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\DOI{10.5802/crbiol.167}
\datereceived{2024-08-31}
\daterevised{2024-10-06}
\dateaccepted{2024-10-09}
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\CDRsetmeta{articletype}{review}

\title{Generating neurons in the embryonic and adult brain: compared
principles and mechanisms}

\alttitle{G\'{e}n\'{e}ration de neurones dans le cerveau embryonnaire
et adulte : principes et m\'{e}canismes compar\'{e}s}

\author{\firstname{Mathilde} \lastname{Chouly}}
\address{Institut Pasteur, Universit\'{e} Paris Cit\'{e}, CNRS UMR3738, Zebrafish Neurogenetics Unit, F-75015 Paris, France}
\address{Sorbonne Universit\'{e}, Coll\`{e}ge doctoral, 75005 Paris, France}

\author{\firstname{Laure} \lastname{Bally-Cuif}\CDRorcid{0000-0001-6611-6274}\IsCorresp}
\addressSameAs{1}{Institut Pasteur, Universit\'{e} Paris Cit\'{e}, CNRS UMR3738, Zebrafish Neurogenetics Unit, F-75015 Paris, France}
\email[L. Bally-Cuif]{laure.bally-cuif@pasteur.fr}

\begin{abstract}
Neurogenesis is a lifelong process, generating neurons in the right
amount, time and place and with the correct identity to permit the
growth, function, plasticity and repair of the nervous system, notably
the brain. Neurogenesis originates from neural progenitor cells (NPs),
endowed with the capacity to divide, renew to maintain the progenitor
population, or commit to engage in the neurogenesis process. In the
adult brain, these progenitors are classically called neural stem cells
(NSCs). We review here the commonalities and differences between NPs
and NSCs, in their cellular and molecular attributes but also in their
potential, regulators and lineage, in the embryonic and adult brains.
Our comparison is based on the two most studied model systems, namely
the telencephalon of the zebrafish and mouse. We also discuss how the
population of embryonic NPs gives rise to adult NSCs, and outstanding
questions pertaining to this transition.
\end{abstract}

\begin{altabstract}
La neurogen\`{e}se est un processus continu qui g\'{e}n\`{e}re des
neurones en quantit\'{e}, en temps et en lieu voulus et avec
l'identit\'{e} correcte pour permettre la croissance, la fonction, la
plasticit\'{e} et la r\'{e}paration du syst\`{e}me nerveux, notamment
du cerveau. La neurogen\`{e}se a pour origine des prog\'{e}niteurs
neuraux (PN), dot\'{e}s de la capacit\'{e} de se diviser, de se
renouveler pour maintenir la population prog\'{e}nitrice, ou de
s'engager dans le processus de neurogen\`{e}se. Dans le cerveau
adulte, ces prog\'{e}niteurs sont classiquement appel\'{e}s cellules
souches neurales (CSN). Nous examinons ici les points communs et les
diff\'{e}rences entre les PN et les CSN, dans leurs attributs
cellulaires et mol\'{e}culaires, mais aussi dans leur potentiel, leurs
r\'{e}gulateurs et leur lignage, dans les cerveaux embryonnaires et
adultes. Notre comparaison est bas\'{e}e sur les deux syst\`{e}mes
mod\`{e}les les plus \'{e}tudi\'{e}s, \`{a} savoir le
t\'{e}lenc\'{e}phale du poisson-z\`{e}bre et de la souris. Nous
discutons \'{e}galement de la fa\c{c}on dont la population de PN
embryonnaires donne naissance aux CSN adultes, et des questions qui
restent en suspens concernant cette transition.
\end{altabstract}

\keywords{\kwd{Neural progenitor}
\kwd{Neural stem cell}
\kwd{Zebrafish}
\kwd{Mouse}
\kwd{Pallium}
\kwd{Notch signaling}}

\altkeywords{\kwd{Prog\'{e}niteur neural}
\kwd{Cellule souche neurale}
\kwd{Poisson-z\`{e}bre}
\kwd{Souris}
\kwd{Pallium}
\kwd{Signalisation Notch}}

\thanks{ANR (Labex Revive), La Ligue Nationale Contre le Cancer (LNCC
EL2019 BALLY-CUIF), Fondation pour la Recherche M\'{e}dicale
(EQU202203014636), CNRS, INSERM, Institut Pasteur,  European Research
Council (ERC) (SyG 101071786 - PEPS), ED Complexit\'{e} du Vivant,
Sorbonne Universit\'{e}, LabEx Revive}

\maketitle

{\vspace*{6pt}}

\twocolumngrid

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\section{Introduction}

There are an estimated 100~billion neurons in the adult human brain,
70~million in the adult mouse, 10~million in the adult zebrafish
\cite{bib1,bib2}. Brain neurons control most body activities,
including sensory, motor, autonomic, emotional and executive functions.
How were they generated, and when? Are there common rules?


The generation of neurons, referred to as neurogenesis, is a multistep
and gradual process that originates from a neural progenitor cell and
ends with the formation of a mature cell capable of network\unskip\break
communication, the neuron. Along the way will occur cell divisions,
commitment to a neuronal fate, entry into the postmitotic state,
detachment and migration away from the progenitor territory, and the
acquisition of neuronal characteristics such as electrical
excitability, neurotransmitter(s) synthesis, an elaborated polar
morphology including axon and dendrites, and connectivity with other
cells (Figure~\ref{fig1}A). The sequential order of these steps is not fixed,
and the neurons generated are highly diverse, in identity, morphology,
plasticity, circuit and function. Increasing this diversity,
neurogenesis is a lifelong process, taking place from embryo to adult.
As such, acquiring a comprehensive understanding of neurogenesis
regulation is an immense task (for recent reviews, see
\cite{bib3,bib4,bib5}).


\begin{figure*}
\vspace*{-3pt}
\includegraphics{fig01}
\vspace*{-4pt}
\caption{Neurogenesis steps and nomenclature. (A) Progression of neural
progenitors along the neurogenic lineage (from left to right). The
relative order of cell cycle exit and neuronal subtype specification is
not fixed. (B) Cell types encompassed by the nomenclature
NP/NSCs, IPs and precursors in the embryonic vs.\  adult brain
and in mouse vs.\  zebrafish.\label{fig1}}
\vspace*{-7pt}
\end{figure*}


The present review will focus on the early steps of the neurogenesis
process, which cover the transition from a progenitor to the
post-mitotic and committed neuronal precursor. These initial events
largely control the location, timing and extent of neuronal generation.
As a driving thread, this review is also a comparative analysis of
embryonic and adult neurogenesis processes, with focus on Notch
signaling, the major regulatory pathway controlling neurogenesis during
a lifetime \cite{bib4,bib6}. This is because general principles are
better extracted from comparisons, and also because current
translational research places high hopes in the manipulation of neural
progenitors or in vitro models of embryonic characteristics to
understand or ameliorate adult pathologies of neurons loss. Finally, it
will largely make use of knowledge gained from the teleost fish model
\textit{Danio rerio} (zebrafish) which, among other practicalities,
offers the unequalled possibility to film neurogenesis ``in
situ'' in vivo in both the embryonic and adult brain
under fully non-invasive conditions, thanks to the transparency of its
embryos and of some pigmentation-deficient adults
\cite{bib7,bib8,bib9,bib10,bib11}. We will, of course, refer to other
models, in particular the mouse, when the latter led to the princeps
discovery or when interspecies comparisons add to the extraction of
principles. As much as extracting shared and divergent neurogenesis
principles between the embryonic and adult brain, we will aim to
identify remaining key questions in the field. 

\section{Neural progenitor cells}

\subsection{Definitions}

There is extensive work, associated with diverse and sometimes subtle
nomenclature differences, to characterize the progenitors at the origin
of neurons in diverse brain locations, time points or species. For
clarity, we will adopt a simple rule here, which essentially reflects
time, during life or along lineage progression. This nomenclature is
placed alongside others in Figure~\ref{fig1}B. In sum, we will refer to neural
progenitors (NPs) when cells can generate both neurons and glial cells
and their long-term neuron-generation potential is either limited
(typically a few weeks in a vertebrate model species such as zebrafish
or mouse) or unknown, to neural stem cells (NSCs) when cells can
generate both neurons and glial cells but their long-term potential is
extensive (typically a few months or more), and to intermediate
neuronal progenitors (IPs) for the proliferating progeny of NPs or
NSCs, that will exclusively generate neurons and will exhaust at short
term. With this nomenclature, neurogenesis occurs in the order
NP~$>$~IP~$>$~neuron or NSC~$>$~IP~$>$~neuron, and typically, NSCs are
an adult cell type, while NPs refer to neural progenitors in the
embryo. Indeed, even if some NPs have been shown to give rise to adult
NSCs and are therefore long-lived (see below), not all of them do, and
it is generally impossible to distinguish between these fates at the
time of observation. 

