JÉSSICA ALVES DE MEDEIROS ARAÚJO
ROLES OF ZBTB20 IN THE SPECIFICATION OF UPPER LAYER NEURONS AND ASTROCYTES IN THE NEOCORTEX
NATAL, RN DECEMBER 2019
JÉSSICA ALVES DE MEDEIROS ARAÚJO
ROLES OF ZBTB20 IN THE SPECIFICATION OF UPPER LAYER NEURONS AND ASTROCYTES IN THE NEOCORTEX
Ph.D. COMMITTEE
Marcos Romualdo Costa (advisor) Ulrich Müller (co-advisor) Cecilia Hedin Pereira (FIOCRUZ-RJ)
João Lacerda de Menezes (UFRJ) Kerstin Erika Schmidt (UFRN) Emelie Katarina Svahn Leão (UFRN)
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE INSTITUTO DO CÉREBRO
PROGRAMA DE PÓS-GRADUAÇÃO EM NEUROCIÊNCIAS
NATAL, RN DECEMBER 2019
Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI
Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial Árvore do Conhecimento - Instituto do Cérebro - ICE
Araújo, Jéssica Alves de Medeiros.
Roles of ZBTB20 in the specification of upper layer neurons and astrocytes in the neocortex / Jéssica Alves de Medeiros Araújo. - Natal, 2019.
146f.: il.
Tese (Doutorado em Neurociências) - Universidade Federal do Rio Grande do Norte, Instituto do Cérebro, 2019.
Orientador: Marcos Romualdo Costa. Coorientador: Ulrich Müller.
1. ZBTB20. 2. Neocortex. 3. Gene expression regulation, developmental. 4. Neuron specification. 5. Astrogliogenesis. I. Costa, Marcos Romualdo. II. Müller, Ulrich. III. Título.
RN/UF/BSICe CDU 612.822
ACKNOWLEDGMENTS
This achievement would not be possible without the support of my family. Primeiramente, agradeço a minha família, pelo apoio, amor e dedicação; pelos inúmeros exemplos de perseverança, força e determinação. Essa e outras conquistas não seriam possíveis sem vocês ao meu lado. Mainha, painho, Jacquelinne, Janaynne, e Angela. Vocês são meu lar! Tia Gorda, obrigada pela amizade, generosidade e acolhimento.
I wish to express my deepest gratitude to Charlie. Thank you for your unconditional support and encouragement and for being there when needed most.
To all of my friends; Erin, Cindy, Desinha, Dyelle, Aninha, Soraia, Andre, Vivi, Pavão, Bryan, Ju, Dani, Bruna, Geissy, Annie, Day, Nathalie, Jeff, Romulo, Vanessa, Kharina and Diego, thank you all for easing my busy mind!
I would like to thank my mentor Marcos for your guidance throughout my scientific journey; during undergrad, master, and Ph.D. It has been a privilege to have worked with you for all these years. I will be eternally grateful for the opportunities you’ve given me. Thank you for your dedication, advice, training, freedom, trust, accountability, wisdom, patience, support, and criticism.
I wish to show my gratitude to all members of Marcos’ lab for being always present even when separated by thousands of kilometers.
To my co-supervisor Uli, thank you for the opportunity to work in such outstanding institutions, the Scripps Research Institute and Johns Hopkins University. Thank you for always challenging me, for investing in my scientific growth, and giving me the freedom to pursue my goals.
To Cristina, thank you for recognizing my potential and supporting me throughout this journey and for so freely giving your time, patience, friendship, and kindness.
I wish to express my gratitude to the current and former members of Uli’s lab for sharing insightful discussions and indispensable assistance during experiments.
Thank you to all the people whose assistance was fundamental in the completion of this project, especially; Ana Espinosa, Liyuan Wang, Diego Coelho, Yi-Ting Chang, Daniel O'Connor, Gabrielle Cannon, and Loyal Goff.
To the members of my committee, Cecilia Hedin, Tarciso Velho, João Menezes, Kerstin Schmidt and Kia Svahn Leão, thank you for sharing your expertise and wisdom.
To Alex Kolodkin and all members of his lab, particularly Joelle and Randal, thank you for the pleasant scientific discussions and shared plasmids.
To core facilities, all technicians and staff scientific, specifically Akaline and Michele, thank you for all the terrific technical assistance.
Finally, I would like to thank all members of The Brain Institute, Scripps Research Institute, and Johns Hopkins University. Thank you all for providing me with an ideal combination of creativity, knowledge, and resources during my joint Ph.D. in Brazil and United States.
RESUMO
A organização dos circuitos neocorticais é fundamental para a percepção sensorial, aprendizado e integração multisensorial. Na área somatossensorial primária (S1), os neurônios da camada IV recebem entradas talâmicas e projetam para neurônios da camada II/III. Esses neurônios superficiais podem conectar outros neurônios dentro de S1 e dentro de outras áreas do hemisfério ipsi ou contralateral, cooperando assim para selecionar uma interpretação consistente com suas várias entradas corticais e subcorticais. Neste trabalho, nós mostramos que a expressão do fator de transcrição Zinc Finger And BTB Domain-Containing Protein 20 (Zbtb20) em progenitores neocorticais é necessária e suficiente para regular a geração e a conectividade de neurônios supragranulares. A deleção condicional do gene Zbtb20 nos progenitores leva a um aumento no número e distribuição radial de neurônios RORβ+ (camada IV) à custa dos neurônios BRN2+ (camadas II/III). Essa mudança na organização laminar do neocórtex é acompanhada por uma expansão da arborização axonal talâmica e da área do barril em S1. Além disso, os neurônios da camada superior aumentam suas projeções axonais intra-hemisféricas, enquanto reduzem a inervação contralateral na ausência da expressão de Zbtb20. Essas alterações também são observadas, embora em menor grau, após a deleção do Zbtb20 nos neurônios pós-mitóticos, indicando que o Zbtb20 atua em estágios sequenciais da progressão da linhagem dos progenitores neocorticais, ajustando os destinos neuronais nas camadas corticais superiores e contribuindo para a organização das projeções axonais dos neurônios calosos (CPN - do inglês, “callosal projection neurons”). Além desses efeitos na especificação de CPNs, também mostramos que o ZBTB20 regula a astrogliogênese de maneira temporal específica. A superexpressão de ZBTB20 em E14, mas não em E16, aumenta a astrogliogênese neocortical, enquanto a expressão de um ZBTB20 negativo-dominante (DN) em E16, mas não em E14, reduz a astrogliogênese. Em conjunto, nossos resultados indicam que o ZBTB20 é um importante regulador da especificação de tipos e subtipo celulares no neocórtex em desenvolvimento.
Palavras-chave: ZBTB20; neocórtex; desenvolvimento; especificação neuronal;
ABSTRACT
Organization of neocortical circuits is critical for sensory perception, learning and multisensory integration. In the primary somatosensory area (S1) layer IV neurons receive thalamic inputs and synapse onto layer II/III neurons. These superficial neurons may connect other neurons within S1 and within other areas in the ipsi- or contralateral hemisphere, thus cooperating to select an interpretation consistent with their various cortical and subcortical inputs. Here we show that expression of the transcription factor Zinc Finger and BTB Domain-Containing Protein 20 (Zbtb20) in neocortical progenitors is necessary and sufficient to regulate the generation and wiring patterns of upper layer neurons. Conditional deletion of the Zbtb20 gene in progenitors leads to an increase in the number and radial occupancy of RORβ+/layer IV neurons at the expense of BRN2+/layers II/III neurons. This change in the laminar organization of the neocortex is accompanied by an expansion of thalamic axonal arborization and barrel area in S1. Furthermore, upper layer neurons increase their intra-hemispheric axonal projections, while reducing contralateral innervation in the absence of Zbtb20 expression. These alterations are also observed, albeit at a lesser extent, after Zbtb20 deletion in post-mitotic neurons, indicating that Zbtb20 act at sequential stages of the lineage progression of neocortical progenitors, fine-tuning neuronal fates in upper cortical layers and contributing to the proper wiring of callosal projecting neurons (CPNs). Besides these effects in CPN fate specification, we also show that ZBTB20 regulates astrogliogenesis in a time-specific fashion. ZBTB20 overexpression at E14, but not at E16, increases neocortical astrogliogenesis, whereas expression of a dominant-negative (DN) ZBTB20 at E16, but not E14, reduces astrogliogenesis. Altogether, our results indicate that ZBTB20 is an important regulator of cell type/subtype specification in the developing neocortex.
