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+ mouse as a tool to investigate cell fate determination in the developing neocortex
Accepted Manuscript Title: Satb2 Cre/+ mouse as a tool to investigate cell fate determination in the developing neocortex Authors: Mateusz Cyryl Ambrozkiewicz, Paraskevi Bessa, Andrea Salazar-L´azaro, Valentina Salina, Victor Tarabykin PII: DOI: Reference:
S0165-0270(17)30266-2 http://dx.doi.org/doi:10.1016/j.jneumeth.2017.07.023 NSM 7799
To appear in:
Journal of Neuroscience Methods
Received date: Revised date: Accepted date:
6-3-2017 18-7-2017 22-7-2017
Please cite this article as: Ambrozkiewicz Mateusz Cyryl, Bessa Paraskevi, SalazarL´azaro Andrea, Salina Valentina, Tarabykin Victor.Satb2 Cre/+ mouse as a tool to investigate cell fate determination in the developing neocortex.Journal of Neuroscience Methods http://dx.doi.org/10.1016/j.jneumeth.2017.07.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Satb2 Cre/+ mouse as a tool to investigate cell fate determination in the developing neocortex Author List: Mateusz Cyryl Ambrozkiewicz1,×; Paraskevi Bessa1,× , Andrea SalazarLázaro1, Valentina Salina2, Victor Tarabykin1,2 ×both
authors contributed equally to presented work
Affiliation: 1Institute of Cell and Neurobiology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Virchowweg 6, 10117 Berlin, Germany 2Institute
of Neuroscience, University of Nizhny Novgorod, pr.Gagarina 24, Nizhny
Novgorod, Russia
Corresponding Author, Lead Contact: Victor Tarabykin, [email protected], [email protected]; Mateusz Cyryl Ambrozkiewicz, [email protected]
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Graphical abstract
Highlights (85 characters/50 words):
Satb2 Cre/+ mouse can be applied to screen for molecules involved in cell fate acquisition
The generation of deeper and upper layer neurons is fundamental for the establishment of brain connectivity. Here, using a Satb2 Cre/+ mouse, Ambrozkiewicz, Bessa et al. describe a high-throughput method to identify molecules involved in cell fate acquisition during cortical development.
Summary (up to 250 words) Background: Generation of different neuronal subtypes during neocortical development is the most important step in the establishment of cortical cytoarchitecture. The transcription factor Satb2 is expressed in neocortical projection neurons that send their axons intracortically as opposed to Satb2-negative neurons that preferentially project to subcortical targets.
New Method: In this report, we present a novel method to carry out large scale screening for molecules that control cell fate in the developing neocortex. It is based on a Satb2 Cre/+ mouse strain that expresses Cre recombinase from the Satb2 locus.
Results: By transfecting neuronal progenitors with a Cre-inducible reporter construct
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by nucleofection or in utero electroporation, we could determine the proportion of cells that become Satb2-positive.
Comparison with existing methods: Compared to genetic tracing or lineage analysis, this method offers a fast, easy-to-perform and reliable way of determining cell fate of newly born neurons.
Conclusions: We demonstrate that the Satb2 Cre/+ mouse can be applied to study factors, such as small molecule inhibitors, sh-RNAs or overexpression constructs, that can alter the proportion of Satb2-positive cells and thus play key roles in differentiation and acquisition of cell fate. Keywords (up to 10): cortical development; neurogenesis; cell fate acquisition; Satb2
Introduction: The mammalian cerebral cortex comprises cells originating from progenitors located in the ventricular zone of the embryonic dorsal forebrain. Essentially, the cerebral cortex consists of two types of neurons. Projection neurons originate from dorsal telencephalic progenitors and are produced either upon asymmetric division of radial glial (RG) progenitors in the ventricular zone (VZ), or neurogenic, symmetric division of transient amplifying cells in the subventricular zone (SVZ) (Dehay and Kennedy, 2007). The second class of neurons that populate the cerebral cortex are interneurons, which originate from ventral ganglionic eminence (GE) progenitor cells (Molyneaux et al., 2007). At the beginning of neurogenesis, at embryonic day 11 in mice (E11.5), the majority of progenitor cells undergo asymmetric divisions, producing one self-renewing RG daughter cell and one postmitotic neuron. This process is termed “direct neurogenesis”. Upper layer (UL) neurons emerge in mice at embryonic day 14-15 (E14.5-15.5) through the process of “indirect neurogenesis.” Here, radial glia at the VZ produce intermediate progenitor cells (IPCs), which mostly contribute to UL neuron formation (Noctor et al., 2002, 2004). However, it has been shown that IPCs can give rise to all projection neuronal
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subtypes (DeBoer et al., 2013; Kowalczyk et al., 2009). Formation of the neocortex relies on precise spatiotemporal control of signaling cascades that orchestrate the generation of deeper layer (DL) and upper layer neurons. UL neurons project intracortically and are the major contributors to the formation of commissural fibers, such as the corpus callosum. DL neurons project subcerebrally and connect higher cognitive centers with other brain regions. Disruption to the acquisition of neuronal fate may lead to dramatic anatomical and physiological consequences, including agenesis of corpus callosum and intellectual disability related syndromes (Edwards et al., 2014). Neurons of UL and DL express different combinations of transcription factors. The transcription factor Special AT-rich sequence-binding protein 2 (Satb2) is expressed in UL neurons and some layer V neurons that project to the contralateral hemisphere. We have previously shown that the deletion of Satb2 in the cortex leads to an ectopic upregulation of the DL-expressed transcription factor Chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (Ctip2) in the UL neurons (Britanova et al., 2005, 2008) Thus, callosally projecting neurons send their axons to sub-cortical targets instead (Britanova et al., 2008; Srivatsa et al., 2014). It is therefore of great importance to understand the molecular mechanisms that govern the acquisition of cellular identities that in turn are responsible for orchestrating the formation of complex structural networks in the brain. Fate mapping technologies provide information about factors contributing to the generation of cellular diversity. The earliest experiments included the application of fluorescent dyes and radioactive labeling to create accurate fate maps, studies of morphogenesis and cell migration between donor and host embryos (DeRuiter, 2012). Later on, fate maps were generated using two genetically engineered alleles expressing Cre recombinase and a reporter fluorescent protein such as green fluorescent protein (GFP) (Brown et al., 2009; Grubb, 2006). In the past few years, various studies have used in vivo fate mapping and genetic lineage tracing analyses in the cerebral cortex in order to test the multipotency and/or the fate-restricted nature of neocortical progenitor cells (Eckler et al., 2015; Guo et al., 2013; Han and Šestan, 2013). In this study, we took advantage of the restricted post-mitotic expression of the transcription factor Satb2 in the developing neocortex. We used a Satb2 Cre/+ mouse, in which exon 2 of the Satb2 gene is replaced by Cre recombinase (Fig. 1A). We
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then transfected progenitors from Satb2 Cre/+ embryonic cortex with the expression vectors: constitutively active pCAG-GFP and Cre-inducible pCAG-flox-stop-floxtdTomato. In the Satb2 Cre/+ mouse, the expression of Cre recombinase is dependent upon transcriptional activation of the Satb2 locus. Once expressed, Cre induces excision of the stop cassette that precedes the tdTomato reporter and permits its expression (Fig. 1A). We then analyzed the populations of cortical progenitors by flow cytometry and immunostaining to quantify the proportion of Satb2-positive cells. We demonstrate the applicability of this approach in screening large compound libraries, si-RNA or CRISPR-Cas9 approaches for causative shifts in post-mitotic neuronal fate.
Results Satb2 Cre/+ mouse model in studies of neuronal cell fate acquisition The main objective of our study was to design an efficient, high-throughput method for identification of molecules involved in neuronal fate establishment in the developing neocortex. For this purpose, we used a Satb2 Cre/+ mouse model where Satb2 locus is disrupted by a Cre expression cassette in one of the alleles. We previously reported that Satb2 is indispensable for determination of UL neuronal fate in the murine neocortex (Britanova et al., 2008). In our approach, we utilized a Cre expression cassette as a reporter of Satb2 expression in developing neurons (Fig. 1A). Cortical cells at E13.5 were isolated from Satb2 Cre/+ embryos and transfected with plasmids encoding for fluorescent reporter constructs: one encoding a Creinducible tdTomato fluorophore and another one, encoding constitutively active EGFP (Fig. S1 for preparing primary cortical cells). To ensure high efficiency of transfection we used nucleofection, a method that relies on direct transfer of DNA to the nucleus (Hamm et al., 2002; Lenz et al., 2003; Nambiar et al., 2003). The cells were maintained in cell culture for 2 days in vitro (2 DIV). During that time, cortical progenitors in the culture give rise to neurons of deeper and upper layer identity. Induction of Satb2 drives the expression of Cre recombinase and enables the concomitant expression of tdTomato exclusively in neurons fated to become upper layer type. Constitutive expression of EGFP in transfected cells provides a baseline level of transfection efficiency against which tdTomato fluorescence can be measured. Using flow cytometry, the proportion of tdTomato-fluorescing, Satb2-
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positive neurons can be quantified and relativized to the total number of transfected cells expressing both tdTomato and EGFP. In a parallel approach, neocortical progenitors were co-electroporated in utero with both plasmids of Satb2 Cre/+ embryos at different developmental stages. Hereafter, we refer to neurons from Satb2 Cre/+ mice that express tdTomato “Satb2 tdTom” and to both vectors, pCAG-GFP and the Cre-inducible, tdTomato-encoding plasmid pCAG-flox-stop-flox-tdTomato as “reporter constructs.
