structure of pallial proliferative layers in rodents (models of Takahashi-Nowakowski-Caviness and Ogawa-Kriegstein-Huttner), carnivores and primates (model of Kennedy-Kriegstein-Huttner)

cell cycle machinery

proneural/antineural machinery

lateral inhibition machinery

additional mechanisms dictating the apical-to-basal progression (intrinsic TFs, intrinsic ligands, extrinsic ligands)

generation of corticocerebral glutamatergic neurons

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UniTs-Neurobiology Degree Neuroembryology II course, AY 2014-2015 A.Mallamaci, SISSA

lesson 3: (1) generation of corticocerebral glutamatergic neurons: cell cycle machinery (2) generation of corticocerebral glutamatergic neurons: proneural/antineural machinery

cell cycle machinery

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control of population kinetics: cell cycle core machinery

the peculiar spatio-temporal expression profiles of cyclinE1 and kip1p27 make these two genes reasonable key candidates controlling dynamics of TC and q parameters in the cortico-cerebral PVE

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Kip1p27 upregulation in the medial rodent cortex promotes cell cycle exit

conditional over-activation of Kip1p27, by double transgenesis p(nes)-rtTA X p(TRE)-Kip1p27 and subsequent doxycyclin administration, in early (E12-14) progenitors leads to: - apparently unchanged cell cycle lenght - neuronogenic rates (Q) selectively increased in the medial cortex - rooting of E14 born neurons to upper layer fates

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medial cx

lateral cx

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regulation of supragranular layer neurons production by cyclinE and p27 in Macaca_I

*

* estimated assuming that cells entering the second half of G1 phase, distinguishable as expressing cyclin E at very high level, had decided to re-enter DNA synthesis

[Dehay and coll., Neuron 2005]

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regulation of supragranular layer neurons production by cyclinE and p27 in Macaca_II

cyclin D1 & Cdk4 promote generation and self-renewal of BPs_I

AP  =  Tbr2(-‐)  within  VZ  BP  =  Tbr2(+)  within  VZ  and  SVZ  N  =  Tbr2(-‐)  within  SVZ  and  Tbr2(±)  in  IZ/CP  

E13.5  +  24hrs  

co-elettroporation of CyclinD1 and Cdk4 into E13.5 neocortex elicited an expansion of the BP compartment, at expenses of the N one; the AP compartment was apparently unaffected

the fraction of co-electroporated cells expressing Tbr2 after 24 hours was upregulated in both VZ and SVZ

[Calegari and coll. (2009): Cell Stem Cell 5, 320–331]

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cyclin D1 & Cdk4 promote generation and selfrenewal of BPs_II

the expansion of the BP compartment was due to:

- accelerated “general” cell cycle progression by electroporated cells

- increased cell-cycle re-entry of BPs, as proven by reduced Tis21-EGFP expression by Tbr2+ electroporated cells  

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long-term morphogenetic consequences of cyclin D1 & Cdk4 overexpression

the BP compartment expansion elicited by cyclinD1/Cdk4 co-electroporation at E13.5 causes:

- a 30% increase of Brn2+ neurons populating layers IV-II of neocortex

- a tangential expansion of the region originating from the electroporated VZ patch, by a factor around 2.5

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proneural-antineural machinery

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proneural genes

They are transcription factor genes of the bHLH (basic helix-loop-helix) family, expressed in periventricular germinal layers of the neural tube and able to trigger neuronogenesis on

Within the early telencephalon, the pallium mainly expresses the Neurogenin genes, Ngn1 and Ngn2, related to Drosophila Biparous, the subpallium expresses Mash1, homologous to the Drosophila achaete-scute

Actually, Mash1 is also expressed, albeit at very low levels, in the hippocampal anlage and then elsewhere in the cortex, where it synergizes with Ngns in regulating progenitor kinetics.

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Results of this analysis suggest that proneural genes (Ngns and Mash1) share three fundamental functional activities:

(a) inhibition of cell cycle progression and promotion of cell cycle exit, plus promotion of precursors translocation from the VZ to the SVZ (i.e. the site where terminal neurogenic mitoses take place)

(b) activation of a basic pan-neuronal differentiation program, including switching on of early, mid and late neuronal differentiation markers (among which: classIII neuro-specific beta tubulin, MAP2, NeuN)

(c) inhibition of astroglial differentiation

Functional dissection of Mash1 and Ngn1/Ngn2 has been performed by:

-  inactivating these three genes via homologous recombination in vivo, in various combinations

-  overexpressing them via DNA electroporation in utero or into organotypic cultures

- overexpressing dominant negative forms of them (e.g., the Ngn2 alleles Ngn2Y241F and Ngn2NRAQ, unable to get phosphorylated at Y241 and to bind to DNA, respectively) in utero or into organotypic cultures

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…for their progression from the SVZ/IZ border to their final location within the CP….