\subsection{Neuroepithelial cells and radial glia}

\subsubsection{Neural progenitors of the embryonic neural tube}


Neural progenitors (NPs) of the embryonic neural tube are apico-basally
polarized cells arranged in a monolayer bordering the neural tube lumen
(ventricle) (Figure~\ref{fig2}A,B) (reviewed in \cite{bib12,bib13}).
Their apical membrane, in contact with the cerebrospinal fluid, is
characterized by several specialized structures, such as a primary
cilium pointing into the lumen, a Cadherin ring, and junctional
complexes \cite{bib14,bib15,bib16,bib17}. The latter include tight
junctions marked by the Zona Occludens~1 (ZO1) protein that connect
with adjacent progenitors at their apico-basal interface and seal the
neural tube lumen. Their basolateral membrane is elongated and connects
with the pial surface of the neural tube. At early developmental stages
(tail bud to a few somite-stage in zebrafish, E7-E8 in mouse), neural
progenitors are neuroepithelial cells (NE) (Figure~\ref{fig2}A),
readily specified from the neuroectoderm. They express transcription
factors that sign both their neural and their progenitor states, such
as Sox2 or Hairy/Enhancer-of-Split (Hes/Her) family members
\cite{bib18,bib19,bib20}. As neurons become generated and the neural
tube thickens, the basolateral membrane of progenitors elongates. The
acquired radial morphology is associated with the expression of
astroglial markers in addition to progenitor markers, hence the name of
radial glia (RG) (Figure~\ref{fig2}B). These markers include
intermediate filaments (Vimentin; Glial fibrillary acidic protein,
GFAP) and the Brain lipid binding protein (BLBP). Both NE and RG~cells
are actively dividing and exhibit the characteristic feature of
interkinetic nuclear migration (INM), where the nucleus transits from
apical to basal positions and back within the cell body as a function
of cell cycle phases \cite{bib12,bib21}. Cytokinesis events occur
apically along the ventricular plane, and INM is believed to avoid
steric hindrance in this location for progenitor division to take
place. Embryonic~NE and~RG are very similar between species, although,
compared to mouse, zebrafish~NE polarize relatively late and express
glial markers almost from their onset \cite{bib22}, the transition from
NE to RG is therefore blurred. 

\begin{figure*}
\includegraphics{fig02}
\bcaption{\label{fig2}Morphology of NPs/NSCs and architecture of the neurogenic
niches in the embryonic vs.\  adult brain and in mouse
vs.\  zebrafish. (A--D) Schematic high magnification
views of the cytoarchitecture of the ventricular zone when observed in
cross sections (main panels), whole forebrain in cross-sections (small
panels), and legends for colors and structures (boxed).
(A,B) NPs at early embryonic stages (A) are neuroepithelial
progenitors (NE), they transform into radial glia (RG) at later
embryonic stages (B).}{1}{ (C,D) NSCs in the subependymal zone of
the mouse adult brain (C) and in the zebrafish pallium (D) are RGs.
Abbreviations: a, activated; AJC, apical junction complex; api mb,
apical membrane; BBB, blood--brain barrier; C, cortex; CSF,
cerebrospinal fluid; D, dorsal; IP, intermediate progenitor; lv,
lateral ventricle; NE: neuroepithelial cell; OPS, oligodendrocyte
progenitor cell; P, pallium; par, parenchyma; PVZ, periventricular
zone; q, quiescent; RG: radial glia; RMS, rostral migratory stream;
SEZ, subependymal zone; SP, subpallium; SVZ, subventricular zone; tc,
tela choroida; V, ventral; ven, ventricle; ven s, ventricular surface;
VZ, ventricular zone.}
\vspace*{-6pt}
\end{figure*}


\subsubsection{Neural stem cells of the adult brain}


Niches of constitutive neurogenesis have been identified in the adult
brain in all vertebrates studied to date (with ongoing controversies
for the human brain), although major differences exist in their
location and extent, activity, and lifetime. For example, these niches
are restricted to the forebrain in mouse, with three major sites
described that are especially active in the young adult: the
ventricular-subventricular zone of the lateral ventricle (vSVZ, also
called sub-ependymal zone, SEZ) (Figure~\ref{fig2}C), the sub-granular
zone of the dentate gyrus of the hippocampus (SGZ), and the ventricular
wall of the hypothalamus~\cite{bib23,bib24,bib25}. In contrast,
neurogenic niches exist in all brain subdivisions in the adult
zebrafish as well as in the retina and are active until a comparably
late age (considering the fact that the two species have the same
lifespan) \cite{bib26,bib27,bib28}. A major niche that has been
extensively studied is located in the pallium (dorsal telencephalon)
(Figure~\ref{fig2}D). We will restrict the following discussion to the
forebrain.


The majority of NSCs across species are radial astroglia, frequently
referred to as RG-like cells~\cite{bib29}. In most niches except the
SGZ, which is not ventricular, they maintain an apical contact with the
brain ventricle and cerebrospinal fluid. During interphase, this apical
membrane bears a primary cilium. The RG basal process contacts the pial
brain surface (in the case of small-sized brain territories) or blood
vessels at specific interfaces contributing to the blood--brain barrier
yet permitting systemic communication to NSCs~\cite{bib7,bib30}. This
basal process is generally extensive and highly branched sub-apically
or deeper into the parenchyma. Non-radial astroglial~NSCs also coexist
with radial~NSCs in the~SGZ, in slightly lower proportions
\cite{bib31,bib32}. Finally, like in the embryo, NE-like cells with
long-lasting neurogenic capacity, hence NSCs, have also been described
in the adult zebrafish brain. These different NSC subtypes share
expression of generic progenitor markers (e.g., Sox2) with variations
for others~\cite{bib33,bib34}. Astroglial markers, such as
cytoskeletal elements (Glial Fibrillary Acidic Protein, GFAP; Nestin;
S100beta in zebrafish), Fatty Acid Binding Proteins (e.g., Brain Lipid
Binding Protein, BLBP), or metabolic enzymes (e.g., Glutamine Synthase,
GS) also characterize RG-like and non-radial glial~NSCs, with some
regional differences~\cite{bib35}. It is important to note, however,
that these markers are often shared between physiologically neurogenic
vs.\  non-neurogenic cells types (e.g., between RG-like cells
and astrocytes in mouse) and should be associated with functional
assays assessing self-renewal and neurogenesis potential, at least at
the population level, to be conclusive.


\subsubsection{Markers and properties}


The overlaps and specificities of markers of NPs, NSCs and NEs are
listed in Figure~\ref{fig3}. The glial markers expressed by
embryonic~RGs encode factors relevant for known astroglial functions,
such as glutamate synthesis and metabolism. This suggests that
embryonic RGs may exert such roles in addition to their progenitor
properties. This point becomes very relevant when considering
adult~NSCs, as a major functional difference is observed in adult
forebrain astroglia between zebrafish and mouse. The zebrafish adult
forebrain is devoid of parenchymal astrocytes~\cite{bib36,bib37}. As a
counterpart, radial glia~NSCs do express markers of mature astroglia,
suggesting that they also are endowed with the dual properties of
progenitor cells and astroglia~\cite{bib38}. In contrast, these
functions are split between NSCs and parenchymal astrocytes,
respectively, in the adult mouse forebrain. Hence, zebrafish NSCs
appear multifunctional while mouse NSCs are specialized stem cells. It
should be noted that several observations argue against the fact that
zebrafish RG NSCs would simply correspond to RGs at an intermediate
state of maturation, which would not have chosen yet between the NSC
and astrocytic fates. First, adult zebrafish RG do exhibit
adult-specific features such as a long quiescence phase (see below).
Second, their markers of astrocytic functions are not or lowly
expressed in RG at juvenile stages, attesting to a maturation since
that stage~\cite{bib39}. Third, the choice towards an astrocytic fate
in fact occurs at a relatively early stage of mouse embryonic
development~\cite{bib40,bib41}. Extramural parenchymal astrocytes in
the adult mouse striatum can however be recruited for neurogenesis
following stroke or lesion, showing that astrocytes have conserved some
neurogenic capacity although not under physiological
conditions~\cite{bib42,bib43,bib44}.


