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Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells.

ABSTRACT: The directed differentiation of forebrain neuronal types from human embryonic stem cells (hESCs) has not been achieved. Here, we show that hESCs differentiate to telencephalic progenitors with a predominantly dorsal identity in a chemically defined medium without known morphogens. This is attributed to endogenous Wnt signaling, which upregulates the truncated form of GLI3, a repressor of sonic hedgehog (SHH). A high concentration of SHH, or the inhibition of Wnt by dickkopf 1 (DKK1) together with a low concentration of SHH, almost completely converts the primitive dorsal precursors to ventral progenitors, which is partially achieved through both downregulation of the truncated GLI3 and upregulation of full-length GLI3 expression. These dorsal and ventral telencephalic progenitors differentiate to functional glutamatergic and GABAergic neurons, respectively. Thus, although hESCs generate dorsal telencephalic cells, as opposed to ventral progenitors in other vertebrates, in the absence of exogenous morphogens, human cells use a similar molecular mechanism to control the dorsal versus ventral fate. The coordination of Wnt and SHH signaling through GLI3 represents a novel mechanism that regulates ventral-dorsal patterning in the development of forebrain neuronal subtypes.

SUBMITTER: Li XJ

PROVIDER: S-EPMC2778748 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells.

Li Xue-Jun XJ Zhang Xiaoqing X Johnson M Austin MA Wang Zhi-Bo ZB Lavaute Timothy T Zhang Su-Chun SC

Development (Cambridge, England) 20091201 23

The directed differentiation of forebrain neuronal types from human embryonic stem cells (hESCs) has not been achieved. Here, we show that hESCs differentiate to telencephalic progenitors with a predominantly dorsal identity in a chemically defined medium without known morphogens. This is attributed to endogenous Wnt signaling, which upregulates the truncated form of GLI3, a repressor of sonic hedgehog (SHH). A high concentration of SHH, or the inhibition of Wnt by dickkopf 1 (DKK1) together wit ...[more]

PMID: 19906872

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Project description:Differentiating cerebellar organoids can be challenging due to complex cell organization and structure in the cerebellum. Different approaches were investigated to recapitulate differentiation process of the cerebellum from human-induced pluripotent stem cells (hiPSCs) without high efficiency. This study was carried out to test the hypothesis that the combination of different signaling factors including retinoic acid (RA), Wnt activator, and sonic hedgehog (SHH) activator promotes the cerebellar differentiation of hiPSCs. Wnt, RA, and SHH pathways were activated by CHIR99021 (CHIR), RA, and purmorphamine (PMR), respectively. Different combinations of the morphogens (RA/CHIR, RA/PMR, CHIR/PMR, and RA/CHIR/PMR) were utilized, and the spheroids (day 35) were characterized for the markers of three cerebellum layers (the molecular layer, the Purkinje cell layer, and the granule cell layer). Of all the combinations tested, RA/CHIR/PMR promoted both the Purkinje cell layer and the granule cell layer differentiation. The cells also exhibited electrophysiological characteristics using whole-cell patch clamp recording, especially demonstrating Purkinje cell electrophysiology. This study should advance the understanding of different signaling pathways during cerebellar development to engineer cerebellum organoids for drug screening and disease modeling. Impact statement This study investigated the synergistic effects of retinoic acid, Wnt activator, and sonic hedgehog activator on cerebellar patterning of human-induced pluripotent stem cell (hiPSC) spheroids and organoids. The results indicate that the combination promotes the differentiation of the Purkinje cell layer and the granule cell layer. The cells also exhibit electrophysiological characteristics using whole-cell patch clamp recording, especially demonstrating Purkinje cell electrophysiology. The findings are significant for understanding the biochemical signaling of three-dimensional microenvironment on neural patterning of hiPSCs for applications in organoid engineering, disease modeling, and drug screening.

Project description:BackgroundThe ventral midbrain contains a diverse array of neurons, including dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and neurons of the red nucleus (RN). Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express Sonic Hedgehog (Shh). However, Shh expression, which is initially confined to the mesencephalic ventral midline, expands laterally and is then downregulated in the ventral midline. In contrast, expression of the Hedgehog target gene Gli1 initiates in the ventral midline prior to Shh expression, but after the onset of Shh expression it is expressed in precursors lateral to Shh-positive cells. Given these dynamic gene expression patterns, Shh and Gli1 expression could delineate different progenitor populations at distinct embryonic time points.ResultsWe employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express Shh (Shh-GIFM) or transduce Shh signaling (Gli1-GIFM) at different time points give rise to different ventral midbrain cell types. We find that precursors restricted to the ventral midline are labeled at embryonic day (E)7.5 with Gli1-GIFM, and with Shh-GIFM at E8.5. These precursors give rise to all subtypes of midbrain dopaminergic neurons and the anterior RN. A broader domain of progenitors that includes the ventral midline is marked with Gli1-GIFM at E8.5 and with Shh-GIFM at E9.5; these fate-mapped cells also contribute to all midbrain dopaminergic subtypes and to the entire RN. In contrast, a lateral progenitor domain that is labeled with Gli1-GIFM at E9.5 and with Shh-GIFM at E11.5 has a markedly reduced potential to give rise to the RN and to SN dopaminergic neurons, and preferentially gives rise to the ventral-medial VTA. In addition, cells derived from Shh- and Gli1-expressing progenitors located outside of the ventral midline give rise to astrocytes.ConclusionsWe define a ventral midbrain precursor map based on the timing of Gli1 and Shh expression, and suggest that the diversity of midbrain dopaminergic neurons is at least partially determined during their precursor stage when their medial-lateral position, differential gene expression and the time when they leave the ventricular zone influence their fate decisions.

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Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells.

Publications

Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells.

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