generated a mouse where Flp recombinase is only active in Cre+ neural progenitors, which is designed to provide a bona fide fate map of genetically defined progenitors when used in conjunction with Flp reporter. To establish the validity of our method, we first sought to test three key parameters, namely, recombination efficiency, absence of leakiness, and lack of Flp-mediated recombination in postmitotic neurons. To test the Nes-LSL-Flpo strain, we crossed it with a ubiquitous Cre driver EIIa-Cre to remove the stop cassette in all cells, resulting in Flp recombinase being only dependent on Nestin expression. These mice were crossed with the Flp reporter RC-FSF-LacZ, which expresses nuclear β-galactosidase (nβGal) upon removal of the frt-flanked stop cassette (FSF) by Flp recombinase. In the EIIa-Cre+, Nes-LSL-Flpo+, RC-FSF-LacZ+ brains, we observed nβGal+ cells throughout the entire brain (Figure 2A). Critically, there was no apparent leakiness observed in the Nes-LSL-Flpo allele in the absence of Cre; no nβGal staining was observed in littermate controls lacking Cre (EIIa-Cre−, Nes-LSL-Flpo+, RC-FSF-LacZ+) or Flp (EIIa-Cre+, Nes-LSL-Flpo−, RC-FSF-LacZ+). Next, we aimed to establish the cellular repertoire and the maximum recombination potential of PRISM in the CNS. Within the brain, neurons, astrocytes, and oligodendrocytes are known to be derived from the Nestin lineage. We examined if our allele could recombine all of these classes of cells. In the cortex (Figure 2B) as well as other regions of the brain (Figure S2), we observed nβGal+ cells that co-expressed the neuronal marker NeuN, the astrocyte marker GFAP, and the oligodendrocyte marker Olig2. Neuronal cell types are more diverse than glial cells (Saunders et al., 2018Saunders A. Macosko E.Z. Wysoker A. Goldman M. Krienen F.M. de Rivera H. Bien E. Baum M. Bortolin L. Wang S. et al.Molecular diversity and specializations among the cells of the adult mouse brain.Cell. 2018; 174: 1015-1030.e16Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, Tasic et al., 2018Tasic B. Yao Z. Graybuck L.T. Smith K.A. Nguyen T.N. Bertagnolli D. Goldy J. Garren E. Economo M.N. Viswanathan S. et al.Shared and distinct transcriptomic cell types across neocortical areas.Nature. 2018; 563: 72-78Crossref PubMed Scopus (347) Google Scholar, Zeisel et al., 2018Zeisel A. Hochgerner H. Lönnerberg P. Johnsson A. Memic F. van der Zwan J. Häring M. Braun E. Borm L.E. La Manno G. et al.Molecular architecture of the mouse nervous system.Cell. 2018; 174: 999-1014.e22Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar) and thus are more likely to be the subject of lineage analysis. To assess the ability of Nes-LSL-Flpo to efficiently recombine neuronal populations, we quantified the percentage of NeuN+ or HuC/D+ cells that are nβGal+ in several regions (Figure S2) in EIIa-Cre+, Nes-LSL-Flpo+, RC-FSF-LacZ+ brains. A high proportion of labeled neurons (NeuN+, nβGal+) were observed in most regions of the brain, with an average of 75.1% (±1.3, n = 3) across all regions analyzed (Figure 2C). Similar results were obtained with HuC/D (Figure S3). For instance, 78.3% (±2.9, n = 3) of HuC/D+ cells in the cortex were nβGal+, as compared with 75.3% (±0.9, n = 3) of NeuN+ cells. HuC/D also labeled some neuronal populations (e.g., Purkinje cells and cochlear neurons) that were not labeled by NeuN, and these neurons appear recombined with similar efficiency (Figure S3). In addition, we observed that a few structures, for example the CA2 region of the hippocampal formation (HPF) and the ventral hypothalamus, appear to have a reduced propensity for recombination (not shown). Altogether, our data clearly show that the Nes-LSL-Flpo allele has the potential to recombine the majority of neurons and can be used to fate