SOXD GENES CONTROL DEVELOPMENTAL AND ADULT NEUROGENESIS IN THE HIPPOCAMPAL
NEUROGENIC NICHE
TESIS DOCTORAL
Lingling Li 2019
Trabajo dirigido por el Dra. Aixa V. Morales.
Departamento de Biología Molecular Facultad de Ciencias
Universidad Autónoma de Madrid
Memoria de Investigación presentada por Lingling Li
Para optar al grado de
Doctor en Biología Molecular por la Universidad Autónoma de Madrid.
Trabajo dirigido por el Dra. Aixa V. Morales (Instituto Cajal-CSIC).
Vº.Bº del director de Tesis,
Aixa V. Morales
La doctoranda,
Lingling Li Insituto Cajal
Departamento de Biología Molecular Facultad de Ciencias
Universidad Autónoma de Madrid
Dr. Aixa V. Morales, Científico Titular del Instituto Cajal (CSIC)
CERTIFICA:
Que la presente tesis doctoral, que lleva por título “SoxD genes control developmental and adult neurogenesis in the hippocampal neurogenic niche” que presenta Lingling Li para optar al grado de Doctor por la Universidad Autónoma de Madrid ha sido realizada bajo mi dirección en el Instituto Cajal (CSIC) y que cumple todos los requisitos paqra su defensa pública, reuniendo, a mi juicio el suficiente rigor científico para optar al grado de doctor.
Madrid, a 22 de Octubre de 2019
Fdo: Dra. Aixa V. Morales Insituto Cajal
Departamento de Biología Molecular Facultad de Ciencias
Universidad Autónoma de Madrid
This thesis has been funded by a Fellowship from the
China Scholarship Council (No.201506300086).
In these 4 years in Spain, I gained more knowledge on neuroscience and a precious abroad experience. Here, I saw the cultural diversity and another way of life-style. Importantly, I met lots of nice friends. I appreciate all of them for their help and support along this travel to arrive to this point.
I thank Dr. Aixa V. Morales, my director of PhD study. I appreciate so much about her supervision on my research work. She gives me a lot of good suggestions of how to work on science. Without her coaching, I could not have reached this step. From her, I got a better understanding of how to properly organize scientific projects and focus on my research direction.
I thank Dr. Juan José Garrido Jurado and Dr. Jose Mª Frade López for their kind help and advices in daily life.
I would like to thank our excellent technician Maria Ciorraga for her assistance on my research. I would like to thank Pilar Jurado and Cristina Medina for their hard work on this project.
I thank my family, in particular, my husband Wei Zhang who has been with me through this challengeable period and always give me good advices and support me. In the rest of my life, we will continue to share joys and tears and built our bright future. Besides, I got lots of supports from my parents, parents-in-law, my brother, brothers-in-law and sister-in-law. It is their understanding and encouragement that promotes me to make progress toward my ideal.
I thank my friends. In particular, Dr. Shaoliang Zhang, Yuwen Chen, Chaska C. Walton, Yu Wang, Ting Wang and Da Guang. Thanks for becoming my friends and sharing a wonderful time in Madrid.
My scientific life will continue with all I learned during these four years, and I believe that tomorrow will be better!
Figure Index... 3
Summary ... 6
Resumen... 8
Presentación ... 10
Introduction ... 13
1. Development of the DG ... 16
1.1 DG development during embryonic stages ... 17
1.2 DG development during postnatal stages ... 18
2. The adult hippocampal neurogenesis ... 19
2.1 Cellular properties of RGLs ... 19
2.2 Neurogenic division and differentiation in adult hippocampal neurogenesis . 21 2.3 Synaptic inputs and outputs of newborn GNs ... 21
3. Comparison between developmental and adult neurogenesis ... 22
3.1 Developmental RGCs and adult RGLs ... 22
3.2 Developmental and adult generated GNs ... 24
3.3 Molecular regulation of developmental and adult neurogenesis in DG ... 24
3.3.1 BMP signaling ... 24
3.3.2 Wnt signaling ... 25
3.3.3 Notch signaling ... 26
4. Developmental origin of adult RGLs ... 26
5. SoxD transcription factors ... 29
5.1 Structures and subgroups of Sox transcription factors ... 29
5.2 Sox transcription factors and neurogenesis ... 29
5.3 Members of the SoxD subgroup ... 31
5.4 Expression and functions of SoxD genes in non-neuronal cells ... 32
5.5 Expression and functions of SoxD genes in the CNS ... 32
Objectives ... 35
Materials and Methods ... 36
1. Reagents ... 37
1.1 Chemicals and recombinant proteins ... 37
1.2 Antibodies ... 38
1.3 Taq man probes for quantitative-PCR ... 39
2. Animals ... 40
3. Tamoxifen administration ... 40
4. Brdu administration... 41
5. Transcardiac perfusion ... 42
6. Tissue preparation ... 42
7. Nissl staining and volumetric analysis of hippocampus and DG ... 42
8. Primary cell culture ... 43
8.1 Quiescence experiment ... 44
8.2 Nucleofection Experiment ... 44
8.3 Proliferation and differentiation experiment with 4-hydroxytamoxifen induced recombined cells ... 44
9. Immunohistochemistry... 45
10. Microscopic analysis ... 45
11. RNA extraction, cDNA synthesis and real-time quantitative PCR ... 46
12. Quantitative analysis in sections ... 46
13. Quantitative analysis in cultured cells ... 47
14. Statistical analyses ... 47
Results ... 48
1.1 Expression of Sox5 and Sox6 in the mouse DG at perinatal and postnatal
stages ... 49
1.2 Expression of Sox5 and Sox6 in the adult DG ... 54
2. Sox5 loss leads to a reduction of DG size during development ... 56
3. Sox5 is required for DG proliferation during the first two postnatal weeks... 60
4. Sox6 is less required for DG proliferation during the postnatal development ... 62
5. Developmental loss of Sox5 could results in alterations in apoptosis in the DG during postnatal development ... 63
6. Sox5 is required for IPCs maintenance during DG development ... 64
6.1 Sox5 is required to control the number of IPCs during DG development... 65
6.2 Sox5 is not required for the proliferation of IPCs during DG development ... 66
6.3 Sox5 is required for cell cycle reentry of IPCs during DG development ... 68
7. Sox5 is required for the control activation of RGLs and its loss leads to the exhaustion of RGL pool in the DG ... 69
8. Sox5 is required for establishment of qRGLs ... 73
9. Sox5 is required for the acquisition of quiescence of hippocampal neural progenitors in vitro... 74
10. Developmental loss of Sox5 induces transcriptional changes in proliferative and quiescent cultured NSCs/NPCs ... 75
11. Sox5 is required for the proper production of GNs during DG development ... 79
12. Sox5 is required for the self-renewal division of a-RGLs ... 79
13. Sox5 is differentially expressed in a-RGLs in comparison to q-RGLs in adult DG ... 81
14. Sox5 and Sox6 are required for activation of q-RGLs in adult DG ... 84
15. Loss of Sox5 and Sox6 reduces adult neurogenesis in adult DG ... 86
16. Sox5 is sufficient to prevent hippocampal NSCs to enter quiescence in vitro and is required to regulate the neuronal specification in vitro ... 89
Discussion ... 93
1. Sox5 controls cell cycle arrest of IPCs in the DG ... 96
2. Sox5 is required for RGLs to acquire a quiescent state during DG development ... 97
3. Sox5 could regulate Id2 to promote quiescence of RGLs ... 99
4. Sox5 could maintain RGLs self-renewal ... 101
5. Sox5 and Sox6 are required for the activation of RGLs in the adult brain ... 102
6. Sox6 is less required than Sox5 in the development of the DG ... 104
Conclusions ... 107
Conclusiones ... 109
References ... 111
Annexes: Research Articles ... 129
Abbreviations
AC-3: activated-caspase3 a-RGL: active radial glial cells BMP: bone morphogenetic protein BMPR: BMP receptor