To test if tdTomato expression is a result of Cre-mediated stop cassette recombination (in other words, to test the threshold of tdTomato expression in Satb2 tdTom
neurons), we performed nucleofection of reporter constructs in E13.5 cortical
cells prepared from Satb2 +/+ (wild type) and Satb2 Cre/+ mice (Fig. 1B-1D). At 2DIV, we quantified Satb2 tdTom neurons using flow cytometry. Expression of tdTomato in wild type neurons (quartiles Q2 and Q4 of the dot plot, Fig. 1B) was marginal and constituted 5±0.2 % of transfected cells (found in quartiles Q1 and Q2, Fig 1B and 1D), as compared to robust tdTomato signals in Satb2 Cre/+ neurons (Fig 1C and 1D), which amounted to 49.2±0.9 % of transfected cells (Table S1). Therefore, we conclude that the quantified fraction of tdTomato-expressing, wild-type cells represents the level of Cre-independent background expression in our method.
At E12.5, the VZ is mainly composed of early neuronal progenitors able to generate both DL and UL neurons. As neurogenesis progresses, E13.5 progenitors become more restricted in their multipotency and shift to the generation of mainly UL neurons. Satb2 is a postmitotic determinant of upper layer neurons. To study the time course of Satb2 activation in cortical primary neurons using our method, we nucleofected cortical cells isolated from Satb2 Cre/+ mice at E12.5, E13.5 and E14.5 with our reporter constructs. At 2DIV, we observed 26.9±0.6 % Satb2 tdTom neurons isolated from E12.5, 45.5±3.3 % Satb2 tdTom cells from E13.5, and 67.3±1.3 % Satb2 tdTom from E14.5 (Fig. 1E, Table S2). Collectively, the increase in the ratio of Satb2 tdTom cells in the culture, quantified using our genetic reporters, mirrors previous observations of Satb2 expression during the progression of embryonic neurogenesis.
After 2 DIV, primary cortical cultures contain a mixture of progenitors and fatecommitted, post-mitotic neurons. To study activation of the Satb2 locus in vivo using our method, we electroporated both reporter constructs in neocortical progenitors at
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E12.5 in Satb2 +/+ and Satb2 Cre/+ embryos in utero. To more closely relate our in vitro observations with the situation in vivo, we counted Satb2 tdTom neurons two days after electroporation. At E14.5, some of the labeled cells remain in a progenitor state, while a fraction began differentiation into neurons (Fig. S2); a scenario that resembles culture conditions for data on Fig. 1E. We observed virtually no tdTomato expression in Satb2 +/+ mice 48 hours post-electroporation (Fig S2A). In Satb2 Cre/+ mice, of all EGFP-expressing cells, we quantified 34.4 ± 4.4% of Satb2 tdTom neurons (Fig. S2B, S2C, Table S3).
Dinaciclib treatment attenuates Satb2-expressing cells As an example application of our model in screening for genes involved in cell fate acquisition, we used the potent pan-cyclin-dependent kinase inhibitor dinaciclib (Johnson et al., 2012; Paruch et al., 2010), and quantified its effects on Satb2 expression in a cortical culture system. Dinaciclib exhibits anti-proliferative effects on multiple cell lines (Chen et al., 2016). Because of Satb2 expression confined to postmitotic neurons, we hypothesized that dinaciclib would halt the cell cycle progression of progenitors present in the culture. To establish a titer of the drug for our assay, we tested the effects of dinaciclib at different concentrations on the number of Satb2 tdTom neurons generated. We transfected cortical cells from E12.5 Satb2 Cre/+ mice with our reporter constructs using nucleofection and applied dinaciclib at final concentrations of 0.2 µM, 1 µM, or an equal volume of DMSO (vehicle solvent for dinaciclib) in the culture medium for 2DIV (Fig. 1F-I). DMSO had no effect on proportion of Satb2 tdTom cells as compared to untreated cultures, as quantified by flow cytometry (Fig. 1I) (Untreated, 32.9±0.1 %; DMSO, 35.3±2.3%; Table S4). Dinaciclib at both tested concentrations reduced the number of Satb2 tdTom cells as compared to DMSO (Fig. 1F-H) (0.2 µM, 3±0.1 %; 1 µM, 2±0.5 %, Table S4). This proof-of-principle experiment shows that our approach can be applied to study compounds that change Satb2 expression in cortical neurons by affecting gene expression or cell signaling.
Dinaciclib increases the number of Ctip2 expressing cortical neurons Cell fate acquisition during cortical neurogenesis is the effect of mutual molecular interplay between Satb2 and Ctip2. The transcription factor Ctip2 marks a subpopulation of DL neurons (layer V) and is critical for specification of cortical, subcerebral projection neurons (Chen et al., 2008; Molyneaux et al., 2005). To
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further characterize the effects of CDK inhibition on neuronal cell fate, we performed quantitative immunocytochemical analysis of Satb2 tdTom cells after dinaciclib treatment. Because of the similar effects of 0.2 and 1 µM dinaciclib on Satb2 tdTom neurons, possibly due to a plateau effect, in the next experiment we expanded the range of drug concentration. Cortical cells from E12.5 Satb2 Cre/+ mice were transfected with our reporter constructs, treated with DMSO or dinaciclib at 0.04, 0.2 or 1 µM dinaciclib and cultured for 2DIV followed by immunostaining for GFP and Ctip2 (Fig. 2A). In line with data obtained after cell sorting, dinaciclib at 0.2 µM and 1 µM significantly decreased the proportion of Satb2 tdTom cells as compared to DMSO treated cells (Fig 2A, 2B Table S5). Furthermore, dinaciclib at 0.2 µM significantly increased the fraction of Ctip2-expressing neurons (Fig. 2A, 2C Table S5, Fig. S4). Interestingly, 40 nM dinaciclib did not affect the number of neurons expressing either Satb2, or Ctip2, suggesting a dose-dependent effect (Fig. 2B and 2C, Table S5). Collectively, our method allows for screening of pharmaceuticals that influence cell fate acquisition. Satb2 tdTom neurons express Cre and Satb2 To corroborate that tdTomato expression is due to Cre-mediated excision of the stop cassette upstream of tdTomato reporter and that tdTomato expression mirrors Satb2 in cortical neurons, we first used an in vitro cortical culture system. We transfected cortical cells from E12.5 Satb2 Cre/+ mouse with the reporter constructs and immunostained primary cultures fixed at 2DIV for GFP, Cre and Satb2 (Fig. 3A; also compare Fig S3 for description of self-made anti-Satb2 antibody). Almost all Satb2 tdTom
cells expressed Cre and Satb2 (Satb2 tdTom and Cre-positive – 97.2±1.8 %;
Satb2 tdTom and Satb2-positive – 91.4±6.8 %) (Fig. 3B, Table S6). Therefore, in our culture system, tdTomato mirrors Cre and Satb2 expression in cortical neurons and can be used as a reliable reporter of Satb2 locus induction. Characterization of Satb2 tdTom neurons in vivo Next, we tested Satb2 tdTom neurons for expression of Cre and Satb2 in vivo. To validate if tdTomato fluorescence in Satb2 tdTom neurons mirrors Cre and Satb2 expression in neurons, we electroporated both reporter constructs in neocortical progenitors at E13.5 and fixed brains at E18.5, when embryonic neurogenesis in murine brains is complete. As observed in our previous in vivo experiments (Fig. S2),
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the expression of tdTomato in Satb2 +/+ neurons was marginal and Cre-independent (Fig. 4A). In Satb2 Cre/+ embryos, nearly all Satb2 tdTom neurons expressed both Cre and Satb2 (Fig. 4B-4D; Satb2 tdTom and Cre-positive – 94.6±4.7 %; Satb2 tdTom and Satb2positive – 95.9±5.3 %; Table S7).