SUMMARY
1 INTRODUCTION ... 1
1.1 Cortical organization and cellular diversity ... 2
1.2 Development of neocortical architecture ... 5
1.2.1 Progenitor fate specification ... 9
1.2.2 Neuronal subtype specification ... 14
1.2.3 Astrocyte development ... 16
1.3 Zbtb20 function during cortical development ... 17
2 AIM ... 20
2.1 Specific aims ... 20
3 METHODS ... 21
3.1 Mouse lines and breeding ... 21
3.2 Mouse genotyping ... 23
3.3 Plasmid constructs ... 23
3.4 Cloning strategies and protocols ... 26
3.5 In utero electroporation ... 28
3.6 Immunohistochemistry ... 29
3.7 Cresyl Violet (Nissl) staining... 30
3.8 Flattened cortex and cytochrome oxidase ... 31
3.9 RNA extraction and qPCR... 31
3.10 Cell culture and Time-lapse Video-microscopy ... 33
3.11 S1 recordings ... 33
3.12 Image analysis and quantification ... 35
3.13 Statistical analysis ... 36
4.1 Screen for transcription factors involved in fate specification ... 37
4.2 ZBTB20 expression correlates with the generation of upper layer neurons and onset of astrogliogenesis ... 41
4.3 Zbtb20 function in astrogliogenesis ... 45
4.4 Zbtb20 function in neuronal specification and axon projection ... 61
4.4.1 Zbtb20 overexpression in neocortical progenitors/post-mitotic neurons at mid-neurogenesis leads to a premature generation of CPNs ... 61
4.4.2 Conditional genetic deletion of Zbtb20 in progenitors leads to laminar disorganization of the neocortex ... 64
4.4.3 Barrel expansion in the primary somatosensory cortex of Zbtb20cKO mice 70 4.4.4 Local field potentials (LFPs) in the barrel cortex of Zbtb20cKO mice ... 72
4.4.5 Interhemispheric connection of upper layer CPNs is compromised in Zbtb20cKO mice ... 75
4.4.6 Cell autonomous and cell extrinsic effect of Zbtb20 in the axonal arborization of upper layer CPNs ... 80
4.4.7 Conditional genetic deletion of Zbtb20 in intermediate progenitors and post-mitotic neurons impairs CPN differentiation ... 82
5 DISCUSSION ... 89
5.1 Summary of results ... 89
5.2 ZBTB20 expression in the neocortex ... 90
5.3 Role of Zbtb20 in gliogenesis ... 90
5.4 Role of Zbtb20 in neurogenesis ... 92
5.5 Role of Zbtb20 in progenitors versus intermediate progenitors and neurons ... 93
5.6 Role of Zbtb20 in the formation of callosal projections and neuronal circuit ... 94
6 CONCLUSION ... 98
LIST OF SCIENTIFIC PAPERS ... 121
LIST OF ABBREVIATIONS ... 122
LIST OF FIGURES ... 125
LIST OF TABLES ... 129
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1 INTRODUCTION
The mammalian neocortex exhibits an amazing cellular diversity, containing numerous cell types that are precisely interconnected and assembled into neural circuits. The neocortex is a region of the forebrain unique to mammals, that is involved in higher functions such as control of motor commands, sensory perception, cognition, and consciousness. The application of high throughput methods to study single-cells has recently begun to unravel the unique molecular signatures associated with the diversity of morphologically and electrophysiologically defined neuronal subtypes, as well as microglial cell heterogeneity (Bayraktar et al. 2018; Marques et al. 2016; Zeisel et al. 2018; Zeisel et al. 2015). Understanding how this cellular diversity and such complexity arise during the development of the neocortex is essential for neuroscience. New studies addressing this question hold promise of identifying critical points in the formation of neocortical architecture that may be involved in the pathogenesis of several neurological and psychiatric disorders.
The generation of cellular diversity in the neocortex requires complex sequential processes, precisely orchestrated by genetic and environmental influences (Miller and Gauthier 2007). Progress has been made during the last two decades in characterizing some of the mechanisms involved in these diversification processes. At early developmental stages, multipotent proliferative radial glial cells residing at the ventricular surface, give rise to intermediate progenitors and, ultimately, post-mitotic glutamatergic neurons that settle in the neocortex following an inside-out pattern (Molyneaux et al. 2007), and GABAergic neurons that disperse widely across the neocortex (Harwell et al. 2015). At the end of embryonic development, the radial glial cells begin to generate macroglial cells (astrocytes, oligodendrocytes and ependymal cells), and the peak of this process takes place during the first 3 weeks after birth in mice (Ge et al. 2012; Kessaris et al. 2006; Kriegstein and Alvarez-Buylla 2009; Spassky et al. 2005).
The study included in this thesis aims to expand our understanding of progenitor diversity and molecular pathways involved in the specification of progenitor cells to generate cellular diversity in the neocortex hoping to provide a stronger foundation for future research on cortical development, cellular differentiation and their implications to
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brain dysfunction. In the present study, using the mouse brain as a model, we identified a genetic program involved in the specification of dorsal progenitor cells to generate a subset of upper layer glutamatergic neurons and astroglial cells in the developing neocortex, and we investigated the direct effect of genetic perturbations of a transcription factor on neural circuit formation within the somatosensory cortex.
1.1 Cortical organization and cellular diversity
The cerebral cortex can be divided into 3 phylogenetically-defined regions: paleocortex, archicortex, and neocortex. The paleocortex is located in the ventrolateral part of the telencephalon and comprising the olfactory piriform cortex. The archicortex includes hippocampus, entorhinal cortex, retrosplenial and subiculum. The paleocortex and archicortex together constitute the allocortex, which consists of three cortical laminae. The neocortex represents the great majority of the cerebral cortex and it is positioned between the two other regions. This region is characterized by six layers, radially organized, which themselves are often subdivided, each containing a heterogeneous population of cells. In its tangential dimension, the neocortex comprises numerous domains, each serving a specific function. These functionally unique subdivisions are distinguished from one another by differences in patterns of gene expression, cellular architecture, and neuronal projections. These properties determine the functional specializations that characterize and distinguish areas in the adult cortex. This study mainly discusses the development of the mammalian neocortex and its connectivity, with special attention to the mouse somatosensory cortex.
The mammalian neocortex is composed of numerous types of cells, including neurons and glial cells that together create complex histological structures. There are two major distinct classes of cortical neurons: excitatory and inhibitory neurons. Most inhibitory neurons extend axons within the cortex making local connections and use γ-aminobutyric acid (GABA) as neurotransmitter. However, a small fraction of GABAergic neurons in the neocortex are long-projecting neurons (Melzer et al. 2017; Tamamaki and Tomioka 2010). Inhibitory interneurons constitute an extremely diverse population of cells comprising 15-20% of all cortical neurons, with very diverse morphologies, connectivity,
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biochemistry, and physiological properties (Lim et al. 2018). These cells are dispersed across all cortical layers (Brandão and Romcy-Pereira 2015).
Excitatory neurons mostly use the neurotransmitter glutamate and account for approximately 75-80% of all neurons in the neocortex, including both pyramidal neurons and spiny stellate neurons, which can be further divided into distinct cell types according to laminar, morphological, electrophysiological properties and patterns of gene expression (Costa and Müller 2015). They can make local connections or extend axons to different cortical areas or to subcortical and subcerebral structures (Douglas and Martin 2004; Molyneaux et al. 2007).
Excitatory neurons can be subdivided into three broad classes that display layer- and subtype-specific differences in the morphology, gene expression and axonal targets: (1) the corticothalamic neurons (CTNs), which are predominantly located within layer VI and project axons to the thalamus; (2) the corticospinal motorneurons (CSMN) that are located within layer V and extend axons toward the brainstem and spinal cord; and (3) the most heterogeneous class, the cortico-cortical and callosal projection neurons (CPNs) that are present in layers II–VI, and extend axons toward targets in the contralateral and ipsilateral cortex, striatum, nucleus accumbens, septum and amygdala subnuclei (Kast and Levitt 2019).
