Impact of c-JUN deficiency on thalamus development in mice and human neural models

Abstract

Background: c-Jun is a key regulator of gene expression. Through the formation of homo- or heterodimers, c-JUN binds to DNA and regulates gene transcription. While c-Jun plays a crucial role in embryonic development, its impact on nervous system development in higher mammals, especially for some deep structures, for example, thalamus in diencephalon, remains unclear.

Methods: To investigate the influence of c-JUN on early nervous system development, c-Jun knockout (KO) mice and c-JUN KO H1 embryonic stem cells (ESCs)-derived neural progenitor cells (NPCs), cerebral organoids (COs), and thalamus organoids (ThOs) models were used. We detected the dysplasia via histological examination and immunofluorescence staining, omics analysis, and loss/gain of function analysis.

Results: At embryonic day 14.5, c-Jun knockout (KO) mice exhibited sparseness of fibers in the brain ventricular parenchyma and malformation of the thalamus in the diencephalon. The absence of c-JUN accelerated the induction of NPCs but impaired the extension of fibers in human neuronal cultures. COs lacking c-JUN displayed a robust PAX6⁺/NESTIN⁺ exterior layer but lacked a fibers-connected core. Moreover, the subcortex-like areas exhibited defective thalamus characteristics with transcription factor 7 like 2-positive cells. Notably, in guided ThOs, c-JUN KO led to inadequate thalamus patterning with sparse internal nerve fibers. Chromatin accessibility analysis confirmed a less accessible chromatin state in genes related to the thalamus. Overexpression of c-JUN rescued these defects. RNA-seq identified 18 significantly down-regulated genes including RSPO2, WNT8B, MXRA5, HSPG2 and PLAGL1 while 24 genes including MSX1, CYP1B1, LMX1B, NQO1 and COL2A1 were significantly up-regulated.

Conclusion: Our findings from in vivo and in vitro experiments indicate that c-JUN depletion impedes the extension of nerve fibers and renders the thalamus susceptible to dysplasia during early mouse embryonic development and human ThO patterning. Our work provides evidence for the first time that c-JUN is a key transcription regulator that play important roles in the thalamus/diencephalon development.

Keywords: c-JUN; AP-1; Cerebral organoids; NPC; Neural development; Thalamus organoids.

Fig. 1

KO mouse embryos at E14.5 exhibited thalamic malformation in the diencephalon. (A, B) H&E staining of cJun^+/- and KO embryos at E14.5 revealed sparse fibers in the ventricular parenchyma and malformation of the thalamus in the diencephalon of the KO embryo. The small inset in the upper-left corner of the image displays the global view of the embryo. H015-1: “H015” was the code number of the mother mouse, and “-1” was the embryo number in the litter, hereinafter the same. (C) IF staining of TUJ1 and CNTN2 showed that WT had normal nerve fibers in the thalamus/diencephalon region, while those of KO were broken down with scattered distribution. a–d, Scale bar: 100 μm. (D) IF staining of TCF7L2 showed strong signals in the thalamus/diencephalon region of WT (a, c), while it was significantly decreased (b) or undetectable (d) in the corresponding areas in the KO groups. Scale bar: 100 μm. (E, F) Statistical analysis of IF staining of TUJ1 and TCF7L2 in the thalamus/diencephalon region of mice embryo section at E14.5. The Y-axis represents the mean gray value of signals. n = 6, the values are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. (G) DiI dye diffusion in the adjacent areas of the diencephalon. The middle image shows a local magnification of the region near the thalamus. The arrows indicate the areas of apparent dye penetration obstruction in KO. The embryo H340-4 c-Jun^-/- at the bottom-right corner was the “small and white” embryo, which was difficult to cut, thus the DiI crystals were implanted into the intact brain parenchyma area through an incision