In conclusion, our method provides a reliable readout of Satb2 locus activation and can be applied in the identification of genes or signaling pathways indispensable for neuronal fate specification during development in vivo.
Discussion
Genetically engineered reporter mice offer a precise and powerful tool for fate mapping and enable the identification of factors that determine cell identity. Using a Satb2 Cre/+ mouse line, we have developed an experimental approach to facilitate the discovery of molecules that play key roles in neuronal differentiation and acquisition of cell fate. In the Satb2 Cre/+ mouse, Cre recombinase is knocked-in to one locus of Satb2, a gene of crucial importance for determination of upper layer neurons and formation of the corpus callosum. Our method relies on transfection of a Creinducible tdTomato reporter construct that mirrors activation of the Satb2 locus and can be quantified by flow cytometry and immunostaining (Fig 1A). We addressed the specificity of our method using Satb2 +/+ mice both in an in vitro system (Fig. 1B) and using in utero electroporation of neocortical progenitors (Fig. 4A and S2A). Based on the distribution of tdTomato fluorescence as measured by flow cytometry, we set the threshold intensity of tdTomato that defines a neuron as Satb2 tdTom.
The observed 5% tdTomato-positive cells in Satb2 +/+ cultures may be the result
of the application of a strict intensity threshold. Additionally, in Satb2 +/+ cortices in vivo, we observe virtually no tdTomato-positive neurons two days post-IUE (Fig. S2A) and marginal (<1%) tdTomato-positive neurons five days post-IUE (Fig. 4A). These tdTomato-positive cells were Cre-negative; it is therefore likely that these cells represent Cre-independent rare artifacts. Moreover, a putative factor involved in neural fate acquisition is expected to engender drastic changes in Satb2-positive
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neuron count as quantified by flow cytometry, which combined with validation using immunocytochemistry and in vivo transfection of reporter constructs allows the reliable discrimination of factors that alter the fate of postmitotic neuron. Additionally, in Satb2 Cre/+ cells, tdTomato fluorescence signals were Cre-dependent and collocalized with Satb2 immunostaining signals (Fig. 3 and Fig. 4). It seems that detection of Cre in Satb2 tdTom cells reflects even slight activation of Satb2 locus due to efficient Cre-mediated activation of tdTomato expression (Meyers et al., 1998). Our approach offers a reliable method for labeling of cells with history of activating Satb2 locus and might be useful in experiments addressing transient versus stable Satb2 expression in neurons or studying the mutual relationship between Satb2 and Ctip2 in neurons.
Our method is based on in vitro transfection using nucleofection and in utero electroporation. Both methods allow for reliable labeling of only a population of cells, due to less than 100% efficiency of transfection. Therefore, in our analyses of method specificity (Fig. 3A, 4B and 4C), we only inspected tdTomato-fluorescing, Satb2 tdTom neurons for Cre- and Satb2- expression. In cortical culture, as well as in the embryonic cortex of Satb2 Cre/+ mice, Cre and Satb2 are additionally expressed cells that are not labelled following transfection with both reporter constructs (Fig. 3 and 4).
The relevance of our proposed method is its application in screening approaches. Here, as a proof of principle experiment, we applied dinaciclib to primary cortical culture prepared from Satb2 Cre/+ mice at E13.5 and quantified its effect on the proportion of Satb2 tdTom cells at 2DIV (Fig. 1F-I and Fig. 2). Dinaciclib, or SCH727965, is a pan inhibitor of cyclin-dependent kinases (CDKs). CDKs are critical for progression of cell cycle and drive the transitions between different stages of mitosis. Dinaciclib-mediated inhibition of CDKs abrogates progression of cell cycle (Chen et al., 2016; Rajput et al., 2016; Senderowicz, 2003). Indeed, in line with post-mitotic Satb2 expression, treatment with dinaciclib at 0.2 and 1 µM consistently led to a decreased proportion of Satb2 tdTom neurons (Fig. 1F-I and Fig. 2). In parallel, we observed increase in the fraction of Ctip2-positive cortical cells after 0.2 µM dinaciclib treatment (Fig 2B, C, E). Decrease in Satb2 tdTom after inhibitor treatment might reflect suppression of the post-mitotic fate of cortical progenitors. Finally, reduction in Satb2
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tdTom
and parallel increase in Ctip2 proportion might reflect disturbances in acquisition
of upper layer fate by developing neurons upon CDKs inhibition.
Our method can therefore be applied both in vitro and in vivo as a high-throughput screening of different compounds (sh-RNAs, CRISPR-Cas libraries, or drug libraries) that regulate genes critical for cell fate determination and act upstream of Satb2 during neuronal development. This mouse model could be particularly useful in in vitro assays where compounds are applied directly into the medium of cortical progenitor cultures isolated from Satb2 Cre/+ mice. Provided approved ethical regulations, small molecule inhibitors could also be injected into the lateral ventricles of developing E12.5 to E14.5 Satb2 Cre/+ embryos, followed by in utero electroporation of reporter plasmids. Moreover, including overexpression plasmids, sh-RNAs, morpholinos, and/or miRNAs in the DNA mixture for transfection in our model enables gene dosage analyses of putative regulators of cell fate acquisition acting upstream of Satb2.
During evolution, the mammalian neocortex has undergone a substantial increase in the proportion of UL neurons, suggesting a possible link between genes are that highly expressed in this subset of neurons and complex cognitive abilities (Edwards et al., 2014). To conclude, the above-presented method could be used for rapid identification of genes and signaling pathways that affect the birth and the function of specific neuronal pools and contribute to an understanding of neuronal development and evolution.
Experimental Procedures
Animal studies and ethical statement All experiments presented in the report were conducted in compliance with welfare guidelines issued by local authorities LaGeSo and Charité University Hospital. All possible efforts were undertaken to minimize suffering. Corresponding permits have been granted: G0079/11. For all experiments presented in this study, Satb2 Cre/+ males were mated to NMRI wild type females, maintained in the animal facility of Charité University Hospital in Berlin, Germany. The date of vaginal plug was counted as E0.5. All mice were sacrificed by administration of lethal dose of pentobarbital.
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Constructs For transfection of cortical primary cells by nucleofection or in utero electroporation of neocortical progenitors we used constructs under the control of β-actin promoter: pCAG-GFP and pCAG-flox-stop-flox-tdTomato. Promoter in both constructs permits for transcription of both reporters in all types of mammalian cells. However, the red fluorescence marker was downstream of stop cassette flanked by loxP sites. Upon Cre-mediated recombination, stop cassette is excised and permits tdTomato expression, specifically in Cre-expressing cells.
Primary cell culture and nucleofection Cortices from E12-E14 were isolated as illustrated on Fig. S1. The tissue was further enzymatically digested with 0.25% trypsin (Gibco) in HBSS solution (Gibco, with MgCl2 and CaCl2) for 30 minutes at 37°C. Next, the tissue was treated with DNaseI (Roche) for 5 minutes at 37°C and the enzymatic digestion was stopped by adding 2 mL of FBS (Gibco). Tissue was briefly centrifuged (1 minute, 1200 rpm) and washed twice with Neurobasal (Life Technologies). Afterwards, tissue was carefully triturated in medium (Neurobasal supplemented with GlutaMax, B27 and penicillin/streptomycin; Life Technologies) 10 times using P1000 pipette tip and then 10 times with P200 pipette tip. Cells were then counted with Neubauer counting chamber and nucleofected according to manufacturer’s protocols (Mouse Neuron Nucleofector Kit, VPG-1001, and Amaxa 2b Nucleofection system, program O-005, Lonza). We used 1 million cells for 1 µg of DNA (both reporter constructs: pCAGGFP, pCAG-flox-stop-flox-tdTomato, mixed at 1:1 ratio). Then, cells were seeded onto plastic or glass bottom 96-well plate, previously coated with 0.1 mg/mL poly-Llysine and 2% laminin (Sigma) at a concentration 100 000/well and maintained for 2DIV at 37°C and 5% carbon dioxide.
Cell treatment Primary cells were treated 2 hours post-seeding. Prior to treatment, dinaciclib (courtesy of David Kaplan) was diluted in cell culture grade DMSO (Sigma). Equal amounts of DMSO and diluted dinaciclib were added directly to the cell media to achieve final concentration of vehicle or drug at 0.04, 0.2 or 1 µM.