Excitatory neurons in the layer IV of the sensorial cortices receive the predominant synaptic input from the thalamus and are mostly connected to nearby neurons forming local circuits (Douglas and Martin 2004). Layer IV neurons can be subdivided further into spiny stellate cells and star pyramidal cells, depending on whether they have an apical dendrite (Zeng and Sanes 2017). Moreover, layer IV neurons develop callosal projections during development that are subsequently eliminated (De León Reyes et al. 2019). On the other hand, cortico‑cortical and callosal projection neurons are mostly concentrated in layer II/III but are also present in deeper layers V and VI. They project to multiple other cortical areas both ipsilaterally and contralaterally, with collateral connections to the striatum (Douglas and Martin 2004; Molyneaux et al. 2007). Thus, it is clear that a diverse population of neocortical projection neurons compose the neocortex and these classes can be further subdivided (Zeng and Sanes 2017).
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Neuronal diversity has been described for more than 100 years, and it is now emerging that glia also exhibits important functional diversity. Actually, Ramón y Cajal and his colleagues extensively documented the morphological diversity of astrocytes and neurons in the human brain (Figure 1), suggesting the presence of cellular diversity. However, for more than a century, the field of astrocyte diversity was largely forgotten (Khakh and Deneen 2019). The diverse and dynamic functions of glial cells orchestrate essentially all aspects of nervous system formation and function: influencing nervous system development, neuronal migration, axon growth and specification, synaptogenesis, neural circuit maturation, synaptic plasticity and homeostasis (Allen and Lyons 2018; Schitine et al. 2015). The main types of glia include astrocytes, oligodendrocytes, ependymal cells, radial glia, and microglia.
Astrocytes are the most numerous glial cell type in the central nervous system and play several key roles on neuronal activity and as neural stem cells in adult neurogenic zones (Wang and Bordey 2008). In the neocortex, most astrocytes are highly ramified with very fine processes that ensheath synapses, blood vessels, and other cells. They are classically divided into two main classes distinguished by morphology, molecular phenotype, and location. Protoplasmic astrocytes are found in gray matter and show many branching processes, which contact and ensheath synapses, and usually have one or two processes in contact with blood vessels. Fibrillary or fibrous astrocytes are found in white matter and they have a star-like appearance, with long and thin processes. Currently, a few markers are used routinely to identify astrocytes, including glial fibrillary acidic protein (GFAP), calcium-binding protein S100β, glutamate–aspartate transporter and glutamate transporter 1 (GLT-1) (Rothstein et al. 1994), and, more recently, aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (Cahoy et al. 2008) and the transcription factor Sox9 (Sun et al. 2017). Astrocyte morphology and gene expression vary substantially among cortical regions (Bayraktar et al. 2018; Emsley and Macklis 2006; Regan et al. 2007), suggesting that astrocyte subpopulations could be specified to exhibit distinct physiologic properties (Chen et al. 2018; Emsley and Macklis 2006; Lin et al. 2017).
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Figure 1 Representation of the morphological diversity of glia cells and neurons in the human cerebral cortex documented by Santiago Ramón y Cajal (1852–1934). Pictures were adapted from Khakh and Deneen, 2019 and Brian Hayes 2018.
1.2 Development of neocortical architecture
During development, multiple progenitor zones contribute to the cellular diversity found in the neocortex. Excitatory neurons and astrocytes are both generated from progenitors of the germinal zone located in the dorsolateral wall of the telencephalon. Inhibitory neurons and Cajal-Retzius cells are generated from progenitors in the ventral telencephalon (Anderson et al. 1997; Miyoshi et al. 2007, 2010) and at multiple locations adjacent to the cortex (Hoerder-Suabedissen and Molnár 2013; Price et al. 1997; Takiguchi-Hayashi et al. 2004), respectively, and migrate tangentially to populate the cortex (Figure 2). Additionally, transient glutamatergic neurons composed of the subplate cells are born in the rostral medial telencephalic wall, ventricular zone (VZ) and subventricular zone (SVZ) (Hoerder-Suabedissen and Molnár 2015) (Figure 2). Proper laminar positioning and development of the mature cortical area map reflect interactions between intrinsic and extrinsic biological mechanisms that together coordinate the neuronal migration from their birthplace to a final location, where they assemble into functional circuits (Cadwell et al. 2019; Kast and Levitt 2019).
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Figure 2 Multiple progenitor zones contribute to the cellular diversity found in the neocortex. A) Scheme of mouse brain at E11, showing several sources of Cajal–Retzius cells (septum, pallial-subpallial boundary and CMTW - caudo-medial telencephalic wall) and subplate (SP) neurons (RMTW - rostro-medial telencephalic wall). B) Illustration of a rostral coronal section, showing origin and generation of SP (VZ and RMTW) and Cajal–Retzius cells (septum and RMTW). C) Illustration of a caudal coronal section showing generation of SP at the SVZ, Cajal–Retzius at the pallial–subpallial boundary, and interneurons in the ventral telencephalon (medial and caudal ganglionic eminence, MGE and CGE). Image from Hoerder-Suabedissen and Molnár et al. 2015.
Studies have addressed two conflicting theories to explain the development of neocortical area map (O’Leary 1989; Rakic 1988). The protomap hypothesis theorizes that cortical progenitor cells are intrinsically specified early in development to give rise to a certain area, and their radially organized neural progeny inherit this spatial information (Bishop, Goudreau, and O’Leary 2000; Rakic 1988; Rubenstein and Rakic 1999). In contrast, the protocortex hypothesis suggests that the cortex is initially homogeneous corresponding to a tabula rasa, originated by equipotent progenitors. Distinct areas are formed in response to extrinsic signals, including incoming thalamocortical axons (Van Der Loos and Woolsey 1973; O’Leary 1989). Currently, there is evidence available supporting both models, and they will be discussed in the following sections.
The early formation of cortical areas is best described by the protomap hypothesis. Data from independent studies demonstrated that normal patterns of cortical region-specific gene expression is stablished in spite of disturbed or absent thalamocortical innervation (Garel, Huffman, and Rubenstein 2003; Miyashita-Lin et al. 1999; Nakagawa,
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Johnson, and O’Leary 1999), and suggested that the process of cortical area formation must depend on patterning mechanisms that operate within the telencephalon.
The size and positioning of cortical areas are controlled by morphogens secreted from the patterning centers at the rostral and caudal ends of the cortical primordium. Expression of fibroblast growth factor 8 (FGF8) in the commissural plate at the rostromedial end of the telencephalon controls rostral neocortical identity (Fukuchi-Shimogori and Grove 2001). Over-expression of FGF8 causes expansion of rostral cortical areas (motor cortex), reduction and a posterior shift of caudal regions (somatosensory and visual cortex), conversely inhibition of Fgf8 signaling causes the opposite effect (Fukuchi-Shimogori and Grove 2001). Additionally, ectopic expression of FGF8 at the caudal end of the cortex elicits duplication of somatosensory barrels (Assimacopoulos et al. 2012; Fukuchi-Shimogori and Grove 2001). At the anterior end several FGF family members including FGF3, 17, and 18 overlap in expression and have been demonstrated to play complementary, yet distinct roles in patterning the cortex (Bachler and Neubüser 2001; Cholfin and Rubenstein 2007, 2008). Furthermore, multiple signaling molecules including Bone Morphogenetic Proteins (BMPs), WNT and their antagonist proteins are secreted from the cortical hem, another key patterning center that extends caudally along the midline of the cortical primordium (Bachiller et al. 2000; Grove et al. 1998; Hébert, Mishina, and McConnell 2002; Kiecker and Niehrs 2001; Nordström, Jessell, and Edlund 2002) (Figure 3).
Figure 3 Morphogens spatially pattern the mouse telencephalon. Illustration of progenitors patterned by the morphogens Wnt and BMPs from the dorsal hem and ventrally patterned by Shh. FGFs are secreted from a rostral signaling center. Lateral and medial ganglionic eminences (LGE and MGE) generate GABAergic interneurons. R, rostral; C, caudal; D, dorsal; V, ventral. Image from Holguera and Desplan 2018.