BLBP: brain-lipid-binding protein bHLH: basic helix-loop-helix Brdu: 5-Bromo-2´-deoxy-uridine CNS: central nervous system CH: cortical hem
CA: cornus amonis Dpi: day post injection DG: dentate gyrus DNE: neuroepithelium DCX: doublecortin
DMEM: dulbecco´s modified eagle´s medium
E: embryonic EtOH: ethyl alcohol
EGF: epidermal growth factor
EGFP: enhanced green fluorescent protein EDTA: ethylenediaminetetraacetic acid ERT2: estrogen ligand-binding domain FBS: fetal bovine serum
FGF2: fibroblast growth factor Fig: figure
GABA: gamma-aminobutyric acid
GCL: granule cell layer
GFAP: glial fibrillary acidic protein GNs: granule neurons
GLAST: astocyte-specific glutamate transporter
Hopx: homeodomain-only protein HMG: high-mobility group
HNE: hippocampal neuroepithelium HF: hippocampal fissure
IPCs: intermediate progenitor cells NEPs: neuroepithelial cells
NPCs: neural progenitor cells NSCs: neural stem cells P: postnatal
Prox1: prospero homeobox protein 1 PFA: paraformaldehyde
PB: phosphate buffer
q-RGL: quiescent radial glial-like cells RT-PCR: real time-polymerase chain reaction
RGCs: radial glial cells RGLs: radial glial-like cells Shh: sonic hedgehog SGZ: subgranular zone
Sox: sex-determing region Y-related high- mobility group box
SVZ: subventricular zone Tbr2: T-box brain protein 2
V-SVZ: ventricular-subventricular zone Wnt: wingless-type
1ry : primary 2ry: secondary 3ry: tertiary
3D: three-dimensional
Figure Index
Figure 1. Schematic representation of inputs and outputs of hippocampus. 16
Figure 2. Schematic representation of hippocampal development from embryonic (E) to postnatal stages (P) with a focus on dentate gyrus development. 17
Figure 3. Summary of developmental stages of adult neurogenesis, intrinsic factors specific for each cell. population and signaling pathway involved in regulating each developmental stage. 20
Figure 4. Summary of developmental stages of adult neurogenesis, intrinsic factors specific for each cell. population and signaling pathway involved in regulating each developmental stage. 23
Figure 5. Schematic representation of structres of Sox family genes. 30 Figure 6. Transgenic mice lines used and genotyping of knockout. 41
Figure 7. Sox5 and Sox6 are expressed in NSCs/NPCs at perinatal stages in the dentate gyrus.
50
Figure 8. Sox5 and Sox6 are expressed in NSCs/NPCs in the DG of mice at P0 and P5. 52 Figure 9. Sox5 is expressed in NSCs/NPCs in the DG of mice at P14 and P30. 53
Figure 10. Sox5 and Sox6 are expressed in NSCs in the SGZ of 2 months old mice hippocampus. 55 Figure 11. Developmental loss of Sox5 alters DG formation at P5. 57
Figure 12. Hippocampal and DG development is compromised by knockout of Sox5. 59 Figure 13. Sox5 is required for NSCs/NPCs proliferation during the development of DG. 61 Figure 14. Developmental loss of Sox6 does not affect DG formation and neurogenesis. 63 Figure 15. Sox5 could be required for cell survival during the development of DG. 64 Figure 16. Sox5 is required for controling the number of IPCs during the development of DG. 66
Figure 17. Sox5 is not required for the proliferation of IPCs during the development of DG. 67
Figure 18. Sox5 is required for the cell cycle reentry of IPCs at P5 and P30. 69
Figure 19. Sox5 is required for the activation of RGLs and maintenance of RGL pool during the development of DG. 71
Figure 20. Developmental deletion of Sox5 induces RGL exhaustion at P90 in the SGZ.
72
Figure 21. Sox5 is required for the maintenance of RGLs in a quiescent state. 74
Figure 22. Sox5 is required for proliferating NSCs/NPCs to enter quiescence in vitro. 76
Figure 23. Developmental deletion of Sox5 induces transcriptional changes in BMP and Wnt signaling pathways in cultured NSCs/NPCs. 78
Figure 24. Sox5 is required for the generation of the correct number of immature GNs during DG development. 80
Figure 25. Sox5 controls the balance between neurogenic division and self-renewal division of RGLs.
82
Figure 26. Sox5 has higher expression in proliferating RGLs both in vivo and in vitro. 83 Figure 27. Tamoxifen induced deletion of Sox5 or Sox6 in RGLs. 85
Figure 28. Sox5 and Sox6 are required for the activation of adult RGLs. 87
Figure 29. Sox5 and Sox6 are required for the generation of IPCs and newborn neurons. 88
Figure 30. Sox5 and Sox6 are not required for the control of proliferation in adult NSCs/NPCs grown in FGF condition. 89
Figure 31. Sox5 is required to maintain proliferating state of cultured hippocampal NSCs/NPCs. 90 Figure 32. Loss of Sox5 or Sox6 promotes neuronal fate in adult NSCs/NPCs in vitro. 91
Figure 33. Summary and predictions of cellular changes resulted by Sox5 deletion from P5 to P90.
97
Figure34. Summary of Sox5 and Sox6 regulation in adult neurogenesis. 102
Summary
During the development of the dentate gyrus (DG), both at embryonic and postnatal stages, radial glial cells (RGCs) and neural progenitors proliferate and generate mature granule neurons, the principal neuron of the DG. In an unique way, in the adult DG, a subpopulation of progenitors with a radial morphology are retained in a quiescent state as adult radial glial-like cells (RGLs) in the subgranular zone (SGZ) of the DG and continue to produce new granule neurons throughout adult life. This raises questions about when and how adult RGLs are generated in the DG, which are essential questions to understand how neurogenic niches are generated and maintained in the adult brain. HMG-box transcription factors of Sox family genes could be at the core of those processes, as many of them have essential regulatory functions in both developmental and adult neurogenesis. In this study, we have focused on SoxD transcription factors (Sox5 and Sox6) in DG neurogenesis, as our laboratory has previously shown that they play a critical role in regulating cell cycle progression in progenitor cells and that they are expressed in the SGZ, both during DG development and in adulthood. We describe now that during DG development both Sox5 and Sox6 are persistently expressed in RGCs/RGLs and that their expression gradually turns off along the progression of those cells towards the neuronal lineage. By conditional deletion of Sox5 and Sox6 from early central nervous system development, we have determined that Sox5 is required for RGCs/RGLs to enter the quiescent state during postnatal development.
Thus, deleting Sox5 expression during development results first in an increase in hippocampal neurogenesis in young adults and then, in mature adults, leads to an exhaustion of RGLs pool. Furthermore, we have found that BMP signaling target, Id2, could be mediating Sox5-regulated quiescence acquisition during DG development. Furthermore, we have found that Sox6 alone is less required than Sox5 for the development of DG, at least during first three postnatal weeks. Interestingly, selective creERT/tamoxifen-induced deletion of SoxD genes in the adult DG, showed that both Sox5 and Sox6 are required for RGLs to transit from quiescent into active proliferative states, and consequently they are needed for adult neurogenesis. Taken together, our results prove that the transition from developmental RGCs into adult RGLs during DG development is regulated by Sox5. These results set up the basis to further explore Sox5 direct targets to understand the molecular mechanism that involve how adult neurogenesis is specifically generated at certain brain areas and how could we modulate the neurogenic process in pathological and ageing brains.