In utero electroporation
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Mouse embryos at determined embryonic stage were subjected to in utero electroporation accordingly to previously published work (Saito, 2006). Briefly, for in utero electroporation, pregnant mice were deeply anaesthetized with vaporized isoflurane/oxygen. To ensure post-operative analgesia, mice received subcutaneous injections of 3 mg buprenorphine (Temgesic). Additionally, for further analgesia and to prevent abdominal cramps, operated females were supplied with 0.05 mg/mL and 6 mg/mL metamizole in drinking water for 3 days after procedure. For electroporation of E12.5 cortical progenitors, 5 pulses of 32 V, each 50 ms long and with 950 ms interval were delivered with a forceps-type electrode. For E14.5 progenitors, 6 pulses of 34 V were applied.
Flow cytometry Prior to analysis, cells at 2DIV were washed with HBSS and incubated with 0.25% trypsin for 5 minutes at 37°C and triturated 10 times with P200 pipette tip. Flow cytometry of cortical cells were performed with BD FACS CantoTM II Flow Cytometer and plots were analyzed by FLowJo v9.8.3. Each measurement was performed using a population of 8000 to 10000 cells, characterized by physical properties of living particles defined based on forward and side scatter laser readouts. The gating thresholds for Alexa 488 and PE-A were set for samples nucleofected with pCAGGFP or pCAG-tdTomato only. Immunostaining and image acquisition For immunostainings, primary cells were maintained on glass-bottom 96 well plates (Cellvis) for 2DIV and fixed with 4% paraformaldehyde (PFA) for 20 minutes. For immunohistochemistry, P0 mouse brains were fixed with 4%PFA for 14 hours and subjected to 10-30% sucrose/PBS gradient, followed by cryosectioning (50 µm). Further fixed cell culture or brain sections were incubated with blocking solution (10% goat serum, 0.5% Triton X-100) for 1 hour in ambient temperature and with primary antibodies solution for 14 hours at 4°C. After washing with PBS, the specimens were incubated with fluorophore-labelled secondary antibody solutions for 4 hours at room temperature (Jackson Immunoresearch). In this study, the following primary antibodies were used at indicated dilutions: anti-GFP (goat, 1:500, Rockland, 600101215), anti-Ctip2 (rat, 1:500, Abcam, ab18465), anti-Cre (rabbit, 1:1000, 257 003, Synaptic Systems), anti-Satb2 (rabbit, 1:200, self-generated). Images of primary cell culture were acquired with the Spinning Disc Confocal microscope (Nikon CSU-
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X), or Leica TCS SP8 confocal, and the immunostained brain sections with Leica SP2 confocal microscope. The antibody against Satb2 (rabbit anti-Satb2) that was used for immunohistochemistry was used at concentration 1:200. The antibody was generated in the lab for the epitope QQSQPTKESSPPREEA. Statistics, Satb2 tdTom quantification and data analysis Statistical analyses of analyzed data were performed using Graph Pad Prism software and Microsoft Excel. All statistical tests and experimental values reported in this study are listed in supplementary tables. For comparison of differences within group means (in dinaciclib treatment experiments), we used one-way ANOVA with Bonferroni post-hoc correction. For determination of proportion of Satb2 tdTom cells using flow cytometry, we quantified the number of tdTomato expressing cells (quartiles Q2 and Q4 on the dot plots) and expressed it relative to the number of GFP expressing cells (quartiles Q1 and Q2). To quantify the of proportion of Satb2 tdTom cells using immunostainings, we quantified number of cells positive of respective staining after setting signal threshold using ImageJ software and ‘Analyze particles’ command (Fig. S4). To analyze the Satb2 tdTom proportion after in utero electroporation, we manually counted the number of neurons positive for respective marker or tdTomato signal with Cell Counter plug-in of ImageJ software. Author Contributions: M.A., P.B. and V.T. designed experiments, M.A., P.B., A.S.L. and V.S. conducted experiments and processed data. M.A. and P.B. wrote the manuscript. V.T. provided funding and critical comments on the project. Acknowledgements: We would like to acknowledge Advanced Medical Bioimaging AMBIO Core Facility at Charité University Hospital for granting microscope access and technical support with imaging of primary cell culture; Britta Eickholt for providing access to nucleofector, necessary for our experiments; David Kaplan for providing dinaciclib for our experiments. The Leica TCS SP8 was funded by BMW/DLR (grant 50WB1421), Center of Space Medicine Berlin. We thank Stephen Horan for helpful comments and editing of the manuscript. This study was supported by a grant from Russian Scientific Foundation (project n 15-14-10021) to V.T. All authors declare no conflict of interest regarding the publication of this report.
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Figure Legend Figure 1. Flow cytometry analysis of cortical cells isolated from Satb2 Cre/+ and wild type mice. (A) The workflow chart of using Satb2 Cre/+ as a model to study cell fate specification in cortical neurons. In Satb2 Cre/+ mouse, Cre is expressed from one Satb2 locus. Primary cortical neurons are prepared from embryonic Satb2 Cre/+ mouse brain and the transfected with reporter plasmids using nucleofection: one encoding EGFP and another one encoding Cre-inducible tdTomato. Upon treatment with compounds (e.g. small molecule inhibitors), or transfection with sh-RNA to knock-down gene of interest, the number of tdTomato-expressing Satb2-positive cells (Satb2 tdTom) is quantified by flow cytometry. (B and C) Example scatter plot of cortical cells after two days in culture isolated from E13.5 Satb2 +/+ (B), or Satb2 Cre/+ (C) mice and transfected with reporter plasmids. (D) Quantification of the proportion of Satb2 tdTom neurons to GFP-expressing neurons from the Satb2 +/+ (wild type) and Satb2 Cre/+ mouse. Results are represented as averages ±S.D. Wild type, Satb2 +/+, n=2 independent measurements from two transfections; Satb2 Cre/+, n=2 independent measurements from two transfections; ***p<0.001, unpaired t-test (Table S1). (E) Quantification of the proportion of Satb2 tdTom neurons prepared from Satb2 Cre/+ mice at E12.5, E13.5 and E14.5. Results are represented as averages ±S.D. Each result is average of at least two independent transfections; ***p<0.001, one-way ANOVA with Bonferroni post hoc test (Table S2). (F-H) Example scatter plots of Satb2 Cre/+ cortical cells nucleofected with reporter plasmids at E13.5 and treated with DMSO (F), 0.2 µM dinaciclib (G), or 1 µM dinaciclib for 2DIV. (I) Quantification of the proportion of Satb2tdTom cells after control treatment, 0.2 µM or 1 µM dinaciclib. Results are represented as averages ±S.D. Each result is mean of two independent measurements; ***p<0.001, one-way ANOVA with Bonferroni post hoc test (Table S4).
Figure 2. Dinaciclib attenuates Satb2 and induces Ctip2 expression in developing neurons. (A) Representative images of 2DIV cortical cells from E12.5 Satb2 Cre/+ mouse transfected with reporter plasmids and treated with DMSO, or dinaciclib at 0.04, 0.2 or 1 µM. Fixed neurons were immunostained for EGFP and Ctip2. Arrowheads point to Satb2 tdTom neurons; stars mark EGFP-positive, Ctip2positive neurons. (B-C) Quantification of Satb2 tdTom, or Ctip2-positive, GFP-positive
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neurons after DMSO or dinaciclib treatment. Results are represented as averages ±S.D. ***p<0.001, *p<0.05, one-way ANOVA with Bonferroni post-hoc test (Table S5). Figure 3. Primary Satb2 tdTom neurons are positive for Satb2 and for Cre recombinase. (A) Representative images of EGFP and Cre immunostaining and tdTomato fluorescence signals in Satb2 Cre/+ cells. Cells were isolated at E12.5, nucleofected with reporter plasmids and fixed at 2DIV. Arrowheads point to Satb2 tdTom
neurons; stars mark EGFP-positive neurons negative for tdTomato. (B)
Quantification of the proportion of tdTomato-positive cells expressing Cre, or Satb2. Results are represented as averages ±S.D. Each result is mean of two independent transfections (Table S6). Figure 4. Satb2 tdTom neurons are positive for Satb2 and for Cre in vivo. (A-B) Representative images of EGFP, Cre or Satb2 immunostaining and tdTomato fluorescence signals in wild type, Satb2 +/+ (A, upper panel), or Satb2 Cre/+ (B, middle and C, bottom panel) cortices of E18.5 mice. Neuronal progenitors were electroporated in utero at E13.5 with reporter constructs. Each image is a maximum projection of a Z-stack through 50 µm thick coronal brain section. Arrowheads point to some Satb2 tdTom neurons positive for Cre (B), or for Satb2 (C). (D) Quantification of Satb2 tdTom neurons positive for Cre, or Satb2. Results are represented as averages ±S.D. (Table S7).
