Sequential phases of cortical specification involve Neurogenin-dependent and -independent pathways

Question 1

Which three TFs act to pattern the telencephalon and establish a cortical identity?

Answer 1

Lhx2, Emx2, Pax6

Question 2

Which TF directly activates Ngn1 and Ngn2?

Answer 2

Pax6

Question 3

E13.5: N2-/- and N1/N2-/- show deregulation in genes expressed in which cell populations?

Answer 3

Downregulation of genes involved in cortical glutamatergic neurons (Tbr1, Tbr2, NeuroD2, NeuroD, Nscl1, Math2) in N2 single mutant. Greater down regulation in N1N2 mutant. No deregulation of genes involved in cortical progenitors (Otx1, Foxg1, Gli3, Pax6, Emx2, Lhx2, Tlx, COUP-TF).

Question 4

Describe what happens to VGLUT1 protein and VGLUT2 transcript levels in N2 and N1/N2 mutants.

Answer 4

Both are decreased in dorsomedial domains in N2 mutant, but persist in lateral cortical neurons/differentiating neurons likely due to persistent N1 expression there (which compensates). Loss of both in double mutant.

Question 5

What do the Ngns activate?

Answer 5

Cortical- and glutamatergic-specific differentiation programs in early-born CP neurons, likely acting downstream of cortical patterning genes.

Question 6

N2 and N1/N2 mutants show UPregulation of which set of genes?

Answer 6

Subcortical genes: ventral telencephalon TFs (Mash1, Dlx1, Dlx2, Dlx5, Brn4), biosynthetic enzymes for GABA (GAD1, GAD2), GABA transporters (GABA-T1), GABA and glycine transporter (GABA/glyT). Ectopic activation of subcortical GABAergic differentiation program.

Question 7

Were the ectopic, ventral like neurons in the PP/CP of cortical or subcortical origin?

Answer 7

Cortical. No inappropriate migration. They were misspecified due to loss of Ngns. Also, GAD1 and VGLUT1 detected in complementary sets of neurons, suggesting neurons choose between these programs.

Question 8

Was ectopic Mash1 expression responsible for specification defects in Ngn mutant cortices?

Answer 8

No; E13.5 similar reduction in transcription of cortical-specific neurons observed in Ngn2 and Ngn2;Mash1 mutants.

Question 9

How do we know that Ngns repress ventral-specific genes in at least a partially Mash1-independent way?

Answer 9

Because in Ngn2;Mash1 mutant, we still see elevated Dlx1, Dlx2, Brn4 levels. If there were Mash1 dependency in this, loss of Mash1 would prevent up regulation of these ventral genes.

Question 10

Is there Mash1 dependence of Ngns on the expression of some ventral markers?

Answer 10

Yes; ectopic expression of Dlx5, GAD1, GAD2, GABA-T1, GABA/glyT in Ngn2 mutant cortices was dependent on Mash1 presence.

Question 11

T/F: In N2 single mutant, you lose N1 expression in rostral cortex.

Answer 11

True. Does this mean N2 activates N1 expression here???

Question 12

E15.5 (mid corticogenesis): in rostral cortex of N2 mutants (where N1 expression is selectively lost), and throughout entire cortex of N1/N2 double mutants, what do we see?

Answer 12

We see gaps in the expression of cortical markers in the DEEP cortical plate (Id2, Robo1, Slit1, Math2, Tbr1). This suggests Ngns are required for proper cortical identity of deep-layer neurons. Not due to loss of neurons or increased cell death.

Question 13

What does VGLUT2 label?

Answer 13

Transiently labels glutamatergic neurons migrating through SVZ.

Question 14

Does the responsiveness of cortical progenitors to ectopic Mash1 change over time?

Answer 14

Likely; increasing Mash1 expression levels was NOT sufficient to respecify cortical neurons born AFTER E14.5. Whereas previously, E13.5 progenitors ectopically expressed some ventral markers in response to ectopic Mash1.

Question 15

What happens to many of the misspecified Dlx1+/GAD1+ neurons?

Answer 15

They do not incorporate into CP; they aggregate into clumps in germinal zone/intermediate zone/marginal zone.

Question 16

What did BrDU labeling at E12 show in mutants?

Answer 16

Wildtype: Accumulation of strong BrdU+ nuclei in layer V!. In mutants, increased darkly-labeled neurons blocked in GZ/IZ and in MZ, corresponding to sites of aggregation of misspecified Dlx1/GAD1+ neurons (confirming that many early-born misspecified neurons were not integrated in CP)

Question 17

What did BrDU labeling at E14 show in mutants?

Answer 17

Wildtype: marked genesis of layer iV and V. In mutants, clear decline in dark nuclei in lower CP, and accumulation in GZ. Subset of neurons destined for layers IV-V aggregated abnormally in deep positions in mutant CPs. Unperturbed upper layer neurons.

Question 18

Specification defects affect neurons in N1/N2 mutants arise during which stages?

Answer 18

Specification defects restricted to cortical neurons born between E12-14.

Question 19

What was revealed when the authors did anterograde tracing of corticofugal projections of layer V/VI in mutant animals?

Answer 19

Strongly reduced axonal numbers in Ngn2 mutant vs wild type at E16. Consistent with selective specification defects in lower-layer neurons. Lower layer neurons in mutants thus have molecular and axonal projection defects.

Question 20

What was the phenotype of P5 Ngn2 mutant cortices?

Answer 20

Smaller neocortex. Striking layer VI defects: reduced Slit1, Tbr1, Id2 expression in rostromedial CP. More modest layer V defects (likely due to Ngn1 persistence). Normal Layer IV (ROR-B), II-II (Oct6, Id2) appearance.

Question 21

Which two factors coordinate to specify the cortical-subcortical boundary?

Answer 21

Pax6 and Tlx

https://dev.biologists.org/content/130/6/1113

Question 22

How did Ngn2 mutants differ from Pax6 and Pax6;Tlx mutants mid-corticogenesis (E15)?

Answer 22

Pax6 and Pax6;Tlx mutants had perturbed cortical regional identity and glutamatergic phenotypes at this stage, whereas defects are rescued in Ngn2 mutants at this time.

Question 23

What happens to many Pax6 mutant CP neurons?

Answer 23

Do not migrate appropriately, becoming trapped in expanded SVZ.

Question 24

What did they notice of neurons trapped in SVZ in E15.5 Pax6 and Pax6;Tlx mutants?

Answer 24

These neurons ectopically expressed Dlx1 and GAD1. This suggests that neurons born during mid-corticogenesis acquire a subcortical phenotype, contrast to normal regional identity of E13.5 born neurons.

Question 25

E18.5 analysis of CP in Pax6 and Pax6;Tlx mutants showed what?

Answer 25

Math2 labeled thinner CP. IZ almost completely devoid of Math2 transcripts, suggesting many CP neurons generated during mid-late corticogenesis did not acquire correct cortical identity or migrate into CP.

Question 26

What can we say about Pax6 and Tlx function in late corticogenesis based on this work?

Answer 26

Pax6 and Tlx both act at late stages of corticogenesis to promote a cortical identity and suppress a subcortical GABAergic phenotype.

Question 27

What was noted about the laminar identities of Pax6 and Pax6;Tlx mutants?

Answer 27

In contrast to Ngn mutants, NO defects in lower layers V and VI. But, layer IV not detectable in double mutants, and absent Cux1/Oct 6 (layer 2-3) absent in double mutants. Both must be mutated to lose upper marker expression, suggesting they cooperate.