Fig. 2

Loss of c-JUN in NPCs promoted neural stem cell induction and accelerated PAX6⁺cell differentiation. (A) Schematic of NPC induction. NIM: neural induction medium, NMM: neural maintain medium, SB: SB431542 (B) RT-qPCR gene expression test revealed that several NPC marker genes were significantly upregulated in the KO groups. (3 independent experiments). The values are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Flow cytometry plots illustrate the distribution of cells stained with PAX6 and NESTIN on day 12, 20, and 25. Differentiated PAX6⁻/NESTIN⁺ subcluster appeared in the KO groups, but were partially rescued in the OE groups on day 25. (D) Statistical analysis of the flow cytometry data showed a significant increase in cells within Q1 quadrant (PAX6⁻/NESTIN⁺ cells) (3 independent experiments). Two-way ANOVA. The values are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001. (E) IF staining showed an increase in PAX6⁻/NESTIN⁺ rosettes (circled with yellow lines) in the KO groups on day 25. Scale bar: 50 μm. (F) Statistical analysis of IF staining showed a significant increase in PAX6⁻/NESTIN⁺ rosettes in the KO groups, which is partially reversed in the OE groups on D25 (n = 12, 4 independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Loss of c-JUN weakened the nerve fiber extension and adherent ability on plates in the neural culture. (A) Schematic of long-period continuous culture and thalamus neuron induction. NMM: neural maintain medium, TPM: thalamus patterning medium. (B) BF view of NPCs on day 28 (left panel) and day 43 (right panel). NPCs in the WT, KO, and OE groups could spread flat on the plates and extended plentiful fibrous axons on D28. In the KO groups, the cell bodies were more likely to aggregate and the axonal fibrous were significantly shrunk on day 43. Scale bar: 100 μm. (C) BF view of curled-up edges of cell colonies in the KO groups but not in the WT or OE group. Cells in the KO groups were easily detached from the culture dish (12-well dish) when subjected to enzymatic digestion. The white film indicated by the arrowheads shows cells shed after digestion. (D) Statistical analysis of the proportion of pro-detached colonies in NPCs on day 43. The KO groups showed a significantly high proportion of pro-detached colonies (3 independent experiments, 9 fields of view per group). *p < 0.05, **p < 0.01, ***p < 0.001. (E) IF staining of TUJ1 and TCF7L2 neurons on thalamus induction D1 and D7, respectively. In order to clearly mark the distribution of nerve fibers, TUJ1⁺ signals are highlighted by yellow lines (the rightmost column) Scale bar: 100 μm. (F) Statistical analysis of the ratio of the signal area of TUJ1 to TCF7L2 in NPCs on thalamus induction D7. The KO groups showed a significantly reduced ratio. This pattern was restored in the OE group. (3 independent experiments, 9 fields of view per group). *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significant. BF, bright-light field

Fig. 4

KO-derived CO showed a robust exterior, but less fiber-connected core. (A) Bright field view of CO induction from WT and KO cell lines., Scale bar: 200 μm for day 8–24; Scale bar: 500 μm for day 30–75. (B–C) KO groups showed a significant increase in the width of the neural ectoderm at day14 (n = 30, 3 independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001. (D, E) The KO groups showed a significantly increased width of the expanded neural cortex-like structures at day30 (n = 30, 3 independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001. (F, G) IF staining of CO on D14 and D30. The KO groups show robust PAX6⁺/NESTIN⁺ closed-ring shape ectoderm and increased width of PAX6⁺/NESTIN⁺ neural cortex-like layer (circled with yellow dashed line in the PAX6 channel). Scale bar: 100 μm. (H) IF staining of CNTN2 in COs at day 50. In WT, CNTN2 could be detected both in the periphery region and the interior (a, b). In the KO groups, CNTN2 was most distributed in the periphery but not in the core (c–f). Scale bar: 500 μm. (I) IF staining of ZO-1, CLAUDIN5 in COs at day 50. WT group showed tight-junction rich signals in the outer neural cortex-like layer as well as in the interior (indicated by the white arrows in b). In the KO groups, the signals were detected mostly on the outer layer, but not in the core. Scale bar: 500 μm. (J) qRT-PCR gene expression test revealed that several NPC/neuron marker genes (LMO3, FOXG1, and TUJ1) were upregulated while tight junction genes (ZO-1) were downregulated in the KO groups during CO induction (3 independent experiments). The values are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

c-JUN KO upregulated the pathways of nervous system development but downregulated the pathways in cell-cell adhesion. (A) Heat map of CO transcriptome sequencing on D12 and D24. (B) PCA analysis revealed the principal components clustered separately according to the two time points, and the divergence was obvious on D24 between the WT and KO groups. (C) Heat map of differentially expressed genes (DEG) in CO D24. (D, E) GO-BP analysis of DEG in CO D24. When compared to WT, the top 10 downregulated and top 10 upregulated biological pathways in KO are listed respectively. (F) Volcano map of DEG in CO day24