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S0165-0270(17)30266-2 http://dx.doi.org/doi:10.1016/j.jneumeth.2017.07.023 NSM 7799
To appear in:
Journal of Neuroscience Methods
Received date: Revised date: Accepted date:
6-3-2017 18-7-2017 22-7-2017
Please cite this article as: Ambrozkiewicz Mateusz Cyryl, Bessa Paraskevi, SalazarL´azaro Andrea, Salina Valentina, Tarabykin Victor.Satb2 Cre/+ mouse as a tool to investigate cell fate determination in the developing neocortex.Journal of Neuroscience Methods http://dx.doi.org/10.1016/j.jneumeth.2017.07.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Satb2 Cre/+ mouse as a tool to investigate cell fate determination in the developing neocortex Author List: Mateusz Cyryl Ambrozkiewicz1,×; Paraskevi Bessa1,× , Andrea SalazarLázaro1, Valentina Salina2, Victor Tarabykin1,2 ×both
authors contributed equally to presented work
Affiliation: 1Institute of Cell and Neurobiology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Virchowweg 6, 10117 Berlin, Germany 2Institute
of Neuroscience, University of Nizhny Novgorod, pr.Gagarina 24, Nizhny
Novgorod, Russia
Corresponding Author, Lead Contact: Victor Tarabykin, [email protected], [email protected]; Mateusz Cyryl Ambrozkiewicz, [email protected]
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Graphical abstract
Highlights (85 characters/50 words):
Satb2 Cre/+ mouse can be applied to screen for molecules involved in cell fate acquisition
The generation of deeper and upper layer neurons is fundamental for the establishment of brain connectivity. Here, using a Satb2 Cre/+ mouse, Ambrozkiewicz, Bessa et al. describe a high-throughput method to identify molecules involved in cell fate acquisition during cortical development.
Summary (up to 250 words) Background: Generation of different neuronal subtypes during neocortical development is the most important step in the establishment of cortical cytoarchitecture. The transcription factor Satb2 is expressed in neocortical projection neurons that send their axons intracortically as opposed to Satb2-negative neurons that preferentially project to subcortical targets.
New Method: In this report, we present a novel method to carry out large scale screening for molecules that control cell fate in the developing neocortex. It is based on a Satb2 Cre/+ mouse strain that expresses Cre recombinase from the Satb2 locus.
Results: By transfecting neuronal progenitors with a Cre-inducible reporter construct
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by nucleofection or in utero electroporation, we could determine the proportion of cells that become Satb2-positive.
Comparison with existing methods: Compared to genetic tracing or lineage analysis, this method offers a fast, easy-to-perform and reliable way of determining cell fate of newly born neurons.
Conclusions: We demonstrate that the Satb2 Cre/+ mouse can be applied to study factors, such as small molecule inhibitors, sh-RNAs or overexpression constructs, that can alter the proportion of Satb2-positive cells and thus play key roles in differentiation and acquisition of cell fate. Keywords (up to 10): cortical development; neurogenesis; cell fate acquisition; Satb2
Introduction: The mammalian cerebral cortex comprises cells originating from progenitors located in the ventricular zone of the embryonic dorsal forebrain. Essentially, the cerebral cortex consists of two types of neurons. Projection neurons originate from dorsal telencephalic progenitors and are produced either upon asymmetric division of radial glial (RG) progenitors in the ventricular zone (VZ), or neurogenic, symmetric division of transient amplifying cells in the subventricular zone (SVZ) (Dehay and Kennedy, 2007). The second class of neurons that populate the cerebral cortex are interneurons, which originate from ventral ganglionic eminence (GE) progenitor cells (Molyneaux et al., 2007). At the beginning of neurogenesis, at embryonic day 11 in mice (E11.5), the majority of progenitor cells undergo asymmetric divisions, producing one self-renewing RG daughter cell and one postmitotic neuron. This process is termed “direct neurogenesis”. Upper layer (UL) neurons emerge in mice at embryonic day 14-15 (E14.5-15.5) through the process of “indirect neurogenesis.” Here, radial glia at the VZ produce intermediate progenitor cells (IPCs), which mostly contribute to UL neuron formation (Noctor et al., 2002, 2004). However, it has been shown that IPCs can give rise to all projection neuronal
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subtypes (DeBoer et al., 2013; Kowalczyk et al., 2009). Formation of the neocortex relies on precise spatiotemporal control of signaling cascades that orchestrate the generation of deeper layer (DL) and upper layer neurons. UL neurons project intracortically and are the major contributors to the formation of commissural fibers, such as the corpus callosum. DL neurons project subcerebrally and connect higher cognitive centers with other brain regions. Disruption to the acquisition of neuronal fate may lead to dramatic anatomical and physiological consequences, including agenesis of corpus callosum and intellectual disability related syndromes (Edwards et al., 2014). Neurons of UL and DL express different combinations of transcription factors. The transcription factor Special AT-rich sequence-binding protein 2 (Satb2) is expressed in UL neurons and some layer V neurons that project to the contralateral hemisphere. We have previously shown that the deletion of Satb2 in the cortex leads to an ectopic upregulation of the DL-expressed transcription factor Chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (Ctip2) in the UL neurons (Britanova et al., 2005, 2008) Thus, callosally projecting neurons send their axons to sub-cortical targets instead (Britanova et al., 2008; Srivatsa et al., 2014). It is therefore of great importance to understand the molecular mechanisms that govern the acquisition of cellular identities that in turn are responsible for orchestrating the formation of complex structural networks in the brain. Fate mapping technologies provide information about factors contributing to the generation of cellular diversity. The earliest experiments included the application of fluorescent dyes and radioactive labeling to create accurate fate maps, studies of morphogenesis and cell migration between donor and host embryos (DeRuiter, 2012). Later on, fate maps were generated using two genetically engineered alleles expressing Cre recombinase and a reporter fluorescent protein such as green fluorescent protein (GFP) (Brown et al., 2009; Grubb, 2006). In the past few years, various studies have used in vivo fate mapping and genetic lineage tracing analyses in the cerebral cortex in order to test the multipotency and/or the fate-restricted nature of neocortical progenitor cells (Eckler et al., 2015; Guo et al., 2013; Han and Šestan, 2013). In this study, we took advantage of the restricted post-mitotic expression of the transcription factor Satb2 in the developing neocortex. We used a Satb2 Cre/+ mouse, in which exon 2 of the Satb2 gene is replaced by Cre recombinase (Fig. 1A). We
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then transfected progenitors from Satb2 Cre/+ embryonic cortex with the expression vectors: constitutively active pCAG-GFP and Cre-inducible pCAG-flox-stop-floxtdTomato. In the Satb2 Cre/+ mouse, the expression of Cre recombinase is dependent upon transcriptional activation of the Satb2 locus. Once expressed, Cre induces excision of the stop cassette that precedes the tdTomato reporter and permits its expression (Fig. 1A). We then analyzed the populations of cortical progenitors by flow cytometry and immunostaining to quantify the proportion of Satb2-positive cells. We demonstrate the applicability of this approach in screening large compound libraries, si-RNA or CRISPR-Cas9 approaches for causative shifts in post-mitotic neuronal fate.
Results Satb2 Cre/+ mouse model in studies of neuronal cell fate acquisition The main objective of our study was to design an efficient, high-throughput method for identification of molecules involved in neuronal fate establishment in the developing neocortex. For this purpose, we used a Satb2 Cre/+ mouse model where Satb2 locus is disrupted by a Cre expression cassette in one of the alleles. We previously reported that Satb2 is indispensable for determination of UL neuronal fate in the murine neocortex (Britanova et al., 2008). In our approach, we utilized a Cre expression cassette as a reporter of Satb2 expression in developing neurons (Fig. 1A). Cortical cells at E13.5 were isolated from Satb2 Cre/+ embryos and transfected with plasmids encoding for fluorescent reporter constructs: one encoding a Creinducible tdTomato fluorophore and another one, encoding constitutively active EGFP (Fig. S1 for preparing primary cortical cells). To ensure high efficiency of transfection we used nucleofection, a method that relies on direct transfer of DNA to the nucleus (Hamm et al., 2002; Lenz et al., 2003; Nambiar et al., 2003). The cells were maintained in cell culture for 2 days in vitro (2 DIV). During that time, cortical progenitors in the culture give rise to neurons of deeper and upper layer identity. Induction of Satb2 drives the expression of Cre recombinase and enables the concomitant expression of tdTomato exclusively in neurons fated to become upper layer type. Constitutive expression of EGFP in transfected cells provides a baseline level of transfection efficiency against which tdTomato fluorescence can be measured. Using flow cytometry, the proportion of tdTomato-fluorescing, Satb2-
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positive neurons can be quantified and relativized to the total number of transfected cells expressing both tdTomato and EGFP. In a parallel approach, neocortical progenitors were co-electroporated in utero with both plasmids of Satb2 Cre/+ embryos at different developmental stages. Hereafter, we refer to neurons from Satb2 Cre/+ mice that express tdTomato “Satb2 tdTom” and to both vectors, pCAG-GFP and the Cre-inducible, tdTomato-encoding plasmid pCAG-flox-stop-flox-tdTomato as “reporter constructs.