\begin{figure*}
\includegraphics{fig03}
\caption{Radial glia markers in the embryonic and adult brain compared
to other progenitors and astroglia in zebrafish and mouse. (A--B)
Distribution of marker proteins between the different neuroepithelial
and astroglial cell types in the embryo (A) and adult (B) in zebrafish
and mouse (no species indicated when the markers are shared in both
species). In B, the yellow trapeze including astrocyte genes in mouse
reflects the expression gradient (low expression to high expression
from top to bottom) of the encoded factors between radial glia-like
cells (bona fide NSCs) and astrocytes (which are not neurogenic under
physiological conditions).\label{fig3}}
\vspace*{-6pt}
\end{figure*}


\subsection{Take home message}

The characterization of neural progenitor subtypes and their comparison
in the embryonic and adult brain lead to a refined understanding of the
NE to RG transition. NE and RG progenitors co-exist in the embryonic
brain, as this transition affects most, but not all, NEs. They also
co-exist at least in the zebrafish brain where they can still be
observed at adulthood. Instead of RG, the adult mouse brain hosts
specialized NSCs (RG-like cells) and parenchymal astrocytes.
Counterintuitively, zebrafish RG NSCs express markers of mature
astroglial function, which are the attribute of astrocytes but not
RG-like NSCs in mouse. 

\section{Lineages and niches}

Genetic tracing studies indicate that adult NSCs originate from
embryonic NPs, as opposed to, for example, the dedifferentiation of
mature neurons or glial cells in the adult brain. However, while
embryonic NPs distribute broadly along the neural tube ventricle, in a
pattern similar between vertebrate embryos in different species, adult
NSCs are restricted to niches and display a species-specific
distribution, much broader in zebrafish than in mouse. Furthermore, at
both embryonic and adult stages, NE and RG cells occupy selective and
exclusive locations. How are these patterns generated and their
temporal evolution controlled between embryo and adult stages?

\begin{figure*}
\includegraphics{fig04}
\bcaption{\label{fig4}Embryonic to adult neurogenesis in zebrafish and mouse. (A)
Temporal progression of neuroepithelial progenitors (NEs, blue), and
radial glia NPs and NSCs (green), represented on schematic
cross-sections (dorsal up) of the forebrain of zebrafish
(A$_{1}$--A$_{3}$, top) and mouse (A$_{4}$--A$_{6}$, bottom)
over a lifetime. Temporal nomenclature: E, embryo; L, larva; J,
juvenile; A, adult; hpf, hours post-fertilization; dpf, days
post-fertilization; E, embryonic day post-fertilization. In the
zebrafish telencephalon, the formation of an initially compact neural
rod (process referred to as ``keeling'') is then followed by the
lateral expansion of the dorsal neural territories and tela choroida
(process referred to as ``eversion''). In contrast, evagination takes
place in mouse to create the central canal. (B--D) Location of
neurogenic niches (RG, green; NE: blue triangles, also indicated by
blue arrows) on schematic sagittal sections of the brain in a
prototypical mouse or zebrafish embryo at mid-embryogenesis
(B) and in the zebrafish (C) and mouse (D)
adult brains (anterior left, dorsal up). Section planes (blue)
correspond to the cross-sections in A.}{3}{ Territorial nomenclature: am,
amygdala; c, cortex; ce: cerebellum; Dc, central pallium; Dd, dorsal
pallium; Dl, lateral pallium; Dm, medial pallium; Dp, posterior
pallium; hi, hippocampus; hyp, hypothalamus; l, lumen; lv, lateral
ventricle; mid: midbrain; MHB: midbrain--hindbrain boundary; fore:
forebrain; hind: hindbrain; nt, neural tube; P, pallium; ob: olfactory
bulb; ot: optic tectum; r1/2, rhombomeres~1 and 2; sp, subpallium; RMS,
rostral migratory stream; SEZ, subependymal zone; SGZ, subgranular
zone; v, ventricle.}
\end{figure*}

\subsection{A (constitutively) neurogenic lineage from radial glia}

Embryonic RG give rise to adult RG NSCs in the adult forebrain of mouse
and zebrafish (Figure~\ref{fig4}). Specific embryonic RG have been
traced genetically using Cre-lox recombination, for example using as
drivers the \textit{Gli1}, \textit{Hopx} or \textit{Nestin} promoters
for SGZ precursors~\cite{bib45,bib46,bib47,bib48}, promoters such as
\textit{Gli1}, \textit{Hopx} (from post-natal day~1 onwards),
\textit{Gfap} or \textit{Nestin} for SEZ precursors
\cite{bib45,bib49,bib50,bib51,bib52,bib53}, and the \textit{her4}
promoter for RG of the zebrafish adult pallium~\cite{bib33}. The
question of whether embryonic RG NPs maintain, or not, a sustained
neurogenic activity during the transition from embryo to juvenile to
adult has been addressed recently. The use of conditional recombination
in zebrafish, together with the fact that the neurons generated in the
pallium over a lifetime neither migrate nor die at a detectable rate,
made it possible to observe that neuronal clones issued from individual
embryonic RG can expand continuously across the pallial parenchyme.
These results demonstrate the existence of RGs that maintain
\textit{her4} expression from embryo to adult and are continuously
neurogenic, showing that a continuous neurogenic lineage ensures the
transition from embryonic RG NPs to adult RGL NSCs \cite{bib33,bib54}.
The same conclusion was later reached in the mouse dentate gyrus for
SGZ NSCs~\cite{bib50}. The SEZ appears to contrast with this situation,
as embryonic RG along the 4th ventricle and destined to populate the
SEZ enter quiescence at an early stage~\cite{bib51,bib52,bib55}.
Whether this reflects a bona fide interruption of neurogenesis between
embryonic stages and adulthood, a heterochronic quiescence entry
between brain territories, or an RG behavior that may have been missed
in other brain territories, remains to be assessed precisely.

\subsection{An embryonic to adult NE lineage at the origin of radial
glia, neurons and growth}

NE populations are positioned at the boundary between neural tube
subdivisions at mid- to late embryogenesis
(Figure~\ref{fig4}B)~\cite{bib56}. A few of them have been reliably
traced in the zebrafish using genetic Cre-lox labeling, and shown to
contribute to the persisting NE NSC populations identified in the
juvenile or adult zebrafish brain. For example, the small
\textit{her9}/\textit{her6}-positive NE population located on the
dorsal midline at the tel-diencephalic junction in the 24-h embryo
contributes to the NE population found along the postero-lateral edge
of the adult pallium (Figure~\ref{fig4}C) \cite{bib33}. Considering the
morphogenetic eversion of the pallium taking place in
teleosts~\cite{bib57}, this location in the adult telencephalon does
indeed correspond to the remnants of the dorsal midline, at the
junction with the choroid plexus. In the adult pallium, this NE pool
expresses \textit{her9}, is highly proliferative, generates neurons and
more of itself by amplification as well as neurogenic RG NSCs that
leave the NE zone to contribute to the progressive enlargement of the
RG population of the pallial ventricle \cite{bib33}. A similar scenario
was described for \textit{her5}-positive NE progenitors at the
midbrain--hindbrain boundary: embryonic NE progenitors located at the
MHB contribute to the NE NSC pool at the adult MHB, which generates
neurons and neurogenic RG NSCs that settle away from the NE pool in an
age-dependent order \cite{bib34}. As such, embryonic and adult NEs are
lineage related, and play multiple functions: they are neurogenic
lifelong generators of RG NSCs, and amplification centers that act as
growth zones. While NE pools are also found in the mouse embryonic
neural tube~\cite{bib58}, their adult equivalents have not been
analyzed as such. The cortical hem, however, is in a homologous
location to the tel-diencephalic dorsal NE pool in zebrafish, and
contributes neurons and scaffold glial cells to the
hippocampus~\cite{bib5,bib59}. Whether a NE remnant can also be found
in this location has not been studied.