map most brain regions. To rule out the possibility that Flp-dependent recombination occurs in postmitotic neurons, we applied PRISM to three vesicular transporter genes that are associated with neurotransmission, which is characteristic of mature neurons. Taken together, the mostly non-overlapping expression of Vgat (Slc32a1), Vglut1 (Slc17a7), and Vglut2 (Slc17a6) covers the vast majority of CNS regions. Interestingly, Vgat and Vglut2 are also expressed early during embryonic development immediately outside the ventricular zone but are not expressed in progenitors (Allen Brain Atlas, not shown). We first validated the recombination pattern of Vgat-Cre, Vglut1-Cre, and Vglut2-Cre using the Cre reporter RC-LSL-LacZ (Figure 2D), and we obtained the expected labeling patterns. Next, we crossed Nes-LSL-Flpo with Vgat-Cre and the Flp reporter RC-FSF-LacZ. As expected, we observed no nβGal+ cells in the brain (Figure 2D), indicating that the PRISM allele does not display recombination in postmitotic GABA neurons. We next crossed Vglut1-Cre and Vglut2-Cre with Nes-LSL-Flpo and the Flp reporter RC-FSF-LacZ and observed little to no cell labeling in both crosses (Figure 2D). These results are in contrast with those obtained when these Cre lines were crossed with the Cre reporter (Figure 2D). For instance, the majority of neurons of the cortex, hippocampus, thalamus, and cerebellum were labeled in the Vglut2-Cre+, RC-LSL-LacZ+ brain (Figure 2D). Since Vglut2 is expressed in many nascent postmitotic neurons even as early as embryonic day (E)11.5 (Allen Brain Atlas, not shown), the lack of labeled cells in the context of PRISM demonstrates the exquisite temporal specificity of this approach. To determine if the PRISM approach was compatible with inducible strategies, we crossed a Sox6-CreERT2 strain with Nes-LSL-Flpo and RC-FSF-tdTomato mice. In such an experiment, tdTomato will be permanently activated in Sox6+ progenitors during a finite time window after induction with tamoxifen. More specifically, pregnant dams were administered tamoxifen (TAM) at E12.5, and embryos were harvested at E18.5. We observed substantial tdTomato labeling in the forebrain, albeit with predictable mosaicism, demonstrating that PRISM is compatible with CreERT2 strains (Figure S3). This feature could be useful for example to test changes in competence of a genetically defined progenitor pool, particularly in situations where late administration of TAM is required, and the driver is expressed in progenitors and postmitotic neurons. Sonic hedgehog (Shh) is involved in the patterning and development of the nervous system (Fuccillo et al., 2006Fuccillo M. Joyner A.L. Fishell G. Morphogen to mitogen: the multiple roles of hedgehog signalling in vertebrate neural development.Nat. Rev. Neurosci. 2006; 7: 772-783Crossref PubMed Scopus (299) Google Scholar). It has been established that, contrarily to Shh-expressing spinal cord and hindbrain progenitors, rostrally located Shh+ progenitors are neurogenic (Alvarez-Bolado et al., 2012Alvarez-Bolado G. Paul F.A. Blaess S. Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions.Neural Dev. 2012; 7: 4Crossref PubMed Scopus (62) Google Scholar, Blaess et al., 2011Blaess S. Bodea G.O. Kabanova A. Chanet S. Mugniery E. Derouiche A. Stephen D. Joyner A.L. Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei.Neural Dev. 2011; 6: 29Crossref PubMed Scopus (71) Google Scholar, Hayes et al., 2011Hayes L. Zhang Z. Albert P. Zervas M. Ahn S. Timing of Sonic hedgehog and Gli1 expression segregates midbrain dopamine neurons.J. Comp. Neurol. 