To test if tdTomato expression is a result of Cre-mediated stop cassette recombination (in other words, to test the threshold of tdTomato expression in Satb2 tdTom
neurons), we performed nucleofection of reporter constructs in E13.5 cortical
cells prepared from Satb2 +/+ (wild type) and Satb2 Cre/+ mice (Fig. 1B-1D). At 2DIV, we quantified Satb2 tdTom neurons using flow cytometry. Expression of tdTomato in wild type neurons (quartiles Q2 and Q4 of the dot plot, Fig. 1B) was marginal and constituted 5±0.2 % of transfected cells (found in quartiles Q1 and Q2, Fig 1B and 1D), as compared to robust tdTomato signals in Satb2 Cre/+ neurons (Fig 1C and 1D), which amounted to 49.2±0.9 % of transfected cells (Table S1). Therefore, we conclude that the quantified fraction of tdTomato-expressing, wild-type cells represents the level of Cre-independent background expression in our method.
At E12.5, the VZ is mainly composed of early neuronal progenitors able to generate both DL and UL neurons. As neurogenesis progresses, E13.5 progenitors become more restricted in their multipotency and shift to the generation of mainly UL neurons. Satb2 is a postmitotic determinant of upper layer neurons. To study the time course of Satb2 activation in cortical primary neurons using our method, we nucleofected cortical cells isolated from Satb2 Cre/+ mice at E12.5, E13.5 and E14.5 with our reporter constructs. At 2DIV, we observed 26.9±0.6 % Satb2 tdTom neurons isolated from E12.5, 45.5±3.3 % Satb2 tdTom cells from E13.5, and 67.3±1.3 % Satb2 tdTom from E14.5 (Fig. 1E, Table S2). Collectively, the increase in the ratio of Satb2 tdTom cells in the culture, quantified using our genetic reporters, mirrors previous observations of Satb2 expression during the progression of embryonic neurogenesis.
After 2 DIV, primary cortical cultures contain a mixture of progenitors and fatecommitted, post-mitotic neurons. To study activation of the Satb2 locus in vivo using our method, we electroporated both reporter constructs in neocortical progenitors at
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E12.5 in Satb2 +/+ and Satb2 Cre/+ embryos in utero. To more closely relate our in vitro observations with the situation in vivo, we counted Satb2 tdTom neurons two days after electroporation. At E14.5, some of the labeled cells remain in a progenitor state, while a fraction began differentiation into neurons (Fig. S2); a scenario that resembles culture conditions for data on Fig. 1E. We observed virtually no tdTomato expression in Satb2 +/+ mice 48 hours post-electroporation (Fig S2A). In Satb2 Cre/+ mice, of all EGFP-expressing cells, we quantified 34.4 ± 4.4% of Satb2 tdTom neurons (Fig. S2B, S2C, Table S3).
Dinaciclib treatment attenuates Satb2-expressing cells As an example application of our model in screening for genes involved in cell fate acquisition, we used the potent pan-cyclin-dependent kinase inhibitor dinaciclib (Johnson et al., 2012; Paruch et al., 2010), and quantified its effects on Satb2 expression in a cortical culture system. Dinaciclib exhibits anti-proliferative effects on multiple cell lines (Chen et al., 2016). Because of Satb2 expression confined to postmitotic neurons, we hypothesized that dinaciclib would halt the cell cycle progression of progenitors present in the culture. To establish a titer of the drug for our assay, we tested the effects of dinaciclib at different concentrations on the number of Satb2 tdTom neurons generated. We transfected cortical cells from E12.5 Satb2 Cre/+ mice with our reporter constructs using nucleofection and applied dinaciclib at final concentrations of 0.2 µM, 1 µM, or an equal volume of DMSO (vehicle solvent for dinaciclib) in the culture medium for 2DIV (Fig. 1F-I). DMSO had no effect on proportion of Satb2 tdTom cells as compared to untreated cultures, as quantified by flow cytometry (Fig. 1I) (Untreated, 32.9±0.1 %; DMSO, 35.3±2.3%; Table S4). Dinaciclib at both tested concentrations reduced the number of Satb2 tdTom cells as compared to DMSO (Fig. 1F-H) (0.2 µM, 3±0.1 %; 1 µM, 2±0.5 %, Table S4). This proof-of-principle experiment shows that our approach can be applied to study compounds that change Satb2 expression in cortical neurons by affecting gene expression or cell signaling.
Dinaciclib increases the number of Ctip2 expressing cortical neurons Cell fate acquisition during cortical neurogenesis is the effect of mutual molecular interplay between Satb2 and Ctip2. The transcription factor Ctip2 marks a subpopulation of DL neurons (layer V) and is critical for specification of cortical, subcerebral projection neurons (Chen et al., 2008; Molyneaux et al., 2005). To
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further characterize the effects of CDK inhibition on neuronal cell fate, we performed quantitative immunocytochemical analysis of Satb2 tdTom cells after dinaciclib treatment. Because of the similar effects of 0.2 and 1 µM dinaciclib on Satb2 tdTom neurons, possibly due to a plateau effect, in the next experiment we expanded the range of drug concentration. Cortical cells from E12.5 Satb2 Cre/+ mice were transfected with our reporter constructs, treated with DMSO or dinaciclib at 0.04, 0.2 or 1 µM dinaciclib and cultured for 2DIV followed by immunostaining for GFP and Ctip2 (Fig. 2A). In line with data obtained after cell sorting, dinaciclib at 0.2 µM and 1 µM significantly decreased the proportion of Satb2 tdTom cells as compared to DMSO treated cells (Fig 2A, 2B Table S5). Furthermore, dinaciclib at 0.2 µM significantly increased the fraction of Ctip2-expressing neurons (Fig. 2A, 2C Table S5, Fig. S4). Interestingly, 40 nM dinaciclib did not affect the number of neurons expressing either Satb2, or Ctip2, suggesting a dose-dependent effect (Fig. 2B and 2C, Table S5). Collectively, our method allows for screening of pharmaceuticals that influence cell fate acquisition. Satb2 tdTom neurons express Cre and Satb2 To corroborate that tdTomato expression is due to Cre-mediated excision of the stop cassette upstream of tdTomato reporter and that tdTomato expression mirrors Satb2 in cortical neurons, we first used an in vitro cortical culture system. We transfected cortical cells from E12.5 Satb2 Cre/+ mouse with the reporter constructs and immunostained primary cultures fixed at 2DIV for GFP, Cre and Satb2 (Fig. 3A; also compare Fig S3 for description of self-made anti-Satb2 antibody). Almost all Satb2 tdTom
cells expressed Cre and Satb2 (Satb2 tdTom and Cre-positive – 97.2±1.8 %;
Satb2 tdTom and Satb2-positive – 91.4±6.8 %) (Fig. 3B, Table S6). Therefore, in our culture system, tdTomato mirrors Cre and Satb2 expression in cortical neurons and can be used as a reliable reporter of Satb2 locus induction. Characterization of Satb2 tdTom neurons in vivo Next, we tested Satb2 tdTom neurons for expression of Cre and Satb2 in vivo. To validate if tdTomato fluorescence in Satb2 tdTom neurons mirrors Cre and Satb2 expression in neurons, we electroporated both reporter constructs in neocortical progenitors at E13.5 and fixed brains at E18.5, when embryonic neurogenesis in murine brains is complete. As observed in our previous in vivo experiments (Fig. S2),
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the expression of tdTomato in Satb2 +/+ neurons was marginal and Cre-independent (Fig. 4A). In Satb2 Cre/+ embryos, nearly all Satb2 tdTom neurons expressed both Cre and Satb2 (Fig. 4B-4D; Satb2 tdTom and Cre-positive – 94.6±4.7 %; Satb2 tdTom and Satb2positive – 95.9±5.3 %; Table S7).