Resumen
Durante el desarrollo del giro dentado (GD), tanto en las etapas embrionarias como postnatales, las células de glía radial (RGC) y los progenitores neurales proliferan y generan neuronas granulares maduras. De una manera única, en el GD adulto, una subpoblación de progenitores con morfología radial se mantienen en estado de quiescencia como células de tipo glía radial (“radial glial-like”, RGLs) adultas en la zona subgranular (SGZ) del GD y continúan produciendo nuevas neuronas granulares a lo largo de la vida adulta. Resulta esencial conocer cuándo y cómo se generan estas células RGL adultas para comprender cómo se generan y mantienen los nichos neurogénicos en el cerebro adulto. La familia de genes Sox, factores de transcripción con dominio HMG, podrían ser fundamentales para entender esos procesos, ya que muchos de ellos son esenciales durante la neurogénesis en el desarrollo y en el cerebro del animal adulto. En este estudio, nos hemos centrado en los factores de transcripción SoxD (Sox5 y Sox6) en la neurogénesis del GD, ya que nuestro laboratorio había demostrado previamente que controlan el ciclo celular en progenitores neurales y que se expresan en las células de la SGZ, tanto durante el desarrollo del GD como en la etapa adulta.En el trabajo actual describimos que, durante el desarrollo del GD, Sox5 y Sox6 se expresan de manera persistente en RGCs / RGLs y su expresión se apaga gradualmente en la progresión de esas células hacia el linaje neuronal. Mediante eliminación condicional de Sox5 y Sox6 en el desarrollo cerebral temprano, hemos determinado que Sox5 es necesario para que las RGCs / RGLs entren en quiescencia en etapas postnatales. La pérdida de función de Sox5 resulta primero en un aumento de la neurogénesis hipocampal en adultos jóvenes y luego, en adultos maduros, conduce a un agotamiento del reservorio de células RGLs. Además, hemos determinado que la proteína Id2 podría estar mediando la entrada en quiescencia inducida por Sox5 durante el desarrollo del GD. Además, hemos determinado que Sox6 no es fundamental para el desarrollo del GD, al menos durante las primeras tres semanas postnatales. Contrariamente a lo observado durante el desarrollo hipocampal, la eliminación selectiva inducida por creERT / tamoxifeno de los genes SoxD en el GD adulto, mostró que tanto Sox5 como Sox6 son necesarios para las células RGLs salgan del estado de quiescencia y se activen entrando en ciclo celular, y en consecuencia son necesarios para la neurogénesis adulta. Tomados en conjunto, nuestros resultados demuestran que la transición de las células de tipo RGC de desarrollo a células RGL de adultos durante el desarrollo de DG está regulada por Sox5. Estos resultados sientan las bases para explorar las dianas directas de Sox5 y así comprender cómo se genera específicamente la neurogénesis adulta en ciertas áreas del cerebro y cómo podríamos modular el proceso neurogénico en el cerebro adulto en situaciones patológicas y durante el envejecimiento.
Presentación
Durante el desarrollo del sistema nervioso central (CNS), las células madre neurales (NSCs) proliferan rápidamente y generan neuronas y glía, procesos conocidos como neurogénesis y gliogénesis, respectivamente. La neurogénesis ocurre desde etapas embrionarias a perinatales en la mayoría de las regiones cerebrales de mamíferos, y está restringida a unas pocas zonas neurogénicas en el cerebro vertebrado adulto. En el giro dentado (GD) del hipocampo adulto, las NSCs residen en la zona subgranular (SGZ) donde se mantienen mayoritariamente en un estado quiescente y presentan una morfología radial similar a la de la glía radial embrionaria, designándolas como “radial glial-like cells” (RGLs).
Las células RGLs continúan produciendo nuevas neuronas granulares a lo largo de la vida adulta que se integran en circuitos neuronales preexistentes y participan en la adquisición, formación y mantenimiento de la memoria. Sin embargo, la cuestión central de cuándo y cómo se generan y establecen las células RGLs en el hipocampo no ha sido resuelta. La característica que mejor define a las células de RGLs adultas es la de la quiescencia, que les permite permanecer durante largos períodos fuera del ciclo celular en fase G1 de manera reversible, evitando el daño al DNA asociado a la proliferación continuada y manteniendo el reservorio de NSCs a largo plazo.
Estudios recientes han demostrado que la primera semana postnatal es una ventana de tiempo crucial para determinar el tamaño de la población de células RGLs adultas y para la adquisición del estado de quiescencia. Por lo tanto, un análisis en profundidad del control molecular de la quiescencia durante el desarrollo del GD es fundamental para entender cómo y cuándo se genera el nicho neurogénico hipocampal que perdurará durante toda la vida adulta.
Los factores de transcripción SoxD (Sox5 y Sox6) son un subgrupo de la familia de genes Sox, que tienen funciones esenciales en la neurogénesis embrionaria, aunque se desconoce su función en el cerebro del animal adulto. Sox5 y Sox6 se expresan en progenitores neurales en las zonas ventricular y subventricular (VZ y SVZ) del sistema nervioso en desarrollo y también en la SVZ del cerebro adulto. Nuestro grupo estableció previamente la función de Sox5 en el control de la salida de ciclo celular de los progenitores neurales durante la transición G1-S, principalmente contrarrestando el efecto proliferativo de la vía de Wnt-β-catenina. En el cerebro adulto, Sox5 y Sox6, junto con Sox21 se expresan en la SVZ de los ventrículos laterales, en la mayoría de las NSCs que expresan Sox2/Nestin y tienen una actividad antiproliferativa ya que son capaces de inducir una respuesta antitumoral a la sobrexpresión de oncogenes en las células progenitoras de SVZ.
Teniendo en cuenta el papel fundamental de Sox5 y Sox6 en el control de la proliferación, planteamos la hipótesis de que Sox5 y Sox6 podrían desempeñar una función relevante en el control de la neurogénesis durante el desarrollo y en la etapa adulta del GD.
En este trabajo, utilizando ensayos de pérdida de función en ratones mutantes condicionales para Sox5 y Sox6, hemos descrito que Sox5 es fundamental en la regulación de la salida del ciclo celular de los progenitores intermedios durante el desarrollo del GD y, lo que es más importante, en la adquisición de la quiescencia en las células RGL durante la primera semana postnatal. Id2, factor de transcripción regulado por la vía de BMP, podría mediar la acción de Sox5 en la regulación de la quiescencia de las RGLs durante el desarrollo del GD. Por el contrario, una vez que el nicho neurogénico adulto se ha establecido, Sox5 es necesario para la activación de las RGLs durante la neurogénesis adulta. Estas dos funciones opuestas de Sox5 en la regulación de la quiescencia y la proliferación de las RGLs durante el desarrollo y la etapa adulta del GD, arrojan nueva luz sobre la comprensión de la generación de las células RGLs, de la adquisición del estado de quiescencia y de la formación del nicho neurogénico hipocampal adulto durante el desarrollo.
Introduction
During development of central nervous system (CNS), neurons and glial cells are continuously generated from neural stem cells (NSCs) and neural progenitors (NPCs), known as neurogenesis and gliogenesis, respectively. As most stem cells, NSCs are defined by their self-renewal capacity and their potential to produce neurons and various glial cells.
NPCs are referred to cells with more limited capacity of proliferation and progeny.
Neurogenesis occurs from embryonic to perinatal stages in most mammalian brain regions (Ming and Song, 2005), and is restricted to a few neurogenic zones in the adult vertebrate brain (Ming and Song, 2011; Obernier and Alvarez-Buylla, 2019). Adult NSCs maintain proliferative capacity and continue to generate new cells mainly in two brain regions: the ventricular-subventricular zone (V-SVZ) of lateral ventricles and the subgranular zone (SGZ) in the dentate gyrus (DG) of the hippocampus. Besides, adult neurogenesis in mammals is also detected in other brain sites such as striatum and hypothalamus (Feliciano et al., 2015;
Inta et al., 2015; Jin, 2016).