\subsection{Sculpting NP and NSC niches}

In the embryonic neural tube, RG and NE NP populations occupy
alternating domains along the antero-posterior axis (and along the
dorso-ventral axis in the hindbrain and spinal cord), with RG pools
located in the center of neural tube subdivisions while NE pools are
located at neural tube boundaries~\cite{bib56}. This organization
corresponds to an alternation between actively neurogenic (RG) domains
and domains of delayed neurogenesis (NEs) which in fact already
pre-exists in the very early neural plate, prior to the
NE~{$>$}~RG transition that affects most NEs at
mid-embryogenesis and is prefigured in the expression domains of
\textit{her}/\textit{Hes} genes. As such, NP activity becomes
patterned according to the general vertebrate body plan, contributing
to the generation of the segmented embryonic neuronal scaffold of the
larva, which controls early larval functions. 


As discussed above, the lineage relation between embryonic and adult
RGs on the one hand, and embryonic and adult NEs on the other hand,
imparts a spatial organization to NSC niches in the adult brain. In
addition, in mouse, many RG populations become exhausted due to
terminal differentiation, leading RGL NSCs to persist in even more
restricted locations (Figure~\ref{fig4}D). A typical example is that of
the cortex: embryonic RG NPs terminate neurogenesis and terminally
differentiate into astrocytes around E18~\cite{bib60}, and this
contributes to isolating the SEZ and SGZ and non-contiguous neurogenic
niches in the forebrain. Together, the spatial organization of adult
NSC niches in the adult brain appears in part inherited from the
lineage relation between NPs and NSCs, plus an exhaustion of some RGL
pools between embryo and adult. This exhaustion varies between species,
it is prominent in mouse and restricted in zebrafish. Of course,
positive local cues (the so-called ``niche'') also promote the
persistence of adult neurogenic niches, such as in the SGZ and SEZ
\cite{bib61}.


\subsection{Take home message}

NSCs are progeny cells of embryonic NPs. A comparative view of NP
spatial organization in the embryo and adult sheds an ontological
perspective on the spatial pattern of adult neurogenic niches, showing
that it integrates a combination of events: spatial events that pattern
the embryonic neural tube and impart progenitor properties that have
long-term consequences on their progeny NSCs, and species-specific
temporal events of local progenitor exhaustion, that further refine NSC
niches to restricted domains in the adult brain.


\section{The embryonic and adult neurogenesis cascade(s)}

A primary molecular cascade controlling neurogenesis commitment is
Notch signaling. It has been extensively studied and reviewed, in
particular for its universal impact on embryonic neurogenesis
\cite{bib4,bib54,bib55}. The idea here is to mostly highlight its shared and
divergent features between embryonic and adult neurogenesis, and to
stress unknowns.


\subsection{Neurogenesis drivers}


In embryonic NPs, major neurogenesis drivers are basic
Helix-Loop-Helix (bHLH) transcription factors of the Neurogenin
(Neurog), Atonal (Ato), Achaete-Scute (Ascl) and NeuroD (specifically
Neurod4)\unskip\break families~\cite{bib62}. These factors trigger the expression
of a cascade of differentiation factors (such as Neurod1, 2 or 6) and
effectors of neuronal functionality or identity (cytoskeletal elements,
axonal differentiation, synaptic components, neurotransmitters etc)
\cite{bib63,bib64,bib65,bib66}. Importantly, they are also the prime
activators of expression of Notch ligand genes, thereby triggering
Notch signaling~\cite{bib67}. Expression of proneural transcription
factors is itself driven by neural Sox proteins such as Sox4 and Sox11,
themselves signing neuronal engagement within permissive domains
positive for Sox2~\cite{bib68,bib69}. Key parameters of this
transcription factors cascade are its timing and tempo relative to
other events such as cell cycle exit, progenitor delamination from the
neural tube ventricle, and migration. The activity of proneural
proteins is also modulated by phosphorylation, which can involve
Cyclin-dependent kinases (Cdk) and hence be linked with cell cycle
length. In particular, lower Cdk levels and cell cycle lengthening with
age as embryogenesis progresses provide a window of opportunity for
lower proneural protein phosphorylation levels and increased neurogenic
efficiency~\cite{bib70}. In addition, proneural proteins are
important counteractors of Notch signaling, discussed below.



\subsection{Notch signaling principles, actors and effectors} 


Notch signaling is a cell--cell communication cascade, the outcome of
which is to jointly regulate the state or fate of communicating cells.
It is based on the interaction between a set of transmembrane receptors
(Notch proteins [Notch1-4 in mammals, Notch1a, 1b, 2 and 3 in
zebrafish]) and transmembrane ligands (Delta proteins [Dll1-4 in
mammals, Dela-Deld in zebrafish] and Jagged proteins [Jag1-2 in
mammals, Jag1a, 1b and 2 in zebrafish]). When expressed by contacting
cells, the interaction between Notch and its ligand triggers the
transmembrane cleavage of Notch to generate an intracellular fragment
(NICD) that translocates to the nucleus, binds its interactor RBPj, and
regulates transcription. This pathway has been extensively reviewed
(for example: \cite{bib71,bib72,bib73,bib74}). 


Multiple levels of regulation exist that involve post-translational
modifications of Notch and its ligands, intracellular trafficking,
degradations and recycling, and mechanical forces, that have been\unskip\break
reviewed \cite{bib75,bib76,bib77,bib78}. An important feature here is
that transcriptional Notch signaling is direct and non-amplified, i.e.,
the receptor itself serves as the transcriptional regulator, and one
molecule of engaged receptor generates one molecule of NICD. Variations
in the outcome are encoded by the Notch receptor used, the specific
ligand--receptor pair that is engaged (see example below), the levels of
available receptors and ligands, the specific dynamics of NICD
interactions with the chromatin, and the cellular context (for example,
the RBPj-bound loci open in a particular cell state, or the set of
interactors available for Notch signaling targets). Another important
feature of the pathway is that Notch signaling is an auto-consolidating
system, as one output of signaling is to (directly or indirectly)
enhance the expression of the receptor and ligand themselves.


General NICD/RBPj transcriptional targets are genes encoding
transcription factors of the E(spl)/Hairy family (Hes and Hey in
mammals, Her and Hey in zebrafish). These are basic helix-loop-helix
(bHLH) transcriptional inhibitors that dimerize or pair with the
ubiquitous bHLH protein~E42 to downregulate target genes at E-boxes
(CANNTG sequences)~\cite{bib63,bib79,bib80}. They themselves are
among their main targets, thus terminating signaling. Some NICD/RBPj
bound sites have been identified in mouse; in the context of embryonic
NPs, they also include microRNA~miR-9, itself an inhibitor of the
neural progenitor state~\cite{bib81}; in adult NSCs, they include the
progenitor gene~Sox2~\cite{bib82}. In the context of embryonic NPs
and neurogenesis, Hes/Her themselves also inhibit expression of
proneural genes. Because proneural proteins also positively control the
expression of Notch ligands, this contributes to the consolidation of
unidirectional Notch signaling. It is to note that, among all these
interactions, very few direct transcriptional regulations have been
formally demonstrated. 


The main process involving trans interaction of Notch and a ligand is
lateral inhibition~\cite{bib83,bib84}. In embryonic NPs, it is
classically initiated by the expression of a proneural factor, which
upregulates expression of a Notch ligand. Signaling to a contacting
Notch-expressing NP drives Hes/Her expression which in turn
down-regulates expression of proneural and Notch ligand genes. This
regulatory mechanism imparts and stabilizes distinct committed
vs.\  progenitor fates to the Notch signaling vs.\ 
receiving cells, respectively. A contrasting trans-regulatory process,
lateral induction, has also been described in embryonic NPs
\cite{bib85,bib86}. There, Notch signaling in the receiving cell
triggers expression of a Notch ligand, which propagates signaling to
the neighboring cell, and so on from one cell to the next. This
regulatory mechanism propagates a cell state across a cell population
and has been described for example to define proneural domains of the
embryonic inner ear~\cite{bib87}. Classically, lateral induction
involves the Jagged ligand and an Hey1 effector, while lateral
inhibition preferably relies on Delta ligands and Hes/Her. Finally,
Notch and its ligands can also interact in cis, i.e., when expressed in
the same cell~\cite{bib88}. Cis interactions decrease the number of
receptors or ligands free to engage in trans interaction, thereby
regulating the directionality and intensity of trans signaling.