..and for their glutamatergic differentiation

moreover, Ngn2 displays five more additional activities:

it is essential for proper pyramidal shaping of cortical projection neurons….

PMI = ratio between the width of the largest process and the total number of processes crossing a sampling circle of fixed diameter

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1

2 3

Ngn1&2 inhibit the ectopic massive activation of Mash1 in the pallium and the consequent spreading of ventral programs to the cortex

Ngn2 (like Ngn1) activates neuronal differentiation genes NeuroD1, NeuroD2 and Nex.

These bHLH genes, active in postmitotic neurons, are supposed to give rise to the functional cascade

and to self-activate.

They would promote late steps of neuronal differentiation in a highly redundant way

NeuroD1 NeuroD2 Nex

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4

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proneural genes perform their activity by forming heterodimers with ubiquitously expressed bHLH transcription factors of the E2 family and recruiting via a p300/CBP bridge the transcription activator complex to E-boxes of target genes

this functional module is common to neuronal differentiation genes as well as to tissue differentiation genes of other families (e.g. MyoD). Moreover, it is very ancient (Drosophila proneural genes achaete-scute and atonal heterodimerize with the ubiquitous product of daughterless and bind to E-boxes in a similar way)

proneural genes inhibit astrogliogenesis competing with Stat dimers for binding to limited p300/CBP available in vivo

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control of neuroblast population kinetics: BMP and Jak/Stat signalling

BMP signalling stimulate neuronogenesis and astrogliogenesis, at early and late corticogenetic stages, respectively.

To achieve these effects, BMP-dependent P-Smad1,4 form complexes with neuronogenic Ngn-E2 and gliogenic P-Stat1,3 dimers, respectively, via a CBP bridge

The higher and higher amounts of pStat1/pStat3 heterodimers available after E14.5-E under CT1/IL6 cytokine stimulation inhibit neuronogenesis, by competing with Ngns for binding to the necessary cofactor CBP/p300

on the other side, BMPs also play a distinct pro-proliferative and anti-neuronogenetic role, by promoting the expression of: (1) Id1,3 (2) Hes5

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phosphorylation of Ngn2 tyrosine 241 is specifically and absolutely necessary to promote pyramidal shaping and radial migration outside germinal layers, as shown by the results of in utero electroporation of the Ngn2Y241F allele, encoding for a dominant-negative, not-phosphorylable form of it.

It is believed that this phosphorylation may allow Ngn2 to bind to a specific co-factor making it competent to transcribe specific genes necessary for these functions

Remarkably, Y241 is peculiar to the mammalian (both eutherian and metatherian) Ngn2 (it is absent in Ngn1 and Ngn3 as well as in avian Ngn2). Its evolutionary appearence might be linked to the appearence of: (1)  the inside-out migratory rule in ancestor mammals (neurons of the avian dorsal telencephalon settle according to the more ancient outside-in rule…) (2) the numerical prevalence of projection neurons with a pyramidal shape in mammalian cortex as compared to sauropsids

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Ids (= “Inhibitors of DNA binding” or “Inhibitors of Differentiation”) as regulators of cortical progenitor kinetics

4 Ids are expressed in the developing early cortex: - Id1, ubiquitous - Id2 and Id4, more abundant laterally than medially - Id3, with a distribution complementary to Id2 and 4

functional characterisation of Id1 and Id3

In the absence of both Id1 and Id3, cortical neuronogenesis rates are abnormally higher, whereby the proliferating pool is exhausted earlier than normal and the embryo is microcephalic

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Ids: mechanisms of action

like their Drosophila extramacrochaete (emc) ortholog, Ids have an HLH (helix-loop-helix) but not a b (basic) domain. They play a pro-proliferative and anti-histogenetic role (anti-neuronogenetic and anti-oligodendrocytogenetic, but not anti-astrogliogenetic), via three main molecular mechanisms:

(a) similarly to Drosophila m. extramacrochaete, all Ids compete for binding to ubiquitous bHLH E proteins (E12, E47, E2-2, HEB) with bHLH proteins of the following families: - Ngns (proneural) - Mash (proneural) - NeuroD (neuronal differentiation) - Olig (oligodendrogenic)

(b) in the specific case of Id2 and Id4 (even Ids), they bind to Rb, stopping it from inhibiting E2F-dependent transcription of genes essential to DNA synthesis

(c) Id1-3 (odd Ids) inhibit Hes1 self-repression, by binding Hes1 and preventing it from binding to the N-box within its promoter, so potentiating Notch signalling within apical neural precursors

(c)

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