The DG of the hippocampus is featured by receiving majority of inputs into the hippocampus (Fig. 1), which contributes to the formation of new episodic memories and the spontaneous exploration of novel environments (Hatami et al., 2018). Remarkably, the DG is characterized by the existence of neurogenesis from development to adulthood. In mice, the generation of DG neurons initiates from late embryonic stages, and most DG granule neurons (GNs) are produced within the first two postnatal weeks (Muramatsu et al., 2007).
Thereafter, the production of GNs slows down significantly (Kempermann, 2011b;Knoth et al., 2010; Lopez-Rojas and Kreutz, 2016; McDonald and Wojtowicz, 2005; Schlessinger et al., 1975). In young adult rats, around 9,000 new GNs are generated whereas adult humans add 700 new neurons in each hippocampus per day, corresponding to an annual turnover of 1.75% of the renewing neuronal population (Spalding et al., 2013).
It has been shown that adult-born GNs (aGNs) can integrate properly in a complex network and process information with functional relevance (Toni and Schinder, 2015).
Decline in the generation of aGNs in the DG correlates with affective and psychiatric disorders (Apple et al., 2017), while increased adult neurogenesis in the DG has been shown to improve memory acquisition, memory formation (Dupret et al., 2008; Saxe et al., 2006;
Shors et al., 2002; Winocur et al., 2006; Zhao et al., 2008), and memory maintenance (Imayoshi et al., 2008; Snyder et al., 2005). In adult human brain, SGZ is thought to contribute GNs to the DG as well (Boldrini et al., 2018; Eriksson et al., 1998; Moreno- Jimenez et al., 2019; Spalding et al., 2013) and adult hippocampal neurogenesis decreases during aging and in Alzheimer patients (Moreno-Jimenez et al., 2019).
each day in the DG, about 6% of the total GNs each month (Cameron and McKay, 2001), 1. Development of the DG
During early embryonic stages of brain development, a pseudostratified neuroepithelium of ectodermal origin ultimately generate neurons and macroglia. When cortical neurogenesis begins, around embryonic day 9 (E9) to E10 in the mouse, neuroepithelial cells gradually acquire features associated with glial cells, named radial glial cells (RGCs) (Kriegstein and Alvarez-Buylla, 2009). RGCs are restricted to the ventricular zone (VZ), a defined region next to the ventricles (Boulder Comm. 1970), and are the major NSCs/NPCs (Anthony et al., 2004). RGCs express many astroglial markers, as glial fibrillary acidic protein (GFAP), astrocyte-specific glutamate transporter (GLAST) and brain-lipid- binding protein (BLBP) (Hartfuss et al., 2001; Kriegstein and Gotz, 2003; Levitt and Rakic, 1980). At the onset of cortical development, RGCs expand their pool through symmetric division before entering a terminal asymmetric, neurogenic division to directly generate cortical neurons (Malatesta et al., 2003; Miyata et al., 2007; Noctor et al., 2001). 10-20% of total cortical neurons are generated by this process (Kowalczyk et al., 2009). Meanwhile, RGCs also give rise to transient amplifying neural progenitors, named intermediate progenitor cells (IPCs). These generated IPCs migrate and establish on the surface of VZ to form a sub-ventricular zone (SVZ). At later embryonic stages of cortical neurogenesis, IPCs expand their pool by symmetric proliferation and generate substantial fraction of cortical neurons, up to 80% of all cortical neurons (Noctor et al., 2007; Kowalczyk et al., 2009;
Figure 1. Schematic representation of inputs and outputs of hippocampus. Adapted from Ming and Song, 2011.
Vasistha et al., 2015). Thus, IPCs are thought as the major source to generating cortical neurons (Haubensak et al., 2004).
The major NSCs/NPCs that generate hippocampal and DG neurons are also originated from the ventricular zone of the telencephalic neuroepithelium and share the expression of GFAP. Different from the formation of the neocortex, the formation of the DG is quite unique, as its development is more protracted than other telencephalic regions and also because a separate group of NSCs/NPCs delaminate from the neuroepithelium and become RGCs, migrating away to form the DG. These RGCs form proliferative hubs and continuously produce IPCs and GNs to form the DG in close proximity to the pial surface.
The proliferative zone of the DG remains active during postnatal stages and eventually these RGCs and neural progenitors become restricted to the SGZ of the DG, and will constitute the adult hippocampal neurogenic niche (Altman and Bayer, 1990; Bayer, 1980a; Bayer, 1980b;
Khalaf-Nazzal and Francis, 2013; Pleasure et al., 2000; Sugiyama et al., 2013).
1.1 DG development during embryonic stages
RGCs of the DG originate from the dentate neuroepithelium (DNE) which is specified at E11.5. The DNE, also called primary (1ry) matrix (Altman and Bayer, 1990), is a restricted area of the medial pallium neuroepithelium that is between the presumptive
Figure 2. Schematic representation of hippocampal development from embryonic (E) to postnatal stages (P) with a focus on dentate gyrus development. HNE: hippocampal neuroepithelium; DNE: dentate neuroepithelium; CH: cortical hem; Hf: hippocampal fissure; Hi: hilus; Gcl: granule cell layer; CA, Cornu Ammonis; MI, molecular layer. Adapted from Morales and Mira, 2019.
fimbria, or cortical hem (CH), and the hippocampal neuroepithelium (HNE) that will generate the hippocampus proper (Fig. 2A). Different from cortical RGCs, hippocampal and dentate RGCs do not express BLBP at embryonic stages (Li et al., 2009; Nicola et al., 2015;
Seki et al., 2014).
Around E14.5, RGCs leave the DNE and migrate along the CH border to the pial side of the cortex, forming a new migratory stream or secondary (2ry) matrix that contains a mixture of RGCs and IPCs at various differentiation stages. The migration of the DG progenitors mainly relies on CH-derived Cajal-Retzius cells (Del Rio et al., 1997; Rickmann et al., 1987) and a radial glial scaffold form by RGCs that bridges the fimbria to the pial side of the cortex. Once they arrive at the hippocampal fissure (HF) around E16.5, RGCs accumulate and form another proliferative hub named the tertiary (3ry) matrix (Fig. 2B).
1.2 DG development during postnatal stages
GNs of the DG originate from all three proliferating matrices. After P0, the migration of RGCs into the hilus ceases. The 2ry matrix forms in the future hilus, which in the following two weeks becomes wider and increasingly diffuse (Nicola et al., 2015).
Intriguingly, a subpopulation of RGCs starts to weakly express BLBP at birth and later rapidly increase in number in the DG (Matsue et al., 2018), indicating that different properties of RGCs are acquired after birth. Although DG development initiates from embryonic stages, the majority of GNs are generated within the first two weeks after birth and are mainly originated from progenitors in the 3ry matrix (Muramatsu et al., 2007). The two blades of granule cell layer (GCL) display the characteristic V-shape that is determined by the Cajal-Retzius cells surrounding the HF and the pial surface (Fig. 2C).
During the first postnatal week, RGCs and IPCs are diffusely spread in the area that will become the GCL. Notably, a group of RGCs gradually enter quiescence during this period and are supposed to become adult NSCs (Berg et al., 2019; Noguchi et al., 2019), but they share same morphology with embryonic RGCs. All of them are positive for GFAP and have round or elongated cell bodies with relatively short processes. From P5 to P14, the mixture of embryonic RGCs and adult NSCs acquire proper radial glial morphologies with a long process toward molecular layer (ML), and gradually confined to the inner side of GCL, the SGZ (Fig. 2D). There they continue to produce new-born GNs during adulthood (Altman and Bayer, 1990; Altman and Das, 1965; Urban and Guillemot, 2014). At P30 (Fig.1E), the early adolescence, adult neurogenesis is thought to established in the SGZ, as ablating
proliferating cells during this period does not result lasting effect on adult NSCs/NPCs (Kirshenbaum et al., 2014).
