Her6 regulates the neurogenetic gradient and neuronal identity in the thalamus

Abstract

During vertebrate brain development, the onset of neuronal differentiation is under strict temporal control. In the mammalian thalamus and other brain regions, neurogenesis is regulated also in a spatially progressive manner referred to as a neurogenetic gradient, the underlying mechanism of which is unknown. Here we describe the existence of a neurogenetic gradient in the zebrafish thalamus and show that the progression of neurogenesis is controlled by dynamic expression of the bHLH repressor her6. Members of the Hes/Her family are known to regulate proneural genes, such as Neurogenin and Ascl. Here we find that Her6 determines not only the onset of neurogenesis but also the identity of thalamic neurons, marked by proneural and neurotransmitter gene expression: loss of Her6 leads to premature Neurogenin1-mediated genesis of glutamatergic (excitatory) neurons, whereas maintenance of Her6 leads to Ascl1-mediated production of GABAergic (inhibitory) neurons. Thus, the presence or absence of a single upstream regulator of proneural gene expression, Her6, leads to the establishment of discrete neuronal domains in the thalamus.

Fig. 1.

The neurogenetic gradient in fish. Glutamatergic neurogenesis spreads in a wave from posterior to anterior in the developing thalamus. Analysis of the dynamic expression of proneural genes during the development of the thalamic complex by in vivo imaging of double transgenic zebrafish and double in situ hybridisations. Upon induction of shh:GFP in the MDO, neurog1:RFP is induced first in the posterior Th (A, arrowheads). Over time, the caudal thalamus is filled with neurog1 positive cells (D and G). At the 20-somite stage, neurog1 mRNA can be detected within the thalamic complex (B). Over time, the expression increases from posterior to anterior and fills the entire cTh at 33 hpf (E and H). At 20 somite stage, her6 expression marks the entire thalamic complex (B and C) and is gradually down-regulated in the caudal part over time. her6 expression is maintained in the PTh, MDO, rTh and absent from the cTh at 33 hpf (H and I). The expression domains of her6 and neurog1 are abutting. In contrast to neurog1, the proneural gene ascl1a is induced from ventral to dorsal within the her6 positive PTh and rTh from the 20-somite stage (C), to 24 hpf (F) and 36 hpf (I). Embryos are shown laterally, white double-headed arrows indicate the increasing width of the cTh. III, third brain ventricle; cTh, caudal Thalamus; MDO, mid-diencephalic organizer; PTh, prethalamus; rTh, rostral Thalamus; ss, somite stage.

Fig. 2.

Her6 is required to repress the proneural gene neurogenin1. In vivo analysis of the loss of Her6 function in double transgenic zebrafish embryos by confocal microscopy. Knock-down of her6 (her6MO) leads to an increase of neurog1 expression in the thalamic complex at 27 hpf (62/86; A and B). At 33 hpf, lack of Her6 function leads to a massive up-regulation of neurog1 in the entire thalamic complex including PTh and MDO (72/91; C and D). The Shh positive MDO is fragmented. Up-regulation of neurog1 leads to a down-regulation of markers, such as dlx4/6:GFP in the PTh at 36 hpf (53/78; E and F). Similarly, the expression of the rTh marker tal1:GFP is completely lacking in her6 morphant embryos 42 hpf (23/28; G and H). By delivering the her6 MO antisense oligomers into a few cells of the rTh by electroporation, neurog1 expression was induced cell-autonomously, as shown by the expression of RFP tagged with a nuclear localisation sequence (5/11; J, higher magnification of the electroporated cell clone marked by arrowheads in J′). Electroporation of a control MO showed no effect (I and I′). III, third brain ventricle; ACeV, anterior cerebral vein; cTh, caudal thalamus; PTh, prethalamus; PTec, pretectum; rTh, rostral thalamus.

Fig. 3.

Maintenance of Her6 is required of fate of the rTh. Mis-expression of her6 represses neurog1 expression and activates rostral thalamic fate. Activation of a heat-shock inducible construct driving her6 and the lineage tracer GFP leads to the cell-autonomous down-regulation of neurog1:RFP at 33 hpf (9/15; B and B′; arrowheads). If Her6 lacks the Groucho interaction motif WRPW, the construct does not alter neurog1:RFP expression in a control experiment and shows a co-localization of GFP and nuclear-localized RFP (8/12; A and A′; arrowheads). Knock-down of grg1 resembles the her6 morphant phenotype (23/43; D and E) and leads to an increase in the caudal expression of neurog1:RFP (white arrowhead) as well as a decrease of shh:GFP in the MDO (yellow arrowhead) at 30 hpf. Clonal overexpression of her6 and the membrane-bound red lineage marker mCherry in the cTh leads to the activation of the GABAergic rTh marker tal1:GFP (14/21; I; arrowhead). Notably, dividing cells at the ventricular surface are only positive for the mCherry lineage marker (asterisk) but become tal1:GFP positive after radial migration (arrowhead). Cells containing the lineage tracer only do not express Tal1 (H). III, third brain ventricle; cTh, caudal thalamus; PTec, pretectum; PTh, prethalamus; MDO, mid-diencephalic organizer; rTh, rostral thalamus.

Fig. 4.

Her6 acts to genetically suppress neurog1. Analysis of ascl1a and irx1b, a pan-thalamic marker, at 30 hpf (A–D) and vglut2.1 and GAD65/67 at 48 hpf (E–H) in her6 and neurog1 morphant embryos. Knock-down of her6 leads to the down-regulation of ascl1a (45/61; A and B) as well as GAD65/67 (22/35; E and F) in the rTh (arrowheads) and in the PTh. Knock-down of neurog1 leads to an unaltered PTh but an increase in width in ascl1a expression (22/34; C) as well as GAD65/67 expression (12/21; G) in the rTh (yellow arrows). Compared to the her6 single knock-down, double knock-down of her6 and neurog1 rescues the prethalamic as well as the rostral thalamic expression domain (ascl1a: 17/22; GAD: 8/17; D and H). Analysis of the MDO in shh:GFP transgenic animals co-expressing ubiquitously the membrane-bound fluorophore mCherry (I–L): Down-regulation of her6 leads to disintegration of the Shh positive MDO (20/26; I and J; arrowheads). In embryos knocked-down for neurog1, the MDO seems unaltered in extension and width compared to the control (8/10; K, arrowhead). Double knock-down of her6 and neurog1 rescues the extend of the shh:GFP expression within the MDO (6/8; L, arrowhead). III, third ventricle; cTh, caudal thalamus; MDO, mid-diencephalic organizer; PTh, prethalamus; rTh, rostral thalamus.