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These signaling centers subsequently induce the expression of transcription factor gradients, which in turn establish the anterior-posterior and mediolateral axes within the ventricular zone (VZ) of the cortex, prior to the arrival of thalamic afferents. Chicken ovalbumin upstream promoter transcription factor 1 (Couptf1) and trans-acting transcription factor 8 (Sp8) are expressed in reciprocal rostromedial-to-caudolateral gradients (Liu, Dwyer, and O’Leary 2000; Sahara et al. 2007; Waclaw et al. 2006; Zhou, Tsai, and Tsai 2001), while empty spiracles homeobox 2 (Emx2) and paired box gene 6 (Pax6) are expressed in the VZ in reciprocal rostrolateral-to-caudomedial gradients (Gulisano 1996; Walther and Gruss 1991) (Figure 4). Manipulation of these transcription factor gradients results in dramatic changes in sizes and positions of cortical areas (Armentano et al. 2007; Bishop, Goudreau, and O’Leary 2000; Bishop, Rubenstein, and O’Leary 2002; Hamasaki et al. 2004; Mallamaci et al. 2000; Zhou, Tsai, and Tsai 2001). Nevertheless, lamination and connectivity of cortical areas appear to develop normally (Armentano et al. 2007; Bishop, Goudreau, and O’Leary 2000; Cholfin and Rubenstein 2007; Grove and Fukuchi-Shimogori 2003), suggesting that the mechanisms patterning the cortex also establish guidance cues necessary for cortical areas to connect with appropriate thalamic nuclei (Leingärtner et al. 2003; Shimogori and Grove 2005).
Figure 4 Transcription factors establish an area identity map. A) Morphogens and signaling molecules induce expression of transcription factor gradients in the VZ, in particular Pax6, Emx2, Sp8 and Couptf1. B) Gradients of expression are shown in schematized wholemount (top) and sagittal (bottom) views. Pax6 and Emx2 are highly expressed rostrolaterally and caudomedially, respectively. Sp8 and Couptf1 are highly expressed rostromedially and caudolaterally, respectively. Image from Greig and Woodworth et al 2013.
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In addition to early patterning of the telencephalon, there is a large body of literature demonstrating the roles of thalamocortical innervation and activity-dependent mechanisms influencing the refinement and the formation of distinct functional areas of the neocortex, providing support for the protocortex hypothesis. One example is the existence of spontaneous thalamic calcium waves that propagate among sensory-modality thalamic nuclei up to the cortex prior to sensory information processing, and that these waves influence the size of specific cortical fields (Moreno-Juan et al. 2017). Another instance is signaling via early-born neurons, the Cajal-Retzius cells. The migration and distribution of Cajal-Retzius cells influence the size and connectivity of higher-order cortical areas in the postnatal cerebral cortex (Barber et al. 2015) and of the subplate neurons controlling radial migration of excitatory neurons (Ohtaka-Maruyama et al. 2018). Bilateral enucleation early in development in primates induces a reduction in the size of primary visual cortical areas and response to alternative sensory modalities, while adjacent cortical areas develop novel cytoarchitecture (Dehay et al. 1996; Dehay et al. 1989; Rakic 1981; Rakic 1988; Rakic, Suñer, and Williams 1991). Recent ablation studies have demonstrated that sensory thalamic input is essential to establish the genetic and functional distinctions between primary and higher-order cortical areas (Chou et al. 2013; Pouchelon et al. 2014).
1.2.1 Progenitor fate specification
During early development, neuroepithelial cells form the telencephalic wall. These cells undergo symmetric cell divisions and expand in number, as well as differentiate into radial glia, creating the ventricular zone (VZ) (Götz and Huttner 2005). Radial glia cells generate outer radial glia and intermediate progenitors, which form the subventricular zone (SVZ) above the VZ (Beattie and Hippenmeyer 2017). Neocortical progenitors in the VZ and SVZ begin to produce excitatory projection neurons around embryonic day 10.5 (E10.5) (Angevine and Sidman 1961). The earliest-born neurons migrate away from the ventricular surface and form the preplate (Marin-Padilla 1978; Raedler and Raedler 1978). Cajal-Retzius and subplate cells are the first neurons to be generated and to migrate to the cortical plate (Angevine and Sidman 1961; Hevner et al. 2003; Meyer et al. 1998; Price et al. 1997). Newly born neurons migrate into the preplate, splitting it into the
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marginal zone and subplate, and creating the cortical plate (Luskin and Shatz 1985; Menezes and Luskin 1994). The cortical plate begins to develop in between these two layers, and projection neurons of the different neocortical layers are generated in a closely controlled temporal order from E11.5 to E19.5 (Angevine and Sidman 1961; Caviness 1982; Caviness and Sidman 1973; Takahashi 1995). As neurogenesis proceeds, later born neurons (layer IV, then layer II/III) arriving at the cortical plate migrate past earlier born neurons (first layer VI, then layer V) in an ‘inside-out’ fashion (Figure 5) (Angevine and Sidman 1961).
Figure 5 Neocortical projection neurons are generated in an ‘inside-out’ fashion. The earliest born neurons form the preplate (PP). Newly born neurons migrate into the preplate, splitting it into the marginal zone (MZ) and subplate (SP), and creating the cortical plate (CP). Later born neurons arriving at the cortical plate migrate past earlier born neurons. Different projection neuron subtypes are born in overlapping temporal waves. CH, cortical hem; Ncx, neocortex; IZ, intermediate zone; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; SVZ, subventricular zone; VZ, ventricular zone; WM, white matter. Image from Molyneaux and Arlotta et al., 2007.
Neocortical VZ and SVZ progenitor cells have distinct morphologies, express different genes and follows a specific pattern of cell division. Radial glia cells retain the
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cell body positioned along the lateral ventricles, extent a long process to the ventricular (apical) surface and a second process to the pial (basal) surface (Figure 6). Radial glia cells are used as a scaffold by some newly born neurons as they migrate into the cortical plate (Rakic 1972). They mainly divide asymmetrically to self-renew, while also giving rise to outer radial glia, intermediate progenitors or neurons (Miyata et al. 2001; Noctor et al. 2001). Outer radial glia cells lack an apical process and undergo asymmetrical divisions to self-renew and generate neurons (Figure 6). Although they were first characterized in the outer SVZ of the developing human cortex (Hansen et al. 2010) and were thought to be present only in gyrencephalic animals (Fietz et al. 2010), they also exist in a small population in the SVZ of rodents (Martínez-Cerdeño et al. 2012; Wang et al. 2011). Intermediate progenitors have a multipolar morphology and are not anchored to either the apical or basal cortical surface (Figure 6). They undergo limited proliferative divisions and divide symmetrically to produce either two new intermediate progenitors or two post-mitotic neurons (Kowalczyk et al. 2009; Miyata et al. 2004; Sessa et al. 2008). Neocortical projection neurons subtypes are sequentially generated by this diverse progenitor types in the VZ and SVZ and migrate radially to establish the laminar structure of the neocortex (Greig et al. 2013; Molyneaux et al. 2007).
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Figure 6 Sequential generation of neocortical projection neuron subtypes. A) Illustration of radial glia (RG) in the ventricular zone (VZ) producing neurons, intermediate progenitors (IPs) and outer RG (oRG). IPs and oRG establish the subventricular zone (SVZ). Cajal–Retzius (CR) cells are generated at multiple locations adjacent to the cortex and migrate tangentially into layer I. Other projection neurons are born in the VZ and SVZ and migrate along radial glial processes to reach their final laminar position. B) Different projection neuron subtypes are born in sequential waves, in the following order: subplate neurons (SPN), corticothalamic projection neurons (CThPN), subcerebral projection neurons (SCPN), Layer IV granular neurons (GN), callosal projection neurons (CPN). NE, neuroepithelial cell. Image from Greig and Woodworth et al 2013.