In conclusion, our method provides a reliable readout of Satb2 locus activation and can be applied in the identification of genes or signaling pathways indispensable for neuronal fate specification during development in vivo.
Discussion
Genetically engineered reporter mice offer a precise and powerful tool for fate mapping and enable the identification of factors that determine cell identity. Using a Satb2 Cre/+ mouse line, we have developed an experimental approach to facilitate the discovery of molecules that play key roles in neuronal differentiation and acquisition of cell fate. In the Satb2 Cre/+ mouse, Cre recombinase is knocked-in to one locus of Satb2, a gene of crucial importance for determination of upper layer neurons and formation of the corpus callosum. Our method relies on transfection of a Creinducible tdTomato reporter construct that mirrors activation of the Satb2 locus and can be quantified by flow cytometry and immunostaining (Fig 1A). We addressed the specificity of our method using Satb2 +/+ mice both in an in vitro system (Fig. 1B) and using in utero electroporation of neocortical progenitors (Fig. 4A and S2A). Based on the distribution of tdTomato fluorescence as measured by flow cytometry, we set the threshold intensity of tdTomato that defines a neuron as Satb2 tdTom.
The observed 5% tdTomato-positive cells in Satb2 +/+ cultures may be the result
of the application of a strict intensity threshold. Additionally, in Satb2 +/+ cortices in vivo, we observe virtually no tdTomato-positive neurons two days post-IUE (Fig. S2A) and marginal (<1%) tdTomato-positive neurons five days post-IUE (Fig. 4A). These tdTomato-positive cells were Cre-negative; it is therefore likely that these cells represent Cre-independent rare artifacts. Moreover, a putative factor involved in neural fate acquisition is expected to engender drastic changes in Satb2-positive
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neuron count as quantified by flow cytometry, which combined with validation using immunocytochemistry and in vivo transfection of reporter constructs allows the reliable discrimination of factors that alter the fate of postmitotic neuron. Additionally, in Satb2 Cre/+ cells, tdTomato fluorescence signals were Cre-dependent and collocalized with Satb2 immunostaining signals (Fig. 3 and Fig. 4). It seems that detection of Cre in Satb2 tdTom cells reflects even slight activation of Satb2 locus due to efficient Cre-mediated activation of tdTomato expression (Meyers et al., 1998). Our approach offers a reliable method for labeling of cells with history of activating Satb2 locus and might be useful in experiments addressing transient versus stable Satb2 expression in neurons or studying the mutual relationship between Satb2 and Ctip2 in neurons.
Our method is based on in vitro transfection using nucleofection and in utero electroporation. Both methods allow for reliable labeling of only a population of cells, due to less than 100% efficiency of transfection. Therefore, in our analyses of method specificity (Fig. 3A, 4B and 4C), we only inspected tdTomato-fluorescing, Satb2 tdTom neurons for Cre- and Satb2- expression. In cortical culture, as well as in the embryonic cortex of Satb2 Cre/+ mice, Cre and Satb2 are additionally expressed cells that are not labelled following transfection with both reporter constructs (Fig. 3 and 4).
The relevance of our proposed method is its application in screening approaches. Here, as a proof of principle experiment, we applied dinaciclib to primary cortical culture prepared from Satb2 Cre/+ mice at E13.5 and quantified its effect on the proportion of Satb2 tdTom cells at 2DIV (Fig. 1F-I and Fig. 2). Dinaciclib, or SCH727965, is a pan inhibitor of cyclin-dependent kinases (CDKs). CDKs are critical for progression of cell cycle and drive the transitions between different stages of mitosis. Dinaciclib-mediated inhibition of CDKs abrogates progression of cell cycle (Chen et al., 2016; Rajput et al., 2016; Senderowicz, 2003). Indeed, in line with post-mitotic Satb2 expression, treatment with dinaciclib at 0.2 and 1 µM consistently led to a decreased proportion of Satb2 tdTom neurons (Fig. 1F-I and Fig. 2). In parallel, we observed increase in the fraction of Ctip2-positive cortical cells after 0.2 µM dinaciclib treatment (Fig 2B, C, E). Decrease in Satb2 tdTom after inhibitor treatment might reflect suppression of the post-mitotic fate of cortical progenitors. Finally, reduction in Satb2
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tdTom
and parallel increase in Ctip2 proportion might reflect disturbances in acquisition
of upper layer fate by developing neurons upon CDKs inhibition.
Our method can therefore be applied both in vitro and in vivo as a high-throughput screening of different compounds (sh-RNAs, CRISPR-Cas libraries, or drug libraries) that regulate genes critical for cell fate determination and act upstream of Satb2 during neuronal development. This mouse model could be particularly useful in in vitro assays where compounds are applied directly into the medium of cortical progenitor cultures isolated from Satb2 Cre/+ mice. Provided approved ethical regulations, small molecule inhibitors could also be injected into the lateral ventricles of developing E12.5 to E14.5 Satb2 Cre/+ embryos, followed by in utero electroporation of reporter plasmids. Moreover, including overexpression plasmids, sh-RNAs, morpholinos, and/or miRNAs in the DNA mixture for transfection in our model enables gene dosage analyses of putative regulators of cell fate acquisition acting upstream of Satb2.
During evolution, the mammalian neocortex has undergone a substantial increase in the proportion of UL neurons, suggesting a possible link between genes are that highly expressed in this subset of neurons and complex cognitive abilities (Edwards et al., 2014). To conclude, the above-presented method could be used for rapid identification of genes and signaling pathways that affect the birth and the function of specific neuronal pools and contribute to an understanding of neuronal development and evolution.
Experimental Procedures
Animal studies and ethical statement All experiments presented in the report were conducted in compliance with welfare guidelines issued by local authorities LaGeSo and Charité University Hospital. All possible efforts were undertaken to minimize suffering. Corresponding permits have been granted: G0079/11. For all experiments presented in this study, Satb2 Cre/+ males were mated to NMRI wild type females, maintained in the animal facility of Charité University Hospital in Berlin, Germany. The date of vaginal plug was counted as E0.5. All mice were sacrificed by administration of lethal dose of pentobarbital.
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Constructs For transfection of cortical primary cells by nucleofection or in utero electroporation of neocortical progenitors we used constructs under the control of β-actin promoter: pCAG-GFP and pCAG-flox-stop-flox-tdTomato. Promoter in both constructs permits for transcription of both reporters in all types of mammalian cells. However, the red fluorescence marker was downstream of stop cassette flanked by loxP sites. Upon Cre-mediated recombination, stop cassette is excised and permits tdTomato expression, specifically in Cre-expressing cells.
Primary cell culture and nucleofection Cortices from E12-E14 were isolated as illustrated on Fig. S1. The tissue was further enzymatically digested with 0.25% trypsin (Gibco) in HBSS solution (Gibco, with MgCl2 and CaCl2) for 30 minutes at 37°C. Next, the tissue was treated with DNaseI (Roche) for 5 minutes at 37°C and the enzymatic digestion was stopped by adding 2 mL of FBS (Gibco). Tissue was briefly centrifuged (1 minute, 1200 rpm) and washed twice with Neurobasal (Life Technologies). Afterwards, tissue was carefully triturated in medium (Neurobasal supplemented with GlutaMax, B27 and penicillin/streptomycin; Life Technologies) 10 times using P1000 pipette tip and then 10 times with P200 pipette tip. Cells were then counted with Neubauer counting chamber and nucleofected according to manufacturer’s protocols (Mouse Neuron Nucleofector Kit, VPG-1001, and Amaxa 2b Nucleofection system, program O-005, Lonza). We used 1 million cells for 1 µg of DNA (both reporter constructs: pCAGGFP, pCAG-flox-stop-flox-tdTomato, mixed at 1:1 ratio). Then, cells were seeded onto plastic or glass bottom 96-well plate, previously coated with 0.1 mg/mL poly-Llysine and 2% laminin (Sigma) at a concentration 100 000/well and maintained for 2DIV at 37°C and 5% carbon dioxide.
Cell treatment Primary cells were treated 2 hours post-seeding. Prior to treatment, dinaciclib (courtesy of David Kaplan) was diluted in cell culture grade DMSO (Sigma). Equal amounts of DMSO and diluted dinaciclib were added directly to the cell media to achieve final concentration of vehicle or drug at 0.04, 0.2 or 1 µM.