\subsection{Embryonic and adult neurogenesis: Notch signaling compared} 


Neurogenesis proper, i.e., the generation of a non-stem committed
progenitor that further progresses towards the neuronal fate, is a
transient event in the life of an adult NSC, which spends most of its
time in quiescence (Figure~\ref{fig5}A). Neurogenesis commitment can
take place in association with NSC activation and division (in one or
both daughter cells from a mother NSC), or independently of division.
The latter case has been described in the zebrafish adult pallium where
quiescent NSCs were observed to lose their astroglial character,
delaminate from the ventricular zone and directly differentiate into
neurons~\cite{bib10,bib11,bib89}. The mechanisms of direct
differentiation remain unknown, and we will focus here on the
generation of IPs from NSCs post-division.


\begin{figure*}
\includegraphics{fig05}
\caption{Cycling states, Notch signaling and fate choices during
embryonic and adult neurogenesis (A). Quiescence (G0) and mitotic
(G1-S-G2-M) cycles in neural progenitors. In the embryo, NPs are
constantly cycling. In the adult brain, NSCs spend most of their time
in quiescence and only infrequently activate to enter the mitotic
cycle. (B) Adult NSC in quiescence, and levels of Hes and Ascl1
proteins (after \cite{bib103}); quiescence is promoted by Notch2/3
signaling. (C) Fate choice in embryonic NPs or activated NSCs
post-division. These choices involve Notch/Delta signaling where Notch1
is the primary Notch receptor. Signaling can be oscillating (top) or,
as is probably the case for intralineage inhibition, directly oriented
(bottom).\label{fig5}}
\end{figure*}


Embryonic RG NPs use Notch signaling when engaged in neurogenesis. The
main proneural factors used in the forebrain are Neurog1 (ventrally)
and Ascl1 (dorsally), and the main signaling players recruited are
Notch1, DeltaA and Her4 in zebrafish (and Notch1, Dll1 and Hes1 in
mouse). Other pathway members can be co-expressed, with partially
redundant roles. For example, in the mouse embryo, it is necessary to
knock-out the three genes~\textit{Hes1}, \textit{3} and \textit{5} to
fully block neurogenesis~\cite{bib90}. Likewise, in the zebrafish
embryo, single mutants have partial phenotypes (e.g.,
\cite{bib91,bib92}). In adult neurogenic niches, dividing NSCs
express Notch1 and Her/Hes factors, and the conditional invalidation of
\textit{Notch1} or \textit{RBPj} in adult NSCs in the mouse SEZ and SGZ
leads to NSC loss and differentiation \cite{bib82,bib93,bib94}.
Likewise, invalidation of \textit{notch1b} in the zebrafish adult
pallium leads to a loss of dividing NSCs and an increased production of
neurons \cite{bib95}. The key proneural factor expressed in the adult
mouse SGZ and SEZ in dividing NSCs is Ascl1, and its direct requirement
for the acquisition of the neuronal fate is discussed (see below). In
the adult zebrafish telencephalon, where neurogenesis is found both in
the subpallium and pallium, expression of \textit{neurog1} and
\textit{ascl1a} is found in subpallial and pallial NSCs
\cite{bib96,bib97,bib98,bib99}. Thus, NPs and NSCs engaged in
neurogenesis largely use\unskip\break similar neurogenesis pathway components,
suggesting that the neurogenesis process involved during terminal
division in NSCs mimics the one at play in embryonic NPs. 


In addition, NPs and NSCs of the embryonic and adult brain rely on the
molecular oscillatory properties of the Notch pathway to promote the
generation of committed progenitors (Figure~\ref{fig5}B,C) (recently reviewed
in \cite{bib100,bib101}. Molecular oscillations are primarily driven
by the self-inhibition of Her/Hes proteins, which inhibit their own
transcription. In embryonic NPs, this leads to an oscillatory
production of Her/Hes proteins with a period of ${\sim}$2--3~h. In
turn, these oscillations drive offset oscillations of proneural
factors. Current evidence in the mouse and zebrafish embryos suggests
that these oscillations of antagonistic fate drivers maintain NP in a
plastic state poised for a fate decision, whether to remain an NP by
stabilizing Notch signaling, or to commit to neurogenesis by
stabilizing proneural expression. Several mechanisms have been
postulated to underlie this final choice. Among them, microRNA-9
(miR-9), itself in a negative transcriptional feedback loop with
Her/Hes factors but whose stability permits accumulation, may serve as
a dose-dependent ``clock'' mechanism that will tilt the equilibrium
towards neuronal commitment when above a threshold \cite{bib102}.
Another parameter is cell cycle length, which increases with time
thereby allowing changes in the activity of proneural factors and/or
cell cycle exit decisions (see below). 


Most interestingly, alternating oscillations of Hes1 and Ascl1 proteins
have also been observed (by means of Hes1-Venus and Ascl1-Venus fusion
proteins using the endogenous \textit{Hes1} and \textit{Ascl1}
loci) in dividing adult NSCs of the mouse SGZ
\cite{bib103,bib104,bib105}. The experimental induction of Ascl1
oscillations drives NSCs to enter the cell cycle and permits
neurogenesis, and Ascl1 itself is absolutely required for NSC
proliferation \cite{bib106,bib107}. Compared to embryonic NPs, it is
less clear in adult NSCs whether Ascl1 itself encodes the neuronal
fate, given that it appears very transiently expressed in IPs
\cite{bib31,bib108}, and that its overexpression rather induces
oligodendrogenesis \cite{bib109,bib110}. It also remains unclear how
oscillations drive proliferation, and how they are initiated and
stopped. Regarding proliferation, the co-expression of Ascl1 and NICD
may render the regulatory elements of proliferation genes accessible to
Ascl1, as observed in embryos \cite{bib111}. Regarding the initiation
of oscillations, it was noted that the \textit{Ascl1} mRNA is in
fact expressed at low levels in most NSCs but is not productive due to
post-translational destabilization of the Ascl1 protein
\cite{bib107}. This block is relieved pre-division, and this release
may be sufficient to initiate a default oscillatory behavior. Finally,
it is interesting to note that Notch1 seems to be the primary Notch
receptor involved in progenitor maintenance during the activated phase
of adult NSCs, both in mouse and zebrafish \cite{bib95,bib112}.
Together, these observations highlight similarities between embryonic
NPs and the activated and neurogenic state of adult NSCs.


\subsection{Cell cycle length and cell cycle exit}


In embryonic NPs, neuronal commitment and cell cycle exit are not
necessarily concomitant but are coupled in several ways. First, the
progressive lengthening of the cell cycle and decreased Cdk activity
during embryogenesis permit the accumulation of non-phosphorylated Sox2
(less active) \cite{bib113} and proneural proteins (more active)
\cite{bib70}, thereby promoting the switch from the progenitor to the
differentiated state. Cell cycle lengthening itself depends on a
Cdk-independent function of mitosis phosphatases such as Cdc25b
\cite{bib114}. In turn, proneural proteins, in addition to commitment,
can control the expression of cell cycle components. This is the case
of Ascl1 in mouse NPs in culture where, depending on the presence or
absence of Notch signaling in its expressing cell, it can activate the
expression of cell cycle-promoting genes (e.g., \textit{CyclinD1})
or of genes inhibiting cell proliferation \cite{bib111}. Second,
Cyclins can also participate in transcription complexes and/or directly
bind DNA to control the expression or activity of commitment proteins.
For example, CyclinD1 can associate with (and inhibit) NeuroD\unskip\break
\cite{bib115}. 

In adult NSCs, direct neuronal differentiation events from quiescent
NSCs have been observed \cite{bib10,bib11,bib89}. The mechanisms of
this fate decision, which temporally uncouples commitment from cell
cycle exit, remain to be identified. For IP-generating NSC divisions,
daughter cell fate choice is generally not made between cycling and
commitment, but rather between quiescence (G0) and commitment. We note,
in the mouse SEZ, the important role of two Cyclin-dependent kinase
inhibitors, p21 and p27. p21$^{\mathrm{Cip}}$, in addition to its cell
cycle-related function, inhibits expression of \textit{BMP2} and
\textit{Sox2}, preventing the exhaustion of SEZ NSCs otherwise taking
place through excessive expansion or astrocytic differentiation,
respectively \cite{bib116,bib117}. These inhibitions involve
Cdk-independent functions of p21, including direct binding to the
\textit{Sox2} enhancer. In epithelial cells in culture, endogenous p21
levels, which accumulate in G2, bias the fate of daughter cells towards
G0 vs.\  G1 \cite{bib118,bib119}. So far however, this was not
reported in adult NSCs. In the SGZ, p27 is necessary for NSC quiescence
and for cell cycle exit of IPs through its regulation of the cell cycle
\cite{bib120}, but it also directly binds to (and inhibits) the
\textit{Sox2} promoter to promote the transition of IPs to
differentiation \cite{bib121}. 