Notably, several lines of evidences support the model that distinct subtypes of excitatory cortical neurons are generated in sequential order: 1) isolated progenitor cells in vitro recapitulate the normal order of neuronal subtype generation, even in a highly simplified environment (Shen et al. 2006), indicating the existence of a cell intrinsic control program. Changes in fate potential over time is also influenced by cell cycle length and the number of divisions undergone by progenitor cells before terminal differentiation (Calegari et al. 2005; Calegari and Huttner 2003; Pilaz et al. 2009). Genetic programs
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regulating proliferation, differentiation, and survival of neural progenitor cells are thus essential aspects of progenitor competence; and (2) ventricular zone progenitors become more hyperpolarized as they generate successive subtypes of neurons in vivo, and manipulations of the membrane potential of these progenitors shift the transcriptional programs to a later developmental state, leading to changes in neuronal subtype identity (Vitali et al. 2018), providing a potential mechanism through which environmental signals might play a role in timing of cortical neurogenesis. In line with this interpretation, there is also evidence indicating that early-born neurons send feedback signals to progenitors controlling the switch to the generation of late-born neurons (Landeira 2017; Parthasarathy et al. 2014; Seuntjens et al. 2009; Toma et al. 2014).
An intrinsic control for the sequential generation of neocortical neurons is also supported by the existence of TFs affecting this process. For example, the transcriptional repressor Forkhead Box transcription factor G1 (Foxg1, formerly known as brain-factor BF-1), regulates neurogenesis in the embryonic telencephalon as well as a number of other neurodevelopmental processes (Kumamoto and Hanashima 2017). Knock-out of Foxg1 leads to severe microcephaly, reduction of the dorsal telencephalic areas and complete loss of ventral telencephalic structures (Xuan et al. 1995). Conditional Foxg1 deletion in neocortical progenitors leads to an excess generation of Cajal-Retzius and decreased generation of later-born neurons (Hanashima et al. 2004). The depletion of the neural progenitor population via premature cell cycle exit and neuronal differentiation cause these abnormalities in Foxg1 mutants (Fasano et al. 2009; Manuel et al. 2011; Martynoga et al. 2005). COUP-TFI and II also play a role in the sequential generation of neuronal subtypes during neocortex development. Double knock-down of Coup-tf I/II in embryonic stem cell-derived neurospheres caused sustained neurogenesis and the prolonged generation of early-born neurons (Naka et al. 2008).
It is noteworthy that the expression and, therefore, functions of both Foxg1 and Coup-tf I/II are under influence of environmental signals, such as cytokines (Naka et al. 2008; Toma et al. 2014). Therefore, an interplay between intrinsic and extrinsic signals might be the most parsimonious explanation to the sequential generation of neuronal subtypes in the developing neocortex. This collaborative mechanism may also help to understand the early plasticity observed at early developmental stages, both at the
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progenitor levels (Landeira 2017; Naka et al. 2008; Toma et al. 2014) and in post-mitotic neurons (Díaz-Alonso et al. 2012; De La Rossa et al. 2013; Rouaux and Arlotta 2013).
1.2.2 Neuronal subtype specification
Upon arrival at the cortical plate, projection neurons undergo further differentiation, showing distinct gene expression profile and axonal projection, that strongly correlates with the laminar position (Harris and Shepherd 2015; Lodato and Arlotta 2015; Molyneaux et al. 2015). The molecular mechanisms governing the acquisition of specific neuronal subtype fates in the neocortex have begun to be revealed over the past two decades. Several studies have contributed to the identification of laminar- and subtype-specific gene expression patterns in the neocortex (Paola Arlotta et al. 2005; Gray et al. 2004; Lein et al. 2007; Magdaleno et al. 2006; Visel 2004). Many of these transcription factors are expressed by early post-mitotic neurons migrating away from the germinal zone and some in progenitor cells (see details bellow), and reciprocal regulation among these genes progressively refines neuronal subtype identity (Figure 7) (Greig et al. 2013; Srinivasan et al. 2012).
Figure 7 Molecular programs direct subtype identity of postmitotic projection neurons. B) Key regulators have been identified to be part of a complex transcriptional network (top). Known interactions are indicated by arrows (bottom). C) Changes in expression of these molecular programs cause a shift in subtype identity, obtaining features of other neuronal classes. The boundaries between corticofugal projection neurons and callosal projection neurons might shift independently of one another (dashed lines). SCPN, subcerebral projection neurons. CThPN, corticothalamic projection neurons. CFuPN, corticofugal projection neurons. CPN, callosal projection neurons. Image from Greig and Woodworth et al 2013.
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Forebrain Embryonic Zinc Finger-Like Protein 2 (Fezf2) is required for the specification of layer V corticospinal motor neurons (CSMNs) and other subcerebral projection neurons, which are absent from Fezf2 null mutant mice neocortex (Molyneaux et al. 2005). Overexpression of Fezf2 by electroporation at E13.5 results in excess production of subcerebral projection neurons that settle ectopically in the germinal zone (Molyneaux et al. 2005). Fezf2 expression in embryonic and early postnatal callosal projection neurons of layer II/III is sufficient to lineage reprogram these cells into layer V/VI corticofugal projection neurons (Rouaux and Arlotta 2013). Although it is not known whether FEZF2 acts in progenitors or postmitotically, Fezf2 mRNA is expressed by a subset of progenitors in the VZ and SVZ and also by postmitotic neurons (Guo et al. 2013).
Another example of a transcription factor important for lineage specification is BAF Chromatin Remodeling Complex Subunit BCL11B, also known as CTIP2. CITP2 acts downstream of FEZF2 (Chen et al. 2008) and is also necessary and sufficient for the specification of CSMNs (Arlotta et al. 2008; Chen et al. 2008). On the other hand, the Special AT-Rich Sequence-Binding Protein 2 (SATB2) seems to function as a suppressor of CTIP2, regulating the acquisition of upper-layer fates including layer IV/RORβ+ and layers II/III Cux1+ neurons (Alcamo et al. 2008).
T-Box Brain Transcription Factor 1 (Tbr1) promotes the identity of corticothalamic neurons while repressing subcerebral fates through reducing expression of Fezf2 and CTIP2 (Mckenna et al. 2011). Similarly, the SRY-Box Transcription Factor 5 (SOX5) is believed to control the sequential generation of distinct corticofugal neuron subtypes (Lai et al. 2008), likely through activation of Tbr1 expression and repression of Fezf2 expression (Greig et al. 2013). Genetic deletion of SOX5 in the developing neocortex impairs differentiation of layer VI corticothalamic neurons, while differentiation of layer V/VI subcerebral projection neurons is accelerated (Lai et al. 2008).
Other noteworthy transcription factors that were not included in this representation of molecular programs controlling neuronal specification (Figure 7) are POU class 3 homeobox 2 (Pou3f2, also known as Brn2) and POU class 3 homeobox 3 (Pou3f3, also called Brn1). These transcription factors have been described to be expressed both post-mitotically and in progenitor cells, governing neurogenesis and subtype identity
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(Dominguez, Ayoub, and Rakic 2013; McEvilly et al. 2002; Sugitani et al. 2002). Brn2 and Brn1 play overlapping roles in the regulation of upper-layer neuronal migration and specification. Absence of Brn1/Brn2 genes in mice led to a dramatic reduction in the number of upper layer neurons and defective neuronal migration (McEvilly et al. 2002; Sugitani et al. 2002). Conversely, overexpression of either Brn1 or Brn2 in early neural progenitors was sufficient to induce the precocious generation of Satb2+ upper-layer neurons, possibly through regulation of neurogenesis by suppressing Notch effector Hes5 and promoting the expression of proneural transcription factors Ngn2 and Tbr1 (Dominguez, Ayoub, and Rakic 2013).
As we will discuss in more detail in a separate chapter, the Zinc Finger and BTB Domain-Containing Protein 20 (ZBTB20) is also expressed by progenitors and post-mitotic neurons and has been associated with the temporal control of neuronal subtype generation and astrogliogenesis in the developing neocortex (Doeppner et al. 2019; Nagao et al. 2016; Tonchev et al. 2016).