2. The adult hippocampal neurogenesis
In 1965, Altman´s pioneer studies first showed the presence of newly generated GNs in the DG of the rat hippocampus (Altman and Das, 1965). Propelled by a general interest and aided by methodological advancements, significant progress has been made to understand the biology of this biological phenomenon, from the identity and location of adult NSCs, proliferation and fate specification of NPCs and neuronal maturation and synaptic integration of newborn GNs in the adult DG (Ming and Song, 2005).
In adult DG, NSCs reside in the SGZ (a narrow layer between the hilus and GCL), are largely maintained in a quiescent state with a radial glia-like morphology. Clonal lineage- tracing analysis identified that these radial glia-like NSCs (RGLs) are able to self-renew and generate both neurons and astrocytes but not oligodendrocytes (Bonaguidi et al., 2011).
During neurogenic cell division, proliferating RGLs could asymmetrically or symmetrically generate IPCs that divide 2-3 times to generate neuroblasts that then become immature GNs.
These new generated GNs then migrate from the SGZ in to the GCL with a short distance and begin their full differentiation into mature GNs (Goncalves et al., 2016). Less than 25%
of newborn immature GNs survive during maturation as majority of the them die within the first four days of their birth (Sierra et al., 2010) or within one to three weeks after birth (Tashiro et al., 2006).
2.1 Cellular properties of RGLs
RGLs (also named Type-1 cells, Kempermann et al., 2015) has been described as the adult NSCs and can be recognized by the expression of several glia-specific markers, such as GFAP, Nestin and Glast and by the expression of the stem/progenitor specific marker Sox2 (Favaro et al., 2009). The majority of RGLs (76%) display a long radial process extending into the ML. These RGLs are more proliferative and more affected by aging, voluntary running and D-serine administration (Gebara et al., 2016). While 24% of RGLs exhibit a shorter radial process and do not proliferate (Gebara et al., 2016), indicating the heterogeneity of RGLs.
As true stem cells, RGLs have the capacity to self-renew, can last for several months and generate different cell types, mostly GNs but also astrocytes during aeging (Bonaguidi et
al., 2011; Calzolari et al., 2015; Ortega et al., 2013; Palmer et al., 1997; Reynolds and Weiss, 1992). Besides, with an anti-mitotic drug to eliminate proliferating precursors, studies have shown that RGLs are largely quiescent in vivo (Doetsch et al., 1999). Quiescence can protect RGLs from DNA damage due to repeated DNA duplication and protects the RGLs pool from depletion. Decreasing GABA release (Song et al., 2012), Wnt inhibitor expression (Jang et al., 2013) or increasing pro-neural gene like Ascl1 expression (Urban et al., 2016) results in the activation of RGLs. Several studies have reported that RGLs can shuttle back and forth between quiescence and activity, dividing for extended periods of time (Bonaguidi et al., 2011; Kempermann, 2011a; Urban et al., 2016).
Moreover, single cell RNA-seq indicated that RGLs could be in a dormant quiescent state or in a primed quiescent state depending on the levels of cell cycle proteins, amongst others proteins (Llorens-Bobadilla et al., 2015; Shin et al., 2015; Fig. 4), suggesting the heterogeneity of quiescent RGL (q-RGL) population. Therefore, the balance between
Signalling pathways
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Figure 3. Summary of developmental stages of adult neurogenesis, intrinsic factors specific for each cell.
population and signaling pathway involved in regulating each developmental stage. Adapted from Yu et al., 2014.
quiescence and activation critically controls the generation of new functional neurons in adult hippocampus.
2.2 Neurogenic division and differentiation in adult hippocampal neurogenesis
The proliferating RGLs can undergo neurogenic division and give rise to IPCs (also known as Type 2a and Type 2b cells, Fig. 3) (Kempermann et al., 2015) through symmetric or asymmetric divisions. Type 2a cells are characterized by expressing Sox2 and Nestin, while type 2b cells express Nestin, Prox1, NeuroD1 and DCX. IPCs have plump and mostly tangential processes, and have irregularly shaped nucleus with dense chromatin. They are highly proliferative and able to respond to physiological stimuli, such as voluntary wheel running (Kronenberg et al., 2003), or pharmacological stimulation through serotonin- dependent mechanisms (Encinas et al., 2006). All type 2 progenitor cells specifically expressTbr2 during adult hippocampal neurogenesis (Hodge et al., 2008).
Once they exit cell cycle, IPCs give rise to neuroblasts that rapidly differentiate into immature GNs. A subset of immature GNs in the DG undergoes programmed cell death in the first one to four days after birth (Buss et al., 2006; Sierra et al., 2010). In adult mice, approximately 30-70% of newly generated GNs are eliminated within the first month after birth during their maturation and integration period, depending on the animal physiological/pathological condition and animal experience (Dayer et al., 2003). The regulation of programmed cell death for the production of GNs is more prominent in the adult stage, as blocking programmed cell death in mice produces no changes in the number of GNs at 2 month but a 2-fold higher number of GNs in 12-month-old animals (Sun et al., 2004). Apart from generating GNs, in aged mice (Encinas et al., 2011) and in kainic acid induced epileptic model mice (Sierra et al., 2015), RGLs are also found to transit into astrocytes after several rounds of neurogenic divisions and that will result in a depletion of the RGL pool.
2.3 Synaptic inputs and outputs of newborn GNs
Newborn GNs complete their development by forming functional synapses and integrating into pre-existing neural circuits. Studies done with retroviruses for birth-dating and labeling have indicated that newborn GNs in young adult mice develop a single primary dendrite with multiple branches that reaches the molecular layer within 7 days and exhibits rapid growth between 7 and 17 days, followed by modest growth for at least two months (Sun et al., 2013). The first functional synaptic inputs onto proliferating neuroblasts appear
within 4 days after birth (Song et al., 2012; Tozuka et al., 2005). Several studies, using paired recording (Markwardt et al., 2011), optogenetics (Song et al., 2012), and rabies virus- based retrograde transsynaptic tracing (Deshpande et al., 2013; Li et al., 2013b; Vivar et al., 2012), have identified multiple interneuron subtypes that innervate newborn GNs within weeks of birth. The first glutamatergic synaptic responses are detected in 11 to 14-day-old newborn GNs, and these responses mature over the following several weeks, accompanied by increased density of dendritic spines (Chancey et al., 2014; Esposito et al., 2005; Ge et al., 2006).
Aside from receiving synaptic inputs, newborn GNs also extend a single axon from the base of cell body that follows a stereotypic pathway through the hilus to reach CA3 pyramidal neurons within 7 days and establishes mature primary projection patterns within 21 days (Sun et al., 2013). In newborn GNs, synaptic structures have been found in both hilus and CA3 within 14 days of being born (Faulkner et al., 2008; Toni et al., 2008). This finding has been confirmed by optogenetic studies in which functional glutamatergic synaptic outputs onto hilar mossy cells, interneurons and CA3 neurons were found through specifically activating newborn GNs in the adult DG (Gu et al., 2012; Toni et al., 2007).
3. Comparison between developmental and adult neurogenesis
In spite of the fact that from developmental to adult neurogenesis, GNs are the only neuronal cell type continuously generated in the DG, there are clear differences between developmental and adult neurogenesis, in relation to NSCs, IPCs and even the generated GNs.