In the context of the relation between cell cycle components and
commitment, one key issue in both embryonic NPs and adult NSCs is to
understand how a different choice between commitment and progenitor
NP/NSC maintenance can be imparted in sister cells, in the case of
asymmetric divisions. Rather than driving self-renewal or commitment,
the factors above may open susceptibility states prone to fate choices,
which are then made in daughter cells following intralineage or
environmental cues (see below).


{\vspace*{-2pt}}

\subsection{Take home message}

{\vspace*{-2pt}}

Notch signaling, Hairy/E(Spl) factors and proneural proteins are major
determinants of the choice between progenitor maintenance and
neurogenesis progression in both embryonic NPs and active adult NSCs.
These pathways are intertwined with cell cycle regulatory components to
coordinate cell cycle exit and lineage progression. The quiescence
state of adult NSCs complexifies this regulation by introducing a
triple choice post-division: to re-divide, to re-enter quiescence, or
to commit.

{\vspace*{-2pt}}

\section{Division modes or how to balance progenitor maintenance and
recruitment}

{\vspace*{-2pt}}

\subsection{Division modes in embryonic NPs and adult NSCs}

{\vspace*{-2pt}}

We will consider here divisions relative to the generation of IPs (or
neurons when there are no intermediates). Hence, NSC/NSC and NP/NP
divisions will be called symmetric amplifying, NSC/IP or NP/IP
asymmetric, and IP/IP symmetric differentiative. 

In the embryo, NP divisions of the three types above were reported in
vivo (Figure~\ref{fig6}A). Typically, NE progenitors divide in a symmetric
amplifying manner, enlarging the NP pool. In contrast, RG NPs divide
asymmetrically to generate one RG and one IP or neuron
\cite{bib60,bib122}. In the zebrafish embryo, this process has been
filmed in the telencephalon and hindbrain \cite{bib123,bib124}. In
addition, at early stages, at least in zebrafish, some NPs divide
following a symmetric differentiative mode, generating two neurons used
to build the first larval neuronal scaffold \cite{bib125}. The
corresponding NPs are therefore lost in the process. 

\begin{figure*}
\vspace*{-4pt}
\includegraphics{fig06}
\vspace*{-4pt}
\caption{Lineage progression and division modes during embryonic and
adult neurogenesis. (A) In the early embryo, NEs divide symmetrically,
while the predominant division mode of RGs is asymmetric self-renewing
to generate one RG and an IP or a neuron daughter. At late
embryogenesis in the mouse, most RGs differentiate into astrocytes, and
some enter quiescence to give rise to adult NSCs. In zebrafish, it is
believed that most neurogenic RGs transit to the NSC state. (B)
In the adult, NSCs undergo NSC/NSC-generating divisions. NSCs also
progressively commit to give rise to NSCs that will generate IPs and,
in turn, neurons. In the zebrafish adult pallium, an upstream
self-renewing and asymmetric NSC/NSC division has been identified
\cite{bib128}, likely responsible for the homeostatic maintenance of the
NSC population. Symmetric divisions are drawn in purple, asymmetric
divisions in red. Abbreviations: a, astrocyte; IP, intermediate
progenitor; n, neuron; NE, neuroepithelial progenitor; aNSC, activated
neural stem cell; qNSC, quiescent neural stem cell; RG, radial
glia.\label{fig6}}
\vspace*{-5pt}
\end{figure*}

Likewise, the three division modes take place in adult NSCs, in both
the mouse and zebrafish forebrain (Figure~\ref{fig6}B) (reviewed in
\cite{bib27}). They have been directly observed using intravital
imaging in the adult mouse SGZ and zebrafish pallium
\cite{bib10,bib11,bib89,bib126,bib127,bib128}. Modeling clonal
dynamics in the zebrafish adult pallium predicts that NSCs choose
stochastically between these division modes, in specific proportions
\cite{bib89}. Intravital imaging tracking validated these
predictions, both regarding proportions and the apparently random order
in which symmetric amplifying and asymmetric divisions occurred in
tracks with multiple divisions \cite{bib128}. In contrast, in the
adult mouse SGZ, intravital imaging revealed non-stochastic choices,
e.g., the fact that symmetric amplifying divisions never followed
asymmetric divisions~\cite{bib127}. 

{\vspace*{-2pt}}

\subsection{Population vs.\  intralineage fate regulations, and
implications}

{\vspace*{-2pt}}

Daughter cell fate choices can be imparted by the mother cell (e.g.,
through the partitioning of fate determinants), organized between
daughter cells post-division through intralineage interactions, or
biased in a non-cell autonomous manner by the surrounding cells or
niche. To distinguish between these regulations, it is necessary to
trace individual NPs/NSCs in situ and modify their signal sending or
receiving properties. A major cell--cell signaling pathway involved is
Notch.


In the embryonic neural tube, the division of individual NE
progenitors has not been tracked; however, the fact that Notch
signaling---at least in the mouse neural
tube---becomes active at a relatively late stage, after the
initial NE amplification phase, strongly suggests that a lateral
inhibition process takes place between adjacent NEs irrespective of the
closeness of their relationship (Figure~\ref{fig5}C, top, and Figure~\ref{fig6}A)
\cite{bib129}. At the RG stage, blocking DeltaA expression in
individual RG of the telencephalon leads to neuronal differentiation of
all cells in each clone after just a few divisions, while control
clones maintain a RG identity \cite{bib123}. This approach prevents
Notch signaling from occurring between clonally related cells but not
with their non-clonally related neighbors (which express Delta, while
cells of the clone express Notch), allowing to conclude that division
asymmetry results from Notch signaling preferentially 
\mbox{occurring} \mbox{between}
\mbox{sister} cells. This has been called ``intralineage regulation''
(Figure~\ref{fig5}C, bottom, and Figure~\ref{fig6}A). It is likely also taking place in
the embryonic hindbrain. Further functional assays demonstrated that it
requires the asymmetric distribution of the Delta ubiquitin ligase
Mindbomb (Mib), itself permitted by the asymmetric distribution of Par3
at division \cite{bib123}, which follows DeltaD-carrying recycling
endosomes, in a complex that necessitates the centrosomal and endosomal
protein~PCM1~\cite{bib130,bib131}. In zebrafish (but not in mouse),
Mib and Par3 are inherited by the future neuron. In a parallel set of
studies, asymmetric distribution of DeltaD between sister cells was
observed during RG division in the embryonic zebrafish hindbrain.
DeltaD is transported by Sara endosomes, which are themselves
partitioned unequally at division~\cite{bib132}. What prevents cells
from interacting with their non-lineage related neighbors is not clear.
It could be that the mechanisms involved in partitioning Notch--Delta
signaling components between sisters at division dominate, in strength
or speed, the establishment of other interactions. Asymmetric
distribution of Dll1 protein was also observed (on static preparations
using immunostaining) in dividing RGs of the mouse embryonic\break cortex.

The mechanisms that account for terminal division asymmetry (NSC/IP or
NSC/neuron) in the adult brain have not been studied in vivo to date.
In cell pair assays in vitro, asymmetric Notch signaling levels can be
observed between mouse SEZ NSC daughters. This study identified that
Pigment epithelium-derived factor (PEDF), possibly released by
endothelial and ependymal cells of the niche, induces NFkB activation,
which itself leads to release of inhibition at Hes1 and EGFR promoters,
permitting Notch signaling and EGFR expression \cite{bib133}. This
mechanism, where differential proximity to PEDF might introduce
asymmetries between sisters, is superimposed on the effect of the Dyrk
phosphatase, which affects the stability of EGFR and decreases
self-renewal \cite{bib134}. In a possible link with this, asymmetric
distribution of the Dll1 protein was observed in GFAP${+}$ NSCs of the
mouse SEZ~\cite{bib135}. In Dll1${+}$ dividing mother cells (at
pre-cytokinesis stages), an asymmetric distribution of Dll1 was seen in
a majority of cases.\looseness=-1


\subsection{Take home message}

The control of the NSC/NP division mode (in relation with the
generation, or not, of an IP or neuron) relies on a combination of
intrinsic determinants, privileged sister-sister interactions or more
general local cues. Notch signaling is an important regulatory pathway
in all scenarios. Its implication in the control of division modes in
adult NSCs in vivo remains to be formally demonstrated.