1.2.3 Astrocyte development
After neurogenesis, neural progenitors in the dorsal telencephalon progressively transition to a gliogenic fate, generating astrocytes (Gorski et al. 2002; Qian et al. 2000). Glial restricted progenitors start to appear around E15.5 (Costa et al. 2009). Intrinsic and extrinsic mechanisms have been proposed to orchestrate the switch from neuron production to astrogenesis (Miller and Gauthier 2007; Rowitch and Kriegstein 2010), including epigenetic regulation (Hirabayashi and Gotoh 2010; Song and Ghosh 2004; Takizawa et al. 2001); NOTCH signaling (Ge et al. 2002; Grandbarbe et al. 2003); JAK-STAT pathway (Kamakura et al. 2004); HES proteins through inhibition of neurogenic bHLH factors (Kamakura et al. 2004; Namihira et al. 2009; Nieto et al. 2001; Tomita et al. 2000); BMPs activate Smad transcription factors (Gross et al. 1996; Mabie, Mehler, and Kessler 1999; Nakashima et al. 2001); cytokines secreted by neurons – particularly members of the interleukin 6 (IL-6) family which activate JAK-STAT pathway, such as leukaemia inhibitory factor (LIF), CNTF and cardiotrophin 1 (CT1 or CTF1) (Barnabé-Heider et al. 2005; Bonni et al. 1997; Koblar et al. 1998; Nakashima et al. 1999; Ochiai et al. 2001); TGFβ-signaling induces differentiation of radial glia into astrocytes (Stipursky
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et al. 2014; Stipursky, Francis, and Gomes 2012); SHP2-MEK-ERK-Rsk pathway and Neureglin-1–ErbB2/ErbB4 pathway are involved in the timing of glial differentiation (Gauthier et al. 2007; Sardi et al. 2006; Schmid et al. 2003).
Astrocytes are generated from multiple sources in the dorsal telencephalon (Schitine et al. 2015). At late stages of embryonic development, cortical protoplasmic astrocytes begin to be generated in a spatially restricted manner from radial glial cell in the VZ that also give rise to columns of cortical projection neurons (Gao et al. 2014; García-Marqués and López-Mascaraque 2013; Magavi et al. 2012). At the end of cortical neurogenesis, a fraction of radial glia cells give rise to astrocytes by direct transformation, an event that is very well documented in different species including in humans (Alves et al. 2002; DeAzevedo et al. 2003; Schmechel and Rakic 1979; Voigt 1989). During this process, after a final asymmetric division, radial glial cells lose their apical process and move toward the pial surface, eventually transforming into astrocytes in the SVZ (Noctor et al. 2004). Another substantial portion of astrocytes seem to derive from intermediate astroglial progenitors in the postnatal SVZ and migrate radially to populate the cerebral cortex (Levison and Goldman 1993; Levison et al. 1993; Luskin and McDermott 1994; Zerlin, Levison, and Goldman 1995). During the first two postnatal weeks after birth, when they reach their destination, the immature cells still proliferate, locally generating the major source of astrocytes in the cortex (Ge et al. 2012). During embryonic and neonatal development, a distinct class of progenitors undergoes cell division in the marginal zone (MZ)/layer I and give rise to astrocytes, oligodendrocytes, and neurons (Breunig et al. 2012; Costa et al. 2007). These marginal zone MZ/layer I progenitors are derived both from dorsal telencephalic Emx1-expressing progenitors and ventral Gsh2- and Nkx2.1-expressing progenitors (Costa et al. 2007). Together with SVZ glial progenitors derived from the ganglionic eminences (Marshall and Goldman 2002; Marshall, Novitch, and Goldman 2005; Minocha et al. 2017), they can be considered as ventral sources of astrocytes in the neocortex.
1.3 Zbtb20 function during cortical development
The Zinc Finger and BTB Domain-Containing Protein 20 (ZBTB20 - also known as DPZF, HOF, ODA8, ZFP288 and ZNF288) belongs to a family of transcription factors with
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an N-terminal BTB/POZ domain and several C-terminal Krüppel-type zinc finger motifs. The BTB/POZ domain permits homo- and heterodimerization as well as protein-protein interactions, while DNA binding is mediated by the zinc fingers. The ZBTB (Zinc finger, Broad-complex, Tramtrack and Bric-à-brac) proteins are encoded by at least 49 genes in mouse and human and commonly serve as transcriptional repressors (Kelly and Daniel 2006; Siggs and Beutler 2012).
Defects in Zbtb20 have been implicated in a wide range of neurodevelopmental disorders, such as autism spectrum disorder, intellectual disability and agenesis of the corpus callosum. Alterations in Zbtb20 gene have been found in patients with Primrose syndrome, chromosome 3q13.31 microdeletion, microduplication syndromes, and major depressive disorder (Casertano et al. 2017; Cleaver et al. 2019; Cordeddu et al. 2014; Davies et al. 2014; Jones et al. 2018; Koul 2014; Mattioli et al. 2016; Mulatinho et al. 2016; Rasmussen et al. 2014). Among them, Primrose syndrome patients presented the most severe phenotype, including macrocephaly, intellectual disability and other behavioral abnormalities, dysmorphic facial features, greater body height compared to the general population, progressive muscle wasting, hearing loss and ectopic calcification of the ears and brain during puberty or early adulthood (Cleaver et al. 2019; Cordeddu et al. 2014). Primrose syndrome is a rare autosomal dominant condition caused by heterozygous missense variants within Zbtb20. All affected codons in Primrose syndrome were located within the C-terminal zinc finger domain, affecting the DNA-binding domain of the transcription factor possibly through dominant-negative action (Cordeddu et al. 2014). However, the molecular mechanism of ZBTB20’s physiological function in the developing brain and implication in these disorders are largely unknown.
ZBTB20 is required for the specification of the hippocampal CA1 field. Ectopic expression of ZBTB20 in projection neurons of the retrosplenial cortex and subiculum induces aberrant CA1-like molecular identity of projection neurons in the midline cerebral cortex (Nielsen et al. 2007; Nielsen et al. 2010; Xie et al. 2010). During development of hippocampal pyramidal neurons ZBTB20 represses several genes that control projection neuron development in the isocortex, including Cux1, Cux2, Fezf2, Foxp2, Mef2c, Rorβ, Satb2, Sox5, Tbr1, Tle4, and Zfpm2 (Nielsen et al. 2014).
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Severe morphological defects of archicortex, reduced life span and growth rate have been reported in Zbtb20 full knock-out mice (Rosenthal 2010). Zbtb20 mRNA exhibits a ventral high-to-dorsal low gradient of expression in medial pallium progenitors at E14, and it’s expressed in upper-layer neurons of the retrosplenial cortex at P4. Loss of Zbtb20 function causes a progressive ventral displacement of retrosplenial cortex and molecularly specified fields in the medial pallium (Rosenthal et al. 2012).
During development of the cerebral cortex, ZBTB20 has recently been associated with the sequential generation of neuronal subtypes (Tonchev et al. 2016) and astrocytes (Doeppner et al. 2019; Nagao et al. 2016). ZBTB20 protein is expressed in the VZ and SVZ from E14 to E18, adult SVZ, astrocytes, and transiently in neurons at early post-natal stages (Mitchelmore et al. 2002; Nagao et al. 2016; Tonchev et al. 2016). Zbtb20 overexpression and knock-down by in utero electroporation at E15.5 promotes and suppresses astrocytogenesis in the somatosensory cortex, respectively (assessed by S100β, GFAP, Aldh1l1-GFP expression at P7), possibly by repression of Brn2 expression in the astrocyte cell lineage (Nagao et al. 2016). Zbtb20 full knock-out showed a reduction of S100β+ astrocytes and normal expression of GFAP in the cerebral cortex at P12 (Doeppner et al. 2019). Zbtb20 loss of function leads to a reduction of the size of L2/3 and an increase of L4, 5 and 6 at early postnatal stages, likely caused by an extension of development window of this cells and migration defects of upper layer neurons (Tonchev et al. 2016).
The findings that ZBTB20 regulates astrogliogenesis by repressing Brn2 expression (Nagao et al. 2016) and its ZBTB20 loss leads to a reduction of layer II/III BRN2+ neurons (Tonchev et al. 2016) are seemingly contradictory. A possible mechanism to conciliate these observations could be that ZBTB20 acts at different stages of the lineage progression of neocortical progenitor to generate upper layer neurons or astrocytes, likely in concert with other players, to regulate cell-fate acquisition. In this work, in order to disentangle the functions of this protein in neocortex development we used a combination of genetic techniques to perturb Zbtb20 expression either in progenitors or post-mitotic neurons. Furthermore, we evaluate the effect of Zbtb20 loss of function in neuronal projections and somatosensory circuit formation.