3.1 Developmental RGCs and adult RGLs
In adult neurogenesis, RGLs that express GFAP constitute the major pool which generates GNs. Interestingly, RGCs persistently express GFAP from early embryonic to postnatal stages (Ganat et al., 2006; Garcia et al., 2004; Seri et al., 2001). RGCs and RGLs are morphologically undistinguishable, as they fill the entire areas of early postnatal neurogenic zones, hilus and SGZ with relatively short processes, and experience a secondary glia transformation to achieve the radial morphology and settle down at SGZ by the end of the second postnatal week (Altman and Bayer, 1990; Brunne et al., 2010; Namba et al., 2005). While at molecular level, RGCs and RGLs exhibit clearly difference. Single cell RNA-sequence analysis showed that RGCs have greater expression of Sox4 and Sox11, while RGLs have increased expression of Notch2 and Padi2 (Hochgerner et al., 2018).
Using advanced time lapse imaging analysis focused on P5 DG, studies have revealed that GFAP-positive NSCs/NPCs divide symmetrically to generate pairs of GFAP-positive cells (45%) or pairs of neuronal-committed cells (45%), whereas a minority (10%) divided asymmetrically to produce GFAP-positive cells and neuron-committed cells (Namba et al., 2011). A different dividing pattern was found in adult hippocampal neurogenic niche. Live imaging analysis focused on Ascl1 expressing RGLs revealed that once RGLs are activated, 79% of them go through asymmetric division to generate one radial and one none-radial cell,
only 13% of them divide symmetrically and produce two RGLs to expand the RGL pool (Pilz et al., 2018). IPCs in the adult hippocampus are capable to undergo as many as six rounds of cell division and thus contribute more than RGLs to GNs production (Liu et al., 2010; Pilz et al., 2018). In contrast, in early postnatal hippocampus, IPCs exhibit relatively lower proliferative ability and quickly differentiate into immature GNs (Namba et al., 2011).
aRGL2 Active mitotic qRGL1
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Cell-to-cell comm.; ion channels and receptors signalling Fatty acid metabolism
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Development Adulthood
Figure 4. Summary of developmental stages of adult neurogenesis, intrinsic factors specific for each cell population and signaling pathway involved in regulating each developmental stage. (Morales and Mira, 2019).
3.2 Developmental and adult generated GNs
In both developmental and adult neurogenesis in the DG, GNs are the only neuronal subtype generated and they conduct major functions in the DG. However, developmentally born GNs (dGNs) show distinct morphology from those aGNs. Although a rapid phase of dendritic outgrowth followed by minimal growth was found in the dendritic elaboration of both dGNs and aGNs (Sun et al., 2013; Zhao et al., 2006), the dendritic elaboration of dGNs is somewhat faster than in aGNs (Kim et al., 2012; Zhao et al., 2006).
In addition, embryonic-born GNs develop at least two primary dendrites, while postnatally-born and adult-born GNs develop one single primary process (Kerloch et al., 2018). In the adult DG, 30-70% of newborn GNs are eliminated by programmed cell death during one month after birth (Brandt et al., 2003; Cameron et al., 1993; Gould et al., 1999;
Mandyam et al., 2007; McDonald and Wojtowicz, 2005; Tashiro et al., 2006) and the remaining neurons experience a long survival time for at least 11 months in mice (Kempermann et al., 2003). In contrast, studies have found that postnatally-born GNs do not experience early cell death during maturation and only around 17% of these neurons die after reaching maturity (Cahill et al., 2017).
3.3 Molecular regulation of developmental and adult neurogenesis in DG
Extrinsic and intrinsic regulations have been studied in both developmental and adult neurogenesis, where they could play similar or different roles. Hippocampus development initiates around E14 and responds to signals from the CH, including Bone Morphogenetic Protein (BMP) and Wnt molecules (Urban and Guillemot, 2014). BMP and Wnt signaling pathways play critical roles in hippocampal development, especially in promoting the proliferation of neural precursors (Caronia et al., 2010; Furuta et al., 1997; Galceran et al., 1999; Lee et al., 2000). In adult stages, both signals continuously regulate NSCs maintenance and differentiation, but in different ways.
3.3.1 BMP signaling
BMPs are critical in both developmental and adult neurogenesis. In early stages, complete knockout of BMP signaling leads to the absence of medio-dorsal structures, including the choroid plexus and the CH, which results in failed formation of the hippocampus (Cheng et al., 2006; Fernandes et al., 2007). During developmental neurogenesis, the regulation of BMP signalling is modulated by the activity of different
type1 BMP receptors (BMPR-I). For instance, BMPR-Ia enhances proliferation in the embryonic telencephalon, while BMPR-Ib induce cell cycle arrest and differentiation (Panchision et al., 2001).
In adult DG, BMPs play important role in regulating the maintenance of the quiescent state of NSCs (Mira et al., 2010). Blocking BMP signaling, by deletion of BMPR-Ia, results in an over-activation of RGLs that ultimately exhausts the RGL pool (Mira et al., 2010). In vitro, BMPs can also promote quiescence in cultured NSCs and progenitors without inducing differentiation, providing a useful model to study the molecular pathways regulating the transition of NSCs between quiescence and activation (Martynoga et al., 2013; Mira et al., 2010; Sun et al., 2011). Recently, increased BMP6 is found to induce an accelerated loss of the RGL pool in the senescence accelerated mouse prone 8 (SAMP8) mice, which mimics the onset of pathological Alzheimer´s disease-like neurodegeneration. In addition, blocking BMP signaling by administrating antagonist Noggin restores hippocampal RGL numbers, neurogenesis, and memory performance in SAMP8 mice (Diaz-Moreno et al., 2018).
Aside from regulating quiescence in RGLs, BMPs are also necessary later on for differentiation and maturation of granule cells (Bond et al., 2014). This dual role of BMPs might be due to differential expression of BMPR-I receptors as previously described during hippocampal development. A study has shown that while BMPR-Ia is expressed in RGLs and its expression turns off in IPCs, neuroblasts and neurons express BMPR-Ib (Mira et al., 2010). Thus, BMP signaling regulate both RGL quiescence and neuroblasts differentiation, but through different receptors in adult DG.
3.3.2 Wnt signaling
Wnts have been reported as hippocampal organizers. During early embryonic development, disruption of Wnt3a inhibits the formation of hippocampus (Lee et al., 2000).
Additionally, disruption of the major downstream effector of canonical Wnt signaling Lef1 or the Wnt receptor Lrp6, also results in severe hippocampal defects (Galceran et al., 1999;
Yoshida et al., 2006), indicating the absolute requirement of Wnt signaling pathway for the correct formation of hippocampus. During DG development, Wnts are also involved in the formation of the glial scaffold which is required for the migration of DG progenitors (Zhou et al., 2004).
Wnt signaling is required as well in postnatal and adult neurogenesis (Ortiz- Matamoros et al., 2013; Varela-Nallar and Inestrosa, 2013; Zhang et al., 2011). Wnts directly
induce neurogenic genes (Neurog2, NeuroD1 and Prox1) in IPCs, and also have functions in synapse formation and the maturation of aGNs (Kuwabara et al., 2009). Several in vitro studies have revealed that canonical Wnt signaling is required for the proliferation of hippocampal progenitors (Lie et al., 2005; Varela-Nallar and Inestrosa, 2013). In addition, deletion of the Wnt inhibitor secreted frizzled related protein 3 (SFRP3) leads to excessive proliferation of RGLs and depletion of the RGL pool (Jang et al., 2013). In aged mice, loss of the Wnt inhibitor Dickkopf-related protein 1 (Dkk1) successfully restored hippocampal neurogenesis (Seib et al., 2013). Wnt3a could cooperate with BMP2/4 to enhance neuronal production in adult rat hippocampus, through the control of Lef1 (Armenteros et al., 2018).