In utero electroporation
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Mouse embryos at determined embryonic stage were subjected to in utero electroporation accordingly to previously published work (Saito, 2006). Briefly, for in utero electroporation, pregnant mice were deeply anaesthetized with vaporized isoflurane/oxygen. To ensure post-operative analgesia, mice received subcutaneous injections of 3 mg buprenorphine (Temgesic). Additionally, for further analgesia and to prevent abdominal cramps, operated females were supplied with 0.05 mg/mL and 6 mg/mL metamizole in drinking water for 3 days after procedure. For electroporation of E12.5 cortical progenitors, 5 pulses of 32 V, each 50 ms long and with 950 ms interval were delivered with a forceps-type electrode. For E14.5 progenitors, 6 pulses of 34 V were applied.
Flow cytometry Prior to analysis, cells at 2DIV were washed with HBSS and incubated with 0.25% trypsin for 5 minutes at 37°C and triturated 10 times with P200 pipette tip. Flow cytometry of cortical cells were performed with BD FACS CantoTM II Flow Cytometer and plots were analyzed by FLowJo v9.8.3. Each measurement was performed using a population of 8000 to 10000 cells, characterized by physical properties of living particles defined based on forward and side scatter laser readouts. The gating thresholds for Alexa 488 and PE-A were set for samples nucleofected with pCAGGFP or pCAG-tdTomato only. Immunostaining and image acquisition For immunostainings, primary cells were maintained on glass-bottom 96 well plates (Cellvis) for 2DIV and fixed with 4% paraformaldehyde (PFA) for 20 minutes. For immunohistochemistry, P0 mouse brains were fixed with 4%PFA for 14 hours and subjected to 10-30% sucrose/PBS gradient, followed by cryosectioning (50 µm). Further fixed cell culture or brain sections were incubated with blocking solution (10% goat serum, 0.5% Triton X-100) for 1 hour in ambient temperature and with primary antibodies solution for 14 hours at 4°C. After washing with PBS, the specimens were incubated with fluorophore-labelled secondary antibody solutions for 4 hours at room temperature (Jackson Immunoresearch). In this study, the following primary antibodies were used at indicated dilutions: anti-GFP (goat, 1:500, Rockland, 600101215), anti-Ctip2 (rat, 1:500, Abcam, ab18465), anti-Cre (rabbit, 1:1000, 257 003, Synaptic Systems), anti-Satb2 (rabbit, 1:200, self-generated). Images of primary cell culture were acquired with the Spinning Disc Confocal microscope (Nikon CSU-
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X), or Leica TCS SP8 confocal, and the immunostained brain sections with Leica SP2 confocal microscope. The antibody against Satb2 (rabbit anti-Satb2) that was used for immunohistochemistry was used at concentration 1:200. The antibody was generated in the lab for the epitope QQSQPTKESSPPREEA. Statistics, Satb2 tdTom quantification and data analysis Statistical analyses of analyzed data were performed using Graph Pad Prism software and Microsoft Excel. All statistical tests and experimental values reported in this study are listed in supplementary tables. For comparison of differences within group means (in dinaciclib treatment experiments), we used one-way ANOVA with Bonferroni post-hoc correction. For determination of proportion of Satb2 tdTom cells using flow cytometry, we quantified the number of tdTomato expressing cells (quartiles Q2 and Q4 on the dot plots) and expressed it relative to the number of GFP expressing cells (quartiles Q1 and Q2). To quantify the of proportion of Satb2 tdTom cells using immunostainings, we quantified number of cells positive of respective staining after setting signal threshold using ImageJ software and ‘Analyze particles’ command (Fig. S4). To analyze the Satb2 tdTom proportion after in utero electroporation, we manually counted the number of neurons positive for respective marker or tdTomato signal with Cell Counter plug-in of ImageJ software. Author Contributions: M.A., P.B. and V.T. designed experiments, M.A., P.B., A.S.L. and V.S. conducted experiments and processed data. M.A. and P.B. wrote the manuscript. V.T. provided funding and critical comments on the project. Acknowledgements: We would like to acknowledge Advanced Medical Bioimaging AMBIO Core Facility at Charité University Hospital for granting microscope access and technical support with imaging of primary cell culture; Britta Eickholt for providing access to nucleofector, necessary for our experiments; David Kaplan for providing dinaciclib for our experiments. The Leica TCS SP8 was funded by BMW/DLR (grant 50WB1421), Center of Space Medicine Berlin. We thank Stephen Horan for helpful comments and editing of the manuscript. This study was supported by a grant from Russian Scientific Foundation (project n 15-14-10021) to V.T. All authors declare no conflict of interest regarding the publication of this report.
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Figure Legend Figure 1. Flow cytometry analysis of cortical cells isolated from Satb2 Cre/+ and wild type mice. (A) The workflow chart of using Satb2 Cre/+ as a model to study cell fate specification in cortical neurons. In Satb2 Cre/+ mouse, Cre is expressed from one Satb2 locus. Primary cortical neurons are prepared from embryonic Satb2 Cre/+ mouse brain and the transfected with reporter plasmids using nucleofection: one encoding EGFP and another one encoding Cre-inducible tdTomato. Upon treatment with compounds (e.g. small molecule inhibitors), or transfection with sh-RNA to knock-down gene of interest, the number of tdTomato-expressing Satb2-positive cells (Satb2 tdTom) is quantified by flow cytometry. (B and C) Example scatter plot of cortical cells after two days in culture isolated from E13.5 Satb2 +/+ (B), or Satb2 Cre/+ (C) mice and transfected with reporter plasmids. (D) Quantification of the proportion of Satb2 tdTom neurons to GFP-expressing neurons from the Satb2 +/+ (wild type) and Satb2 Cre/+ mouse. Results are represented as averages ±S.D. Wild type, Satb2 +/+, n=2 independent measurements from two transfections; Satb2 Cre/+, n=2 independent measurements from two transfections; ***p<0.001, unpaired t-test (Table S1). (E) Quantification of the proportion of Satb2 tdTom neurons prepared from Satb2 Cre/+ mice at E12.5, E13.5 and E14.5. Results are represented as averages ±S.D. Each result is average of at least two independent transfections; ***p<0.001, one-way ANOVA with Bonferroni post hoc test (Table S2). (F-H) Example scatter plots of Satb2 Cre/+ cortical cells nucleofected with reporter plasmids at E13.5 and treated with DMSO (F), 0.2 µM dinaciclib (G), or 1 µM dinaciclib for 2DIV. (I) Quantification of the proportion of Satb2tdTom cells after control treatment, 0.2 µM or 1 µM dinaciclib. Results are represented as averages ±S.D. Each result is mean of two independent measurements; ***p<0.001, one-way ANOVA with Bonferroni post hoc test (Table S4).
Figure 2. Dinaciclib attenuates Satb2 and induces Ctip2 expression in developing neurons. (A) Representative images of 2DIV cortical cells from E12.5 Satb2 Cre/+ mouse transfected with reporter plasmids and treated with DMSO, or dinaciclib at 0.04, 0.2 or 1 µM. Fixed neurons were immunostained for EGFP and Ctip2. Arrowheads point to Satb2 tdTom neurons; stars mark EGFP-positive, Ctip2positive neurons. (B-C) Quantification of Satb2 tdTom, or Ctip2-positive, GFP-positive
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neurons after DMSO or dinaciclib treatment. Results are represented as averages ±S.D. ***p<0.001, *p<0.05, one-way ANOVA with Bonferroni post-hoc test (Table S5). Figure 3. Primary Satb2 tdTom neurons are positive for Satb2 and for Cre recombinase. (A) Representative images of EGFP and Cre immunostaining and tdTomato fluorescence signals in Satb2 Cre/+ cells. Cells were isolated at E12.5, nucleofected with reporter plasmids and fixed at 2DIV. Arrowheads point to Satb2 tdTom
neurons; stars mark EGFP-positive neurons negative for tdTomato. (B)
Quantification of the proportion of tdTomato-positive cells expressing Cre, or Satb2. Results are represented as averages ±S.D. Each result is mean of two independent transfections (Table S6). Figure 4. Satb2 tdTom neurons are positive for Satb2 and for Cre in vivo. (A-B) Representative images of EGFP, Cre or Satb2 immunostaining and tdTomato fluorescence signals in wild type, Satb2 +/+ (A, upper panel), or Satb2 Cre/+ (B, middle and C, bottom panel) cortices of E18.5 mice. Neuronal progenitors were electroporated in utero at E13.5 with reporter constructs. Each image is a maximum projection of a Z-stack through 50 µm thick coronal brain section. Arrowheads point to some Satb2 tdTom neurons positive for Cre (B), or for Satb2 (C). (D) Quantification of Satb2 tdTom neurons positive for Cre, or Satb2. Results are represented as averages ±S.D. (Table S7).
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Figures
Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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