20 2 AIM
The main purpose of this project is to identify and characterize the molecular pathways involved in the specification of neocortical progenitor cells to generate upper layer neurons and macroglial cells.
2.1 Specific aims
Evaluate the expression of transcription factors in the proliferative zones of the dorsal telencephalon at different developmental stages;
Pinpoint transcription factors expressed by progenitor cells that could be involved in the specification of upper layer neurons and/or astrocytes in the developing neocortex;
Characterize the function of the candidate transcription factor by in utero electroporation and using conditional knock-out mice models.
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3 METHODS
3.1 Mouse lines and breeding
All mice experimentation were performed in accordance with the United States of America animal welfare regulations and were approved by The Scripps Research Institute (protocol number 08-0030) and The Johns Hopkins University (protocol number M016M273 and MO19255) Institutional Animal Care and Use Committee (IACUC).
C57BL/6J wild-type (WT) mice were obtained from Charles River Laboratories for overexpression experiments and breeding with genetically modified mice described below. The date of the vaginal plug detection was designated embryonic day E0.5, and the date of birth P0 (postnatal day 0).
Emx1-Cre (B6.129S2-Emx1tm1(cre)Krj) (Gorski et al. 2002), Ai9 (Madisen et al. 2010), DCX-Cre (Harris et al. 2014), NEX-Cre (Goebbels et al. 2006), Aldh1l1-eGFP (Tg(Aldh1l1-EGFP)OFC789Gsat) (Gong et al. 2003), and FLPe (B6.Cg-Tg(ACTFLPe)9205Dym/J) (Rodríguez et al. 2000) mice have been previously described.
Emx1-Cre mice express Cre recombinase in Emx1-expressing cells of the developing forebrain. Emx1 is expressed in both progenitor cells (as early as E10.5) and postmitotic neurons of the medial, dorsal, and lateral pallia. In this transgenic mouse strain, excitatory neurons and glia cells are recombined in the hippocampus, neocortex and piriform cortex (Gorski et al. 2002).
Nex-Cre mice express Cre recombinase under control of regulatory sequences of NEX, a gene that encodes a neuronal basic helix-loop-helix (bHLH) protein. The coding region of NEX (on exon 2) was replaced by a Cre expression cassette. NEX is expressed in progenitor cells in the SVZ and newly generated neurons of the dorsal telencephalon prior to their migration. In this transgenic mouse, Cre-mediated recombination is prominent in neocortex and hippocampus (Belvindrah et al. 2007; Goebbels et al. 2006; Wu et al. 2005).
Dcx-Cre mice express Cre recombinase under control of regulatory sequences of doublecortin (Dcx), a microtubule-associated protein expressed in immature neurons (Harris et al. 2014).
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Ai9 is a Cre reporter mouse line containing the CAG promoter followed by a loxP-flanked (‘floxed’) stop cassette and a fluorescent marker gene, tdTomato (Madisen et al. 2010). Removal of the stop cassette is induced by Cre recombination, allowing expression of tdTomato. This reporter mouse is useful in imaging and tracking specific cell populations in vivo, such as fate mapping of progenitor cells transfected with Cre by in utero electroporation.
FLPe is a mouse strain expressing the site-specific flippase (FPL) recombinase driven by the human ACTB regulatory sequences. The ACTB promoter region drives expression in the germline. FLP recognizes a pair of FLP recombinase target (FRT) sequences that flank a genomic region of interest and mediates site-specific excisional recombination (Rodríguez et al. 2000).
Aldh1l1-eGFP is a BAC (bacterial artificial chromosome) transgenic mice, in which enhanced green fluorescent protein (eGFP) is driven by the regulatory sequences of aldehyde dehydrogenase 1 family, member L1 (Aldh1l1) gene (Gong et al. 2003). Aldh1l1 is expressed in glial progenitors, postnatal neural stem cells, and adult astrocytes (Anthony and Heintz 2008; Cahoy et al. 2008; Foo and Dougherty 2013).
Zbtb20 knockout-first allele with conditional potential embryonic stem (ES) cells (Zbtb20tm1a(EUCOMM)Hmgu) were obtained from the EUCOMM European Conditional Mouse Mutagenesis Program (ES clone HEPD0822_1_A11). It was designed to insert LoxP sites flanking a critical exon of the Zbtb20 gene, with upstream elements including a neomycin-resistance cassette (PGK-neo) and LacZ reporter flanked by two FRT sites. ES cells were transplanted into mouse blastocysts by The Scripps Research Murine Genetics Core (La Jolla, CA, United States) to produce transgenic mice. Heterozygous F1 mice (Zbtb20flox-neo/+) were mated with FLPe mice (The Jackson Laboratory) to remove the PGK-neo cassette and LacZ reporter. The resulting offspring were subsequently mated to C57BL/6J mice to remove the FLPe transgene. Crossing heterozygous mice generated Zbtb20flox/flox mice. Zbtb20flox/flox mice were crossed with Cre lines that were heterozygous for Zbtb20flox/+ to generate Cre Zbtb20flox/flox (conditional knockout or cKO), and Cre Zbtb20flox/+, Zbtb20flox/flox and Zbtb20flox/+ (control).
23 3.2 Mouse genotyping
Genotyping was carried out using purified DNA preparation from mouse tail biopsies followed by polymerase chain reaction (PCR). Tails were digested overnight at 55°C with proteinase K (0.1mg/mL) in lysis buffer (10 mM EDTA, 0.5% SDS, 100 mM NaCl, 10 mM Tris-HCl, pH 8). Precipitation of DNA was performed with isopropanol, the DNA pellet was washed with ethanol to remove salts, air-dried and resuspended in water.
PCR genotyping was performed according to previously described protocols by the Jackson Laboratory. For genotyping of Emx1-Cre, DCX-Cre, NEX-Cre, Aldh1l1-EGFP and FLPe were used a standard master mix containing Taq DNA Polymerase (#MB042-EUT-L, SYD Labs), buffer (#MB042-(#MB042-EUT-L, SYD Labs), 0.2mM dNTPs (#N0447L, New England Biolabs), and 0.25µM each primer (all primers used here were listed in Table 1). For the PCR genotyping of Ai9 and Zbtb20 mutant mice, we used 2X green mix (GoTaq® Green Master Mix, #M712C, Promega) containing DNA Polymerase, dNTPs, MgCl2 and reaction buffer.
DNA amplicon was examined for size and quality through electrophoresis, using 1% to 2% agarose gel in TAE (0.04M Tris-acetate, 0.001M EDTA) with ethidium bromide (0.5 μg/mL) to visualize the DNA under ultraviolet light. A DNA molecular weight marker, 2-Log DNA Ladder (#N3200L, New England Biolabs), was used to identify the approximate size of the PCR product. The length of PCR products or amplicons are listed in Table 1.
3.3 Plasmid constructs
pCIG2 or pCAG-IRES-GFP has been described (Hand et al. 2005). It contains an internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) expression cassette, under the control of a CMV-enhancer and a chicken β-actin promoter (pCAG).
pCAG-YPet was a kind gift from Randal A. Hand. This construct contains a yellow fluorescent protein (YPet) under the control of pCAG.
pBlueScriptIISK+ (Agilent Technologies) was used to subclone fragments. It was a kind gift from Vinicius Toledo Ribas.
The piggyback (PB) transposon system was used to permanently label electroporated cells, in order to avoid dilution of reporter proteins after several rounds of
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cell division. For this purpose, cells were co-electroporated with pPBCAG-mRFP, and PB-GLASTase or PB-CBAase (Chen and LoTurco 2012).
PB-CBA-Klf15 and PB-CBA-Zbtb20 were cloned by Cristina Gil-Sanz, a former member of Müller’s lab. Coding sequences for Klf15 and Zbtb20 were amplified from mouse cDNA by PCR. Isoform 1 of Klf15 and Zbtb20 including the 9 first nucleotides before the ATG (Kozak sequence) were amplified. Restriction enzymes sites were added to the primer sequence, and fragments were cloned into PB-CAG-EGFP (Chen and LoTurco 2012). AgeI and NotI sites were added to Klf15 forward and reverse primers, respectively. EcoRI and BglII sites were added to Zbtb20 primers.
pCIG-DCX or pDCX-Ires-GFP has been described (Franco et al. 2011). This construct contains a characterized promoter fragment from the Dcx gene (Wang et al. 2007) controlling the expression of IRES-EGFP. Coding sequences for Zbtb20 and Klf15 were inserted between the Dcx promoter and IRES-EGFP to allow expression in immature neurons (see cloning details in the next two paragraphs).