3.3.3 Notch signaling
Notch signaling acts mostly as an antineurogenic pathway maintaining the pool of proliferating neural progenitors in different organisms and context including brain development and adult neurogenesis (Imayoshi and Kageyama, 2011; Kageyama et al., 2008;
Urban and Guillemot, 2014). Loss of Notch signaling mediator RBPJk in early stages results in proliferation defects and premature differentiation of embryonic NSCs (Imayoshi et al., 2010). During hippocampal development, absence of the Notch ligand Jagged1 results in decreased proliferation and disrupted maintenance of NSCs without affecting the formation of DG (Breunig et al., 2007; Lavado and Oliver, 2014).
RGLs are shown to respond to Notch signaling, indicated by the expression of the Notch transcriptional targets Hes1 and Hes5 (Imayoshi et al., 2010; Lugert et al., 2010). Hes proteins are transcription factors that act as potent repressors of gene expression (Imayoshi and Kageyama, 2014) and that exhibit cyclic expression, which drives cyclic expression of their targets, such as Neurog2 and Ascl1 (Imayoshi et al., 2013; Shimojo et al., 2008).
Conditional inactivation of RBPJk in RGLs results in increased neuronal differentiation of RGLs in adult hippocampus and, later on, in the depletion of Sox2-expressing RGLs (Ehm et al., 2010). A recent study has shown that Ascl1 oscillations, which depend on Hes1 oscillations, regulate the activation of RGLs, while high Hes1 expression, and resultant Ascl1 suppression, promotes quiescence in NSCs (Sueda et al., 2019).
4. Developmental origin of adult RGLs
Evidences from molecular, cellular and functional analysis have shown that adult neurogenesis in the DG is a unique process. However, questions as when and how adult neurogenesis is established in the DG are still unclear.
The main questions in relation to the origin of adult neurogenesis are how and when the population of adult RGLs emerges during development. One of the characteristics that better distinguish adult RGLs from developmental RGCs is the acquisition of the quiescence state, by which RGLs could remain out of the cell cycle inthe SGZ for long periods of time, avoiding the exhaustion of the neurogenic pool.
In the generation of the adult neurogenic niche of the ventricular-subventricular zone (V-SVZ), the origin of adult NSCs (named B1 cells) was first studied (Obernier and Alvarez- Buylla, 2019). During embryonic development, RGCs in neocortex generate the majority of forebrain neurons (Obernier and Alvarez-Buylla, 2019). During embryonic development, RGCs in neocortex generate the majority of forebrain neurons (Merkle et al., 2004).
Meanwhile, at mid-embryonic stages (E13.5 to E15.5), a subpopulation of RGCs divides and generates the embryonic progenitors of B1 cells (pre-B1 cells) (Fuentealba et al., 2015).
These pre-B1 cells upregulate the cell cycle inhibitor p57 and remain relatively quiescent until they are reactivated in postnatal life (Furutachi et al., 2015). Vascular cell adhension molecule 1 (VCAM1) is expressed in these pre-B1 cells and loss of VCAM1 expression results in the depletion of the adult B1 cells pool (Hu et al., 2017). During late-embryonic stages (E15.5 to E17.5), RGCs adopt a quiescent state and transit into adult B1 cells, as they acquire a core transcriptional identity of adult B1 cells (Yuzwa et al., 2017).
In the DG, several groups have revealed important aspects on the origin of adult RGLs.Using fate-mapping analysis in Gli1-creER mice, Pleasure´s group has found that the ventral hippocampus generate a group of Sonic Hedgehog (Shh)-responding progenitors at embryonic stages that relocate into the dorsal hippocampus and are long-term RGLs (Li et al., 2013a). They suggested that progenitors from the ventral hippocampus are the origin of RGLs. However, the contribution of these cells at adult stages has not been estimated yet.
Recently, the group of H. Song has proposed a ¨continuous¨ model to explain the generation of adult RGLs based on tracing Homeodomain-only protein (Hopx) positive cells during the development of the DG (Berg et al., 2019). In this study, they have found that Hopx is specifically expressed in embryonic NPCs in the DG and then in most adult qRGLs. Using long-term clonal lineage-tracing, population fate-mapping and transcriptome analysis for DG Hopx+
As for adult NSCs in the SVZ, different studies have tried to reveal the time when quiescence is established during DG development. Ablation of dividing stem cells in mice progenitors, they have found a common neural precursor population that exhibits constant lineage specification from an early embryonic stage and continuously contributes to embryonic, early postnatal and adult DG neurogenesis (Berg et al., 2019).
for 1 week during adolescence (around P30) or adulthood does not have a lasting effect on the DG stem cell lineage (Kirshenbaum et al., 2014), suggesting that adult neurogenesis is established earlier on. However, Youssef and colleagues found that the adult RGL pool is depleted following ablation of proliferating cells during the first postnatal week of DG development, but not during the third postnatal week (Youssef et al., 2018), indicating that the critical time window for determining the size of RGL pool is during the first two postnatal weeks. Similarly, using long Brdu label-retention assay, it has been established that quiescence is acquired during the first postnatal weeks by finding that most of RGLs in P30 DG were generated from cell divisions that occurred during first two postnatal weeks, peaking from P3 to P7 (Berg et al., 2019). Additionally, the group of Pleasure observed that the Shh-responding progenitors, which have been proved to behave as adult RGLs, are significantly expanded their pool before entering quiescence during the first postnatal week, indicating the presumptive RGLs would also experience an expansion of their population during first postnatal week development of DG (Noguchi et al., 2019).
Furthermore, a large-scale single-cell RNA-seq analysis of DG cells has been performed throughout development into adulthood and offers new insight about the developmental origin of adult NSCs (Hochgerner et al., 2018). The data relies on a vast number of cells from perinatal, juvenile and adult DG, and on the use of two complementary platforms to rule out batch effects. Through comparison of cells from different developmental stages, they found many genes are significantly differentially expressed in between RGCs and RGLs, suggesting different molecular identity of RGCs and RGLs. In addition, through molecular identification, they observed no RGLs at P5 and no RGCs at P18, suggesting RGCs drastically switch to an adult RGL state during the second postnatal week (Hochgerner et al., 2018).
Apart from that, a fast maturation of GNs and mossy cells around the third postnatal week was described in this study. In adult neurogenesis, quiescence of RGLs can be regulated by the input from contra- and ipsi-lateral mossy cells and by long-range GABAergic projections from the medial septum (Bao et al., 2017; Yeh et al., 2018).
Previously, it has been suggested that the establishment of the commissural fiber tract of the DG around P15 (Fricke and Cowan, 1977; Ribak et al., 1985) might have an impact on DG development (Nicola et al., 2015). Therefore, it is conceivable that the postnatal changes in connectivity also contribute to the formation of the adult neurogenic niche in the DG and the generation of adult RGLs (Morales and Mira, 2019).
Taking together all the above, the first two postnatal week is indicated to be critical for adult RGL establishment.
5. SoxD transcription factors
5.1 Structures and subgroups of Sox transcription factors
Sox genes encode for a group of transcription factors with a high-mobility group (HMG)-type DNA-binding domain. The HMG domain allows Sox proteins to bind the minor groove of target DNA and additionally regulate transcriptional initiation of nearby promoters (She and Yang, 2017). The first described member of Sox family was sex-determining region Y (SRY) discovered in 1990 (Sinclair et al., 1990), the inactivation of which underlies sex development disorders (DSDs) (Gonen and Lovell-Badge, 2019). Since then, 20 different genes encoding SOX proteins have been identified in mice and humans (Schepers et al., 2002). Based on phylogenetical analysis of HMG box domains, SOX proteins have been divided into several subgroups named from SOX A to H (Kumar and Mistri, 2019) (Fig.