\section{Lineage progression, conserved and changing progenitor
properties over time}

\subsection{Commitment status along lineage progression during adult
neurogenesis}

Our recent work based on intravital imaging to track NSC behavior in
the adult zebrafish pallium points to another level of complexity in
the control of the NSC division mode. Indeed, symmetric NSC/NSC
divisions were observed to generate differently fated NSC daughters
(Figure~\ref{fig6}B). Specifically, NSCs negative for the expression of
the Notch ligand DeltaA (\textit{deltaA}$^{\mathit{neg}}$) divide to
generate one \textit{deltaA}$^{\mathit{neg}}$ daughter NSC seemingly
identical to its mother, and one daughter that switches on expression
of deltaA (\textit{deltaA}$^{\mathit{pos}}$) and will further divide at
higher frequency to finally generate IPs~\cite{bib128}. The divisions
of \textit{deltaA}$^{\mathit{neg}}$ NSCs are therefore asymmetric in
terms of NSC fate, splitting daughter NSCs between self-renewal and
lineage progression. The mechanisms controlling this asymmetric
division mode remain unknown. Furthermore, the division of
\textit{deltaA}$^{\mathit{pos}}$ NSCs was seen to generate daughters
with progressively increasing levels of \textit{deltaA} expression,
possibly indicating progressive neurogenesis commitment during
divisions. These levels could differ between daughters, which may also
signify different commitment levels. Although our understanding remains
very limited, and heavily relies on our limited readouts of cell state
or identity, these observations together raise the fundamental question
of whether bona fide symmetric NSC divisions exist at all during adult
neurogenesis.

\subsection{The notion of self-renewal}

The notion of self-renewal is associated with the definition of a NSC,
hence largely with the adult context. Probing for it is however limited
by our experimental readouts. Whether molecular, cellular or
functional, these readouts are only very partial accounts of a cell's
state. It is within the frame of these technical limitations that
\textit{deltaA}$^{\mathit{neg}}$ NSCs of the adult zebrafish pallium
are currently considered to self-renew (Figure~\ref{fig6}B)
\cite{bib128}, but more precise analyses of these cells in the
long-term would be needed. In this context, ageing is an interesting
aspect to study. Zebrafish pallial NSCs are described to activate less
frequently in aged animals \cite{bib136,bib137}, but NSC state under
precise ageing conditions remains largely unexplored at the molecular
level. Changes in activation frequency and potential have also been
reported in aged NSCs in mouse~\cite{bib138,bib139,bib140,bib141}.
However, self-renewal proper has not been studied in either species.


Embryonic NPs, except for those that will ultimately give rise to
adult NSCs, are bound to exhaust after the generation of neurons. This
can occur after a fixed number of asymmetric divisions, as described in
the mouse embryonic cortex where RG undergo on average 8 divisions to
generate 8--9 neurons~\cite{bib142}. It is difficult to imagine that
daughter RGs arising at each division are strictly identical to their
mother until the end, as some mechanism must register each round of
cell division, and in fact, the duration of the cell cycle increases
over time. In the mouse embryonic cortex and retina, the transcription
factors expressed by NPs also change over time and divisions
\cite{bib143,bib144}, akin to the transcription factor series deployed
to generate different neuronal subtypes during nerve cord or optic lobe
neurogenesis during fly development \cite{bib145,bib146}.

\subsection{Quiescence and quiescence instatement}

A striking feature of adult NSCs, compared to embryonic NPs, is their
prolonged quiescence (Figure~\ref{fig5}A)~\cite{bib147}. Thus, to initiate the
neurogenesis process, adult NSCs first need to exit the quiescence
state, and their quiescence/activation balance is an additional level
of control compared to embryonic neurogenesis. 


Adult NSCs are stalled in G0, in which they spend most of their time.
In the adult mouse SGZ for example, tracing RG-like NSCs shows a mean
duration between two cell cycles (for dividing NSCs) varying between 1
and 90~days \cite{bib127}, and this amounts to a doubling time of
124~days for the highly quiescent NSC subpopulation in the zebrafish
pallium \cite{bib128}. Several pathways were identified to \mbox{control}
the \mbox{quiescence/activation} balance, among which Notch2/3 signaling, BMP
signaling, and Id factors promote quiescence
\cite{bib95,bib148,bib149,bib150,bib151,bib152,bib153}, while mTORC1
and EGF signaling and the transcription factor~Ascl1 promote
activation \cite{bib154,bib155,bib156,bib157,bib107}. In
detail, the Notch ligand DeltaA is expressed by IPs in the adult
zebrafish pallial niche, and IPs signal via Notch to prevent activation
in contacting NSCs \cite{bib158,bib159}. As mentioned above,
\textit{deltaA} is also expressed in the subpopulation of NSCs
engaged in neurogenesis, and these cells also maintain their quiescence
via Notch signaling, possibly tempered by a cis-regulation by DeltaA
\cite{bib128}. In zebrafish, BMP4, produced by pallial neurons, acts
in concert with Notch, possibly via the induction of expression of Id1
\cite{bib150}. Id4 plays a similar role in mouse \cite{bib149}.
Notch signaling and Ascl1 are particularly interesting to consider in
the context of this review, as they have been extensively studied in
embryonic progenitors and permit a direct comparison. While Notch2/3
maintain quiescence, Notch1 is expressed in activated NSCs and
maintains their progenitor potential, as the selective abrogation of
Notch1 function triggers neuronal differentiation \cite{bib93}. In
parallel, low levels of Ascl1 transcription are found in quiescent
NSCs, while higher and oscillatory expression of Ascl1 is sufficient to
trigger activation \cite{bib103,bib106,bib127}. Because the
involvement of Notch1 and Ascl1 oscillations are reminiscent of
neurogenesis control in embryonic NPs, these observations suggest that,
at adult stage, activated NSCs engaged in neurogenesis recapitulate an
embryonic process, and that new regulators have been superimposed on
this initial embryonic regulatory cascade to encode the quiescence
state in adult NSCs. 


Quiescence duration is highly variable between NSCs, with overall much
longer doubling times for NSCs of the
\textit{deltaA}$^{\mathit{neg}}$ self-renewing pool
(123~days) than for the
\textit{deltaA}$^{\mathit{pos}}$ neurogenic pool
(28~days) in the adult zebrafish pallium \cite{bib128}. In addition,
NSCs activate more or less rapidly upon Notch blockade, suggesting the
existence of different quiescence depths \cite{bib95}. Thus,
compared to embryonic NPs, adult NSCs not only display a quiescence
phase but different quiescence durations and/or depths. The regulation
of this heterogeneity is only beginning to be unraveled. It is a
complex task given that, in vivo, quiescence parameters such as
duration are linked with NSC progression along the lineage, hence these
aspects are difficult to disentangle. 


Finally, it remains important to understand how quiescence is instated
in NPs that transit to NSCs during life. Classically, quiescence is
measured at the population level as the proportion of NPs found in the
non-activated state (PCNA$^{\mathrm{neg}}$ or
MCM2/5$^{\mathrm{neg}}$) at any given time within a population.
With such measurements, quiescence became detectable between larval
day~5 and 8 in the zebrafish pallium (50\% PCNA$^{\mathrm{neg}}$
RGs) to progressively increase during the juvenile period to finally
reach adult levels (typically 5\% of PCNA$^{\mathrm{neg}}$ or
MCM2/5$^{\mathrm{neg}}$ NSCs)~\cite{bib160}. Individual
NPs/NSCs have not been tracked, however, and they divide
asynchronously. Thus, whether the increased proportion of
PCNA$^{\mathrm{neg}}$ or MCM2/5$^{\mathrm{neg}}$ NSCs with
age reflects the progressive increase of quiescence duration in each
cell over time, or the asynchronous entry of individual cells into an
adult-like duration of quiescence, cannot be concluded at this point.
If, as suggested above, quiescence is the result of the recruitment
over time of an additional molecular pathway (such as Notch2/3) by NPs,
another open question is what triggers the expression of this pathway.