For the generation of pDcx-zbtb20-Ires-GFP, the vector pCIG-DCX was cut with SmaI and EcoRI. The Zbtb20 sequence was extracted from PB-CBA-Zbtb20. This construct was cut with BglII, blunted to allow non-compatible ends to be joined (SmaI and BglII), and then cut with EcoRI. Vector (pDCX-Ires-GFP) and insert (Zbtb20) were connected by a ligation reaction, generating pDcx-zbtb20-Ires-GFP construct.
pDcx-Klf15-Ires-GFP was generated by Gibson assembly method. pCIG-DCX was cut with EcoRI, and Klf15 was amplified by PCR. Vector (DCX-Ires-GFP) and insert (Klf15) were connected by Gibson assembly reaction, generating pDcx-Klf15-Ires-GFP.
pDcx-Zbtb20-Ires-Cre was generated by Gibson assembly method. pCIG-DCX was cut with EcoRI and NotI. The fragments Zbtb20, Ires, and Cre were individually amplified by PCR. Vector (pDCX) and inserts (Zbtb20, Ires, and Cre) were connected by Gibson assembly reaction, generating pDcx-Zbtb20-Ires-Cre.
pDcx-Klf15-Ires-Cre was generated by Gibson assembly method. pCIG-DCX was cut with EcoRI and NotI. The fragments Klf15-Ires and Cre were individually amplified by PCR. Vector (pDCX) and inserts (Klf15-Ires, and Cre) were connected by Gibson assembly reaction, generating pDcx-Klf15-Ires-Cre.
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pCAG-Cre-Ires-GFP or pCIG2-NLSCre was cloned by Isabel Martinez-Garay, a former member of Müller’s lab. The N-terminal nuclear localization sequence (NLS) and the coding sequence of Cre recombinase (CRE) were inserted into pCIG2 between XhoI and EcoRI.
To generate pCAG-Zbtb20-Ires-GFP construct, pCIG2 (pCAG-IRES-GFP) and pDcx-zbtb20-Ires-GFP were cut with EcoRI and NotI. Vector (pCAG) and insert (Zbtb20-IRES-GFP) were connected by a ligation reaction.
pCAG-zbtb20wt-Ires-Cre was cloned using restriction enzymes. The CAG promoter was first subcloned into pBlueScriptIISK+. pBlueScriptIISK+ and PBCAG-eGFP (Chen and LoTurco 2012) were cut with SpeI and EcoRI. Vector (pBlueScriptIISK+) and insert (pCAG) were connected by a ligation reaction, generating pCAG-pBlueScriptIISK+ construct. Dcx-zbtb20-Ires-Cre and pCAG-pBlueScriptIISK+ were cut with EcoRI and SacI. Vector (zbtb20-Ires-Cre) and insert (pCAG) were connected by a ligation reaction, generating pCAG-zbtb20wt-Ires-Cre construct.
Dominant negative mutations of Zbtb20 and Cre recombinase were expressed under the control of pCAG, through pCAG-zbtb20DN-Ires-Cre constructs. Human dominant negative mutations described by Cordeddu et al., 2014 were cloned by PCR amplification using mega primers. First, the Zbtb20 fragment was cut from CAG-zbtb20wt-Ires-Cre and subcloned into pBlueScriptIISK+ using the restriction enzymes SmaI and EcoRI. Vector (pBlueScriptIISK+) and insert (Zbtb20wt) were connected by a ligation reaction. The product pBlueScriptIISK+Zbtb20wt was used to introduce point mutations at Zbtb20 sequence. The dominant negative mutations (henceforth called DN) were generated by PCR using mega primers containing the Zbtb20 point mutation. Zbtb20DN was amplified through three rounds of PCR (Table 6 - Primers for cloning). The constructs pBlueScriptIISK+ with Zbtb20 DN were used to clone Zbtb20 DN back into CAG-Ires-Cre using the restriction enzymes, SpeI and XhoI. Thereby, CAG-zbtb20DN-Ires-Cre constructs were generated. All the constructs were sequenced to confirm the point mutations.
We cloned seven different Zbtb20 DN mutations (Figure 8) described in patients with Primrose syndrome by Cordeddu and colleagues (2014), all mutations were located within the C-terminus zinc finger domain: in the first zinc finger (1768, 1771 and 1787),
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second zinc finger (1861), or in the linker connecting these motifs (1802, 1805 and 1811). The numbers used here refer to the altered nucleotide position in the longer transcript variant sequence (NCBI Reference Sequence: NM_001164342.1). These mutations affect the DNA-binding domain of the transcription factor possibly through dominant-negative action (Cordeddu et al. 2014). We used the Zbtb20 DN mutation 1768 in all electroporation experiments, that correspond to the following nucleotide and amino acid change, respectively: 1768A>C and Lys590Gln (Figure 8). This DN mutation exhibited the strongest effect in reduced binding to DNA, impaired function in repressing luciferase reporter expression under the control of the promoter for AFP (encoding α-fetoprotein) known to contain a ZBTB20 recognition site and had a dominant-negative impact on wild-type ZBTB20 (Cordeddu et al. 2014).
Figure 8 Zbtb20 dominant negative mutations described in patients with Primrose syndrome by Cordeddu and colleagues (2014).
3.4 Cloning strategies and protocols
DNA construct maps were analyzed, and cloning strategies designed using SnapGene 4.0.4.
Restriction enzymes and the appropriate buffer (New England Biolabs) were used to digest DNA plasmids or PCR products, according to the manufacturer's protocol (//nebcloner.neb.com/#!/redigest). The resulting fragments were analyzed by agarose gel electrophoresis.
Vectors digested with only one restriction enzyme were dephosphorylated with calf intestine alkaline phosphatase (CIP) to prevent self-ligation of linearized plasmid DNA according to the manufacturer's protocol (New England Biolabs).
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To clone fragments into blunt-end vectors, 5´overhangs of fragments were filled in with dNTP and 3´overhangs were removed by T4 DNA polymerase (#M0203S, New England Biolabs) in the appropriate buffer (NEBuffer™ 2.1 #B7202S, New England Biolabs) with 1mM dNTPs (#N0447L, New England Biolabs). The reaction was incubated at 12°C for 15 minutes and then inactivated at 75°C for 20 minutes.
DNA fragments were PCR amplified from mouse genomic DNA using Phusion High-Fidelity DNA Polymerases (#F549L, Thermo Fisher Scientific). Some PCR products were designed to contain specific restriction enzyme sites compatible with the desired vector. In such cases, the PCR fragments were digested with the corresponding enzyme before ligation. In absence of restriction enzymes sites compatibility or difficult cloning, the PCR product containing 3´A overhangs was cloned into pGEM-T Easy vector (Promega) as described by the manufacturer.
Mega primer-mediated cloning strategy was used to introduce point mutations at Zbtb20 sequence, through three rounds of PCR using multiple forward and reverse priming oligonucleotides (Table 6 - Primers for cloning).
Insert and vector with compatible overhangs were ligated for two hours at room temperature or overnight at 4°C using T4 DNA Ligase (Thermo Fisher Scientific). The concentration of insert and vector was calculated based on the size of each fragment: Insert concentration (ng) = size (bp) X 0.03
Vector concentration (ng) = size (bp) X 0.01
Gibson assembly method was used to clone multiple DNA fragments into a vector, and to clone fragments with restrictions sites inside the fragment of interest. The strategy was designed using NEBuilder Assembly Tool (//nebuilder.neb.com/#!/) and Gibson Assembly cloning kits (#GA1100-50, Synthetic Genomics) were used following the manufacturer's protocol.
Heat shock transformation was performed using DH5α competent E. coli. All plasmids used here contained an antibiotic resistance gene, either ampicillin or kanamycin. The transformation was plated onto LB agar plates containing the appropriate antibiotic and incubated overnight at 37°C. Isolated colonies were selected and inoculated in LB liquid containing antibiotics. Bacterial cultures were incubated overnight at 37°C in a shaking incubator.