5). Members from the same subgroup share an overlapping function due to similar biochemical properties and co-expression in the same cell subtype (Wegner, 2010). Members from different subgroups have distinct biological functions even though they could recognize the same DNA consensus motif. This is mainly due to the fact that different SOX transcription factors have different affinity for particular flanking sequences next to consensus SOX sites and also they can have different binding cofactors adjacent to the cognate sequence of a co-motif (Hutchins et al., 2013).
5.2 Sox transcription factors and neurogenesis
Several subgroups of Sox factors have been reported to be sequentially expressed and involved in neural progenitors and neuronal cell lineages from embryonic to adult stages (Reiprich and Wegner, 2015; Bergsland et al., 2011). Sox2, a member of SoxB1 subgroups, is well known for its master roles in maintaining pluripotent embryonic stem cells and establishing neuroectodermal fate (Sarkar and Hochedlinger, 2013; Zhao et al., 2004).
Together with its relatives Sox1 and Sox3, all three SoxB1 members are broadly expressed in neural progenitors (Bylund et al., 2003; Graham et al., 2003) and they keep these progenitors in an undifferentiated state through counteracting proneural bHLH proteins, which are needed for neuronal differentiation (Bylund et al., 2003). Deletion of Sox2 in the mouse embryonic brain leads to completely loss of neural progenitors and neurogenesis during postnatal development (Favaro et al., 2009). In adult hippocampus, Sox2 is also
Figure 5. Schematic representation of structres of Sox family genes. Adapted from Bowles et al., 2000.
expressed in most RGLs and neural progenitors (Ferri et al., 2004). Conditional knockout of Sox2 in RGLs results in a depletion of the RGLs pool and impaired hippocampal neurogenesis (Favaro et al., 2009). Sox1 is also expressed in activated RGLs in the adult hippocampus (Venere et al., 2012), suggesting its potential role in regulating the activated pool of RGLs.
Aside from SoxB1 members, SoxE member Sox9 is initially expressed in neural progenitors around E10 in mouse spinal cord and then its expression broadly overlaps with that of SoxB1 genes (Scott et al., 2010; Stolt et al., 2003). Sox9 is required for the generation and maintenance of multipotent NSCs in embryos and in cultured neurospheres, and promote glial generation (Scott et al., 2010). Additionally, combination of Sox2 and Sox9 are essential to determine a NSC phenotype (Scott et al., 2010). Moreover Sox9 has a gliogenic function as embryonic loss of Sox9 in the spinal cord results in a transient increase in motoneurons and interneurons at the expenses of astrocytes (Stolt et al., 2003) and in adult SVZ, Sox9 also behaves as an inhibitor of neuronal differentiation (Cheng et al., 2009).
During neuronal fate specification, SoxB1 expression is downregulated and SoxB2 member Sox21 is induced to promote differentiation. In the ventricular zone, the induction of Sox21 depends on bHLH proteins and it is initiated in proliferating neural progenitors expressing SoxB1 proteins (Sandberg et al., 2005). In adult neurogenesis, Sox21 regulates the transition of IPCs from type2a to type 2b by repressing the transcriptional expression of the Hes5 gene (Matsuda et al., 2012).
Another Sox subgroup that has been extensively studied in neurogenesis is the SoxC subgroup, especially Sox4 and Sox11. They are highly expressed in newly specified neuroblasts during spinal cord development (Bergsland et al., 2006; Hoser et al., 2008) and in adult neurogenic niches (Haslinger et al., 2009; Mu et al., 2012). Overexpression of SoxC in spinal cord reveals their critical importance in the establishment of pan-neuronal protein expression (Bergsland et al., 2006; Jacob et al., 2018). Similar function of SoxC is also reported in adult neurogenesis, where they activate the pan-neuronal differentiation program of the proliferating cells (Haslinger et al., 2009).
5.3 Members of the SoxD subgroup
SoxD subgroup is comprised by Sox5, Sox6 and Sox13 in most vertebrates. They are characterized by containing two highly conserved functional domains: the Sox family- specific HMG box DNA-binding domain and the leucine zipper motif which allows for homo- and hetero-dimerization and consequently endows added flexibility in binding site selection (Lefebvre, 2010). The structures of the three SoxD genes and proteins are highly identical to each other. Human Sox5 and Sox6 genes are located in paralogous chromosomal regions on 12p12.1 and 11p15.3-15.2, respectively, and are more closely related to each other than to Sox13, located on 1q32 (Lefebvre, 2010). In human, Lamb-Schaffer syndrome is caused by heterozygous translocations and microdeletions disrupting Sox5. Patients from this syndrome show global developmental delay, intellectual disability, hypotonia, autistic- like features, and mild facial dysmorphism and skeletal malformations (Lamb et al., 2012) (Zawerton et al., 2019). In other patients with this syndrome additional loss-of-function variants, including nonsense ones, were subsequently reported (Nesbitt et al., 2015).
Members of SoxD group are widely expressed from embryonic to adult tissue in both non-neuronal and neuronal cells. Accordingly, they have shown critical regulations of many cellular processes. The functional redundancy of SoxD genes has been revealed in contexts where they have similar expression profiles.
5.4 Expression and functions of SoxD genes in non-neuronal cells
Extensive studies in chondrocytes have revealed important functions of Sox5 and Sox6. They are reported to be co-expressed with Sox9 in all chondrogenic sites of mice embryos (Lefebvre et al., 1998). Either Sox5 or Sox6 knockout mice exhibit relatively mild skeletal phenotypes, whereas Sox5/Sox6 double-knockout mice show severe chondrodysplasia, resulting in impaired skeletal growth and ossification (Smits et al., 2001).
Molecular analysis has proposed that Sox5/Sox6 binds to distinct but near sites from those of Sox9, and induced expression of Sox5/Sox6/Sox9 combination (SOX trio) could induce chondrocyte differentiation even in nonchondrogenic cells as they corporately activate major chondrocyte markers (Han and Lefebvre, 2008; Ikeda et al., 2005; Lefebvre et al., 1998; Liu and Lefebvre, 2015).
Sox5 is expressed in melanoblasts, and deletion of Sox5 can partially rescue the strongly reduced melanoblasts generation in Sox10 heterozygous mice (Stolt et al., 2008).
Sox6 is expressed in erythroid cells and promotes cell survival and proliferation in synergy with erythropoietin signaling and acts beyond erythropoietin signaling to facilitate erythroid maturation (Dumitriu et al., 2006; Yi et al., 2006). During cardiac and skeletal muscle differentiation, Sox6 facilitates the proper switch from slow to fast skeletal muscle fibers in late fetuses (Hagiwara et al., 2005). The analysis of fetuses with Sox13 gain-of-function and loss-of-function mutations has revealed that Sox13 critically controls the emergence of gamma-delta T cells in the thymus, while opposing alph-abeta T cell differentiation (Melichar et al., 2007). In conclusion, SoxD genes are capable of using various mechanisms to either enhance or repress transcription to modulate such varied processes as cell proliferation, survival and differentiation. As most Sox genes, SoxD genes exhibit redundant functions especially when they are expressed in the same cell population.
5.5 Expression and functions of SoxD genes in the CNS
SoxD transcription factors are also expressed in neural progenitors in the VZ and SVZ of the developing nervous system (Azim et al., 2009; Martinez-Morales et al., 2010; Stolt et al., 2006).
In the developing spinal cord, Sox5 and Sox6 are co-expressed in many neural epithelial cells (NEPs) and controlled multiple aspects of oligodendrocyte development (Stolt et al., 2006). Deletion of either Sox5 or Sox6 specifically induces premature appearance of Sox10-positive oligodendrocytes. Sox13 can also contribute to the inhibition