\subsection{Take home message}

Lineage progression involves intrinsic changes in NP/NSC properties,
affecting their commitment level, the identity of the neurons they
generate, or, in the adult brain, the depth or duration of quiescence.
These changes occur concomitantly and are experimentally difficult to
disentangle. Another question that remains open is their relationship
with cell division. Steps in lineage progression are often observed to
take place concomitantly with cell division, e.g., with at least one
daughter appearing to differ from its mother. However, whether lineage
progression also occurs between cell divisions remains to be studied.


\section{Environmental/systemic/large-scale populational regulation}

NPs/NSCs and the neurogenesis process are controlled by cell-intrinsic
molecular and cellular events, as well as local cell--cell interactions,
as discussed until now. On a larger spatial scale, NPs/NSCs are also
embedded in a niche, which is a key regulator of their fate. 


An important component contacting NPs and NSCs in both embryos and
adults is the cerebrospinal fluid, largely produced by the choroid
plexuses and containing a spatially heterogeneous mix of bioactive
molecules (notably hormones and growth factors), peptides and
neurotransmitters that are either filtered from the blood or secreted
by the choroid plexuses themselves, the supracommissural organ,
tanycytes, or neurons. NPs/NSCs themselves release factors or vesicles
into the CSF~\cite{bib161,bib162,bib163}. Many of these factors,
whether brain-borne or blood-borne, in turn impact the proliferation or
commitment of NPs/NSCs or downstream steps of the neurogenesis process.
Steroid hormones, thyroid hormone, insulin, cortisol, growth hormone,
FGF/EGF etc, have been the focus of intense study in this context.
Hormones vary between the embryonic and adult contexts, also because in
embryos some are mother-derived (released during pregnancy in mammals,
or deposited in the oocytes in fish for example). They will not be
discussed here as they do not affect the principles of neuron
generation as such, but they are clear modulators of neurogenesis at
the systemic level. 


Additional to this external, systemic niche, our recent work in the
zebrafish adult pallium led us to postulate the existence of a
medium-scale ``intrinsic niche'' created, via spatiotemporal feedbacks,
by NSCs and IPs~\cite{bib164}. By restricting NSC activation in the
vicinity of lineage intermediates (IPs), a specific number of activated
NSCs is permitted at any given time, and is positioned in physical
space within the NSC population. Spatial modeling suggests that this
regulation is important to avoid spatial drifts in neuronal production
in the long-term~\cite{bib159}. At this point, the demonstrated
interactions are local, and whether and how they propagate across the
NSC population is expected to depend on the relation between the
frequency and duration of NSC states competent for activation and the
half-life of IPs. Long sub-apical protrusions have also been observed
in \textit{deltaA}$^{\mathit{pos}}$ cells that could signal at a
distance~\cite{bib128}. An interesting aspect of these findings is
that spatial signaling is temporally linked with lineage progression.
Thus, in the asynchronous adult NSC population where IPs are also
short-lived (and leave the germinal population upon differentiation),
this allows a self-perpetuating system to emerge where lineage
termination allows lineage initiation from another NSC. The
spatio-temporal coordination of NSC fate decisions, other than their
activation, remains to be tested, as well as the relevance of other
signaling pathways, whether relying on cell--cell interactions,
reaction--diffusion processes, or mechanical information. Finally, in
addition to this spatio-temporal regulation at the individual cell
level, it is interesting to wonder whether the intrinsic niche also
includes spatio-temporal feedbacks at a clonal level. Such regulations
akin to competition processes have been described in non-follicular
epidermal SCs of the mouse adult skin~\cite{bib165}. 


In the embryonic central nervous system, the occurrence of
cross-interactions between NPs to control the position of neuron
generation in physical space is a well-known property of lateral
inhibition. In contrast to the adult pallial NSC pools, obviously, it
affects neurogenesis commitment rather than quiescence exit. In
addition, Notch-Delta signaling via horizontal protrusions exhibited by
NPs were recently described to space out neuronal differentiation in
the zebrafish embryonic hindbrain, showing that lateral inhibition can
also generate longer-distance patterns~\cite{bib166}.




\subsection{Take home message}

In addition to systemic or local signals from non-NPs/NSCs, the fact
that NPs/NSCs are often found in groups allows inter-progenitor
interactions. This ``intrinsic niche'' is responsible for the
organization of neurogenesis progression in physical space over time. 

\section{Conclusion}

The core principles and molecular regulators of neurogenesis are shared
between the embryonic and adult central nervous systems, with some
adaptations in spatial organization and speed. The latter is much
slower in the adult compared to the embryonic brain, due to a number of
factors including the existence of an NSC quiescence phase and a
lengthened progenitor commitment phase. Overall, the neurogenesis
process is better understood, because experimentally more easily
tractable, in the embryonic than in the adult brain. But the next step
and recurrent remaining issue is to understand transitions: how are
cell identities gradually changed, how is cycling behavior modified (to
introduce quiescence), how are lineages prolonged (to introduce
transient progenitor states) in the transition from embryo and adult?
Are these transitions abrupt or gradual, what are their regulators, and
how is a new equilibrium reached that will characterize the adult
stage? Future work along these lines will be highly informative for
understanding how dynamic pattern and tissue generation concert over
time to result in tissue homeostasis. 


\section*{Abbreviations}

\begin{inftab}
\begin{tabular}{ll}
\tbody
bHLH &  Basic helix-loop-helix domain  \\
BLBP &  Brain Lipid-Binding Protein \\
BMP &  Bone morphogenetic protein  \\
Cdk &  Cyclin-dependent kinase  \\
CSF &  Cerebrospinal fluid  \\
Dll1 &  Delta-like 1  \\
EGF &  Epidermal growth factor \\
EGFR &  Epidermal growth factor receptor \\
FGF &  Fibroblast growth factor  \\
GFAP &  Glial fibrillary acidic protein \\
GS &  Glutamine synthase  \\
Hes/Her &  \parbox[t]{6cm}{\raggedright Hairy/Enhancer-or-Split transcription factor}\vspace*{2pt} \\
INM &  Interkinetic nuclear migration  \\
IP &  Intermediate neuronal progenitor \\
MHB &  Midbrain--hindbrain boundary  \\
NE &  Neuroepithelial cell   \\
NICD &  Notch intracellular domain  \\
NP &  Neural progenitor  \\
NSC &  Neural stem cell  \\
PEDF &  Pigment epithelium-derived factor \\
RG &  Radial glia  \\
SEZ &  \parbox[t]{6cm}{\raggedright Sub-ependymal zone of the lateral ventricle}\vspace*{2pt} \\
SGZ &  \parbox[t]{6cm}{\raggedright Sub-granular zone of the dentate gyrus of the hippocampus}\vspace*{2pt} \\
ZO1 &  Zona Occludens~1
\end{tabular}
\end{inftab}


\section*{Methods}
Images in Figures~\ref{fig2}, \ref{fig4}, \ref{fig5} and \ref{fig6}
were generated using BioRender (with a license from Institut Pasteur).

\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Author contributions} 
Conceptualization and writing---original draft---:
LB-C; review \& editing: MC, LB-C; illustrations: MC. 

\section*{Funding} 
Work in the LB-C laboratory was funded by the ANR (Labex Revive), La
Ligue Nationale Contre le Cancer (LNCC~EL2019 BALLY-CUIF), the
Fondation pour la Recherche M\'{e}dicale (EQU202203014636), CNRS,
INSERM, Institut Pasteur and the European Research Council (ERC) (SyG
101071786 - PEPS). MC was a recipient of a 3-year PhD student
fellowship from ED Complexit\'{e} du Vivant, Sorbonne Universit\'{e},
Paris, complemented for one year by LabEx Revive.

\CDRGrant[LNCC]{LNCC~EL2019 BALLY-CUIF}
\CDRGrant[FRM]{EQU202203014636}
\CDRGrant[ERC]{SyG 101071786 - PEPS}

\section*{Acknowledgments} 
We thank the ZEN team for generating some of the important scientific
data mentioned in this review, and for regular discussions.

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