2011; 519: 3001-3018Crossref PubMed Scopus (41) Google Scholar, Hayes et al., 2013Hayes L. Ralls S. Wang H. Ahn S. Duration of Shh signaling contributes to mDA neuron diversity.Dev. Biol. 2013; 374: 115-126Crossref PubMed Scopus (20) Google Scholar, Joksimovic et al., 2009aJoksimovic M. Anderegg A.M. Roy A. Campochiaro L. Yun B. Kittappa R. McKay R. Awatramani R. Spatiotemporally separable Shh domains in the midbrain define distinct dopaminergic progenitor pools.Proc. Natl. Acad. Sci. USA. 2009; 106: 19185-19190Crossref PubMed Scopus (80) Google Scholar, Joksimovic et al., 2009bJoksimovic M. Yun B.A. Kittappa R. Anderegg A.M. Chang W.W. Taketo M.M. McKay R.D.G. Awatramani R.B. Wnt antagonism of Shh facilitates midbrain floor plate neurogenesis.Nat. Neurosci. 2009; 12: 125-131Crossref PubMed Scopus (148) Google Scholar, Nouri and Awatramani, 2017Nouri N. Awatramani R. A novel floor plate boundary defined by adjacent En1 and Dbx1 microdomains distinguishes midbrain dopamine and hypothalamic neurons.Development. 2017; 144: 916-927Crossref PubMed Scopus (14) Google Scholar). However, as Shh continues to be expressed postnatally (Carney et al., 2010Carney R.S.E. Mangin J.M. Hayes L. Mansfield K. Sousa V.H. Fishell G. Machold R.P. Ahn S. Gallo V. Corbin J.G. Sonic hedgehog expressing and responding cells generate neuronal diversity in the medial amygdala.Neural Dev. 2010; 5: 14Crossref PubMed Scopus (48) Google Scholar, Harwell et al., 2012Harwell C.C. Parker P.R.L. Gee S.M. Okada A. McConnell S.K. Kreitzer A.C. Kriegstein A.R. Sonic hedgehog expression in corticofugal projection neurons directs cortical microcircuit formation.Neuron. 2012; 73: 1116-1126Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Ihrie et al., 2011Ihrie R.A. Shah J.K. Harwell C.C. Levine J.H. Guinto C.D. Lezameta M. Kriegstein A.R. Álvarez-Buylla A. Persistent sonic hedgehog signaling in adult brain determines neural stem cell positional identity.Neuron. 2011; 71: 250-262Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, Lewis et al., 2004Lewis P.M. Gritli-Linde A. Smeyne R. Kottmann A. McMahon A.P. Sonic hedgehog signaling is required for expansion of granule neuron precursors and patterning of the mouse cerebellum.Dev. Biol. 2004; 270: 393-410Crossref PubMed Scopus (231) Google Scholar), its expression clouds putative lineage obtained using Shh-Cre. We sought to decipher Shh lineages by comparing Shh-Cre cumulative fate maps with PRISM-generated fate maps. We harvested postnatal day 21 (P21) brains of Shh-Cre mice crossed with the Cre reporter RC-LSL-LacZ (Figures 3A–3L). This resulted in nβGal labeled cells in numerous regions of the brain (Figures 3A–3L). To determine which one of these cell populations originated from Shh+ progenitors, we compared these brains with P21 brains obtained with PRISM (Figures 3A′–3L′). We found that, although there was concordance between both Shh-Cre and PRISM experiments, some regions (see below) are overrepresented in the Shh-Cre fate map implying that the cells populating these regions are not part of the Shh-Cre lineage. We describe these distinct populations along the rostrocaudal axis below. Comparing Shh-Cre and PRISM fate maps in the forebrain, we observed fewer nβGal+ cells in the cortex of PRISM brains (Figure 3). In the cortex, Shh-Cre-based labeling should be a composite of postmitotically labeled neurons (Harwell et al., 2012Harwell C.C. Parker P.R.L. Gee S.M. Okada A. McConnell S.K. Kreitzer A.C. Kriegstein A.R. Sonic hedgehog expression in corticofugal projection neurons directs cortical microcircuit formation.Neuron. 2012; 73: 1116-1126Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) as well as interneurons derived from preoptic area (POA) progenitors (Gelman et al., 2009Gelman D.M. Martini F.J. Nóbrega-Pereira S. Pierani A. Kessaris N. Marín O. The embryonic preoptic area is a novel source of cortical GABAergic interneurons.J. Neurosci. 