Fig. 6

The disturbed expression pattern of thalamus/diencephalon marker TCF7L2 inc-Jun KO embryos and CO. (A) A diagram of the CO section and mouse embryo section; the red color area in the embryo section shows the thalamus in diencephalon. (B) WT-derived CO D50 could detect TCF7L2 signals, implying primary diencephanlic cells near the central area (a, b), while TCF7L2 was hardly detected in the KO groups. Image b is a partially enlarged image of the white arrow-indicated area in the image (a) Scale bar: 500 μm for a, c, d; 200 μm for (b) (C) WT embryos at E14.5 could detect TCF7L2 signals, implying thalamus in the diencephalon (a, b, c), while KO embryos appeared different in the staining cases: H213-5 showed TCF7L2 signals in the diencephanlic area (d); H312-9 and H312-10 have negligibly detected TCF7L2 signals (e, f). Another KO embryo E14.5 H037-8 from another litter showed undetectable TCF7L2 signals in the corresponding area (g). Image c is a partially enlarged image of image b. Scale bar: 500 μm for a, b, d–g; Scale bar: 100 μm for (c) (D) Statistical analysis of the proportion of TCF7L2⁺ cells detectable samples in embryos at E14.5. (E) Statistical analysis of the proportion of TCF7L2⁺ cells in COs on day 50

Fig. 7

c-JUN KO-derived ThO showed a hindered sphere in diameter and sparse internal fibers. (A) Global overview in the bright field of ThO induction from WT, KO, and OE cell lines. day 8–35, Scale bar: 200 μm. (B) IF staining of ThO revealed robust TUJ1⁺/TCF7L2⁺ cells in the WT and OE groups on D30, while the KO groups showed a decreased density. Scale bar: 100 μm. (C) Statistics analysis of the diameter of ThO in WT, KO, and OE under a bright-field view on days 8, 16, and 30. The KO groups showed a significantly smaller diameter (n = 9, 3 independent experiments). (D, E) Statistics analysis of IF staining of TCF7L2 and TUJ1 in ThOs in WT, KO, and OE on day 30. The KO group showed a significantly reduced TCF7L2 and TUJ1 signals (n = 6, 3 independent experiments). (F) IF staining of NESTIN and CNTN2 in ThO on day 30. Scale bar: 100 μm. (G, H) Statistics analysis of IF staining of NESTIN and CNTN2 in ThOs in WT, KO, and OE on day 30. The NESTIN signal in KO groups was decreased, whereas there was no significant difference in the CNTN2⁺ signal among three groups. Scale bar: 100 μm. (n = 6, 3 independent experiments). Values are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8

ATAC-seq revealed that c-JUN KO restrained the thalamus gene expression in ThO. (A) Schematic of the characteristic signal distribution in the telencephalon and diencephalon regions in the early embryonic neural tube. ANR, anterior neural ridge; IsO, isthmic organizer; rFB, rostral forebrain; cFB, caudal forebrain; MB, midbrain; HB, hindbrain; (B, C) Schematic representation of the characteristic gene expression patterns from a lateral view (B) and the frontal section (C). The solid line in B represents the plane of the frontal section in C. tel, telencephalon; di, diencephalon; mes, mesencephalon; met, metencephalon; myel, myelencephalon; 3rd V, third ventricle; mz, mantle zone; vz, ventricular zone; pTec, pretectum; pTh, prethalamus; Th, thalamus; (D, E) ATAC-seq revealed that chromatin accessibility was reduced in genes related to thalamus development and axon projections in the KO groups and restored after over-expression but no difference (FOXG1, SIX3) or slight reduction (RAX) in the genes relate to telencephalon development. Lanes 1–9 (blue or red) were ATAC-seq data and lanes 10–11 (pink) were CUT&Tag data. (F) Volcano plot of DEG from RNAseq in ThOs D25. Multiple genes were significantly differentially expressed in the KO group in thalamus organoid development on D25. (G, H) GO analysis of RNA-seq data obtained from ThO D25, c-JUN KO to WT. fibroblast growth factor…: fibroblast growth factor receptor signaling pathway (2); mitochondrial electron…: mitochondrial electron transport, NADH to ubiquinone (2)