2009; 29: 9380-9389Crossref PubMed Scopus (192) Google Scholar). Consistent with these studies (Gelman et al., 2009Gelman D.M. Martini F.J. Nóbrega-Pereira S. Pierani A. Kessaris N. Marín O. The embryonic preoptic area is a novel source of cortical GABAergic interneurons.J. Neurosci. 2009; 29: 9380-9389Crossref PubMed Scopus (192) Google Scholar, Harwell et al., 2012Harwell C.C. Parker P.R.L. Gee S.M. Okada A. McConnell S.K. Kreitzer A.C. Kriegstein A.R. Sonic hedgehog expression in corticofugal projection neurons directs cortical microcircuit formation.Neuron. 2012; 73: 1116-1126Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), we observed labeled cells in the cortex, many of them located in layer 5, in the Cre fate map (Figure 3A). Notably, an 80.3% reduction of labeled cells was observed in the PRISM fate map (52.0 ± 3.5 versus 10.2 ± 1.2 nβGal+/mm2, p < 0.0005, n = 3; Figure 3A′), reflecting the exclusion of postmitotic cells expressing Shh. The co-expression of nβGal and the neuronal marker NeuN (95.3% ± 3.3% of nβGal+ are NeuN+) as well as the morphology of the labeled cells observed with the Flp reporter RC-FSF-tdTomato, confirmed that these were indeed neurons (Figure S4). In the PRISM experiment, 32.9% (±3.6%, n = 3) of nβGal+ cells in the MOp and 27.4% (±1.5%, n = 3) in the SSp were parvalbumin (PV)+, indicating that these belonged to the PV+ interneuron class (Figure S4). In contrast to the cortex, the basal forebrain and striatum appear to contain about the same number of labeled cells in both Shh-Cre and PRISM fate maps (Figures 3B, 3B′, 3C, and 3C′). Based on NeuN expression and morphology (Figure S4), striatal nβGal+ cells are neurons that tend to be located in the lateral striatum, and 24.6% (±3.5%, n = 3) of these were PV+ (Figure S4). In another region of the basal ganglia, the external segment of the globus pallidus (GPe), we observed a 97.5% reduction of nβGal+ cells in the PRISM fate map compared with the cumulative Cre-driven fate map (213.8 ± 29.0 versus 5.3 ± 1.3 nβGal+/mm2, p < 0.005, n = 3; Figures 3D and 3D′). In this region, occasional labeled cells were PV+ or Npas1+ in the PRISM fate map (not shown), overall consistent with studies showing only a small contribution from the POA to the GP (Abecassis et al., 2020Abecassis Z.A. Berceau B.L. Win P.H. García D. Xenias H.S. Cui Q. Pamukcu A. Cherian S. Hernández V.M. Chon U. et al.Npas1+-Nkx2.1+ neurons are an integral part of the cortico-pallido-cortical loop.J. Neurosci. 2020; 40: 743-768Crossref Scopus (21) Google Scholar, Marin et al., 2000Marin O. Anderson S.A. Rubenstein J.L. Origin and molecular specification of striatal interneurons.J. Neurosci. 2000; 20: 6063-6076Crossref PubMed Google Scholar). Thus, even in a single macrostructure, such as the basal ganglia, the expression of Shh in both progenitors and postmitotic cells, obfuscates traditional lineage analysis but can be disentangled using the PRISM approach. In the thalamus, we also observed distinct labeling patterns between the traditional Shh-Cre and PRISM fate maps. For instance, in the PRISM fate map, we observed a significant reduction (96.7%) with nβGal+ cells virtually absent from medial nuclei of the thalamus (6.7 ± 2.0 nβGal+/mm2) as compared with Cre fate map (200.9 ± 23.5 nβGal+/mm2; p < 0.005, n = 3; Figures 3E and 3E′; e.g., intermediodorsal nucleus, central medial, and paracentral nuclei). In contrast, the number of nβGal+ cells in the lateral thalamus was similar (Figures 3G and 3G′). These cells were mostly GFAP+ glia, possibly remnants of the zona limitans intrathalamica (ZLI), a Shh-expressing region in the developing embryo parsing the thalamic and pre-thalamic regions (Figure S4). In the hypothalamus, we observed similar labeling between the Shh-Cre and PRISM fate maps, as exemplified with the subthalamic nucleus where no significant difference was observed in the number of nβGal+ cell (716.9 ± 76.7 versus 706.7 ± 49.6 nβGal+/mm2, p > 0.05, n = 3; Figures 3H and 3H′), consistent with previous conclusions (Alvarez-Bolado et al., 2012Alvarez-Bolado G. Paul F.A. Blaess S. Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions.Neural Dev. 2012; 7: 4Crossref PubMed Scopus (62) Google Scholar, Nouri and Awatramani, 2017Nouri N. Awatramani R. A novel floor plate boundary defined by adjacent En1 and Dbx1 microdomains distinguishes midbrain dopamine and hypothalamic neurons.Development. 2017; 144: 916-927Crossref PubMed Scopus (14) Google Scholar). The vast majority of nβGal+ cells were NeuN+ (99.0% ± 0.6%). In more rostral sections of the hypothalamus, however, somewhat fewer cells were observed in PRISM fate maps (not shown), likely due to reduced recombination efficiency in this region (see below). In the amygdala, we observed a similar number of potential interneurons in the basolateral complex (34.2 ± 7.2 versus 39.6 ± 7.2 nβGal+/mm2, p > 0.05, n = 3). However, the number of nβGal+ cells in medial nucleus of the amygdala was reduced by 94.6% in the PRISM fate map (883.6 ± 58.1 versus 48.0 ± 4.8 nβGal+/mm2, p < 0.0001, n = 3; Figures 3F and 3F′), suggesting that these neurons predominantly express Shh postmitotically. Indeed, Shh expression in the developing amygdala has been previously reported (Sousa and Fishell, 2010Sousa V.H. Fishell G. Sonic hedgehog functions through dynamic changes in temporal competence in the developing forebrain.Curr. Opin. Genet. Dev. 2010; 20: 391-399Crossref PubMed Scopus (74) Google Scholar). In the midbrain, the distribution of labeled cells was similar between the Shh-Cre and PRISM fate map (Figure 3). In both experiments, we observed numerous nβGal+ neurons located in the red nucleus (Figures 3J and 3J′), substantia nigra pars compacta (SNc; Figures 3I and 3I′), and ventral tegmental area (VTA), consistent with previous studies (Blaess et al., 2011Blaess S. Bodea G.O. Kabanova A. Chanet S. Mugniery E. Derouiche A. Stephen D. Joyner A.L. Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei.Neural Dev. 2011; 6: 29Crossref PubMed Scopus (71) Google Scholar, Joksimovic et al., 2009aJoksimovic M. Anderegg A.M. Roy A. Campochiaro L. Yun B. Kittappa R. McKay R. Awatramani R. Spatiotemporally separable Shh domains in the midbrain define distinct dopaminergic progenitor pools.Proc. Natl. Acad. Sci. USA. 2009; 106: 19185-19190Crossref PubMed Scopus (80) Google Scholar, Nouri and Awatramani, 2017Nouri N. Awatramani R. A novel floor plate boundary defined by adjacent En1 and Dbx1 microdomains distinguishes midbrain dopamine and hypothalamic neurons.Development. 2017; 144: 916-927Crossref PubMed Scopus (14) Google Scholar). In these latter midbrain dopaminergic nuclei, we found that the majority of tyrosine hydroxylase (TH)+ cells were nβGal+. In contrast, hypothalamic and olfactory dopamine neurons were nβGal− (Figure 4). This highlights a developmental dissimilarity between different dopamine clusters across the anterior-posterior axis that could underpin later observed transcriptomic differences (Hook et al., 2018Hook P.W. McC" @default.
• W3036633926 created "2020-06-25" @default.
• W3036633926 creator A5001017141 @default.
• W3036633926 creator A5019539721 @default.
• W3036633926 creator A5037114097 @default.
• W3036633926 creator A5062174429 @default.
• W3036633926 creator A5064586683 @default.
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• W3036633926 date "2020-06-01" @default.
• W3036633926 modified "2023-09-28" @default.
• W3036633926 title "PRISM: A Progenitor-Restricted Intersectional Fate Mapping Approach Redefines Forebrain Lineages" @default.
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