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. 2025 May 2;28(6):112569.
doi: 10.1016/j.isci.2025.112569. eCollection 2025 Jun 20.

Impact of ATF6 deletion on the embryonic brain development

Loc Dinh Nguyen  1 , Ly Huong Nguyen  1 , Dat Xuan Dao  1 , Tsuyoshi Hattori  1 , Mika Takarada-Iemata  1 , Hiroshi Ishii  1 , Takashi Tamatani  1 , Hiroshi Kawasaki  2 , Yohei Shinmyo  3 , Kenta Onoue  4 , Shigenobu Yonemura  4   5 , Jun Zhang  6 , Masato Miyake  6 , Seiichi Oyadomari  6 , Kazutoshi Mori  7 , Osamu Hori  1
Affiliations

Impact of ATF6 deletion on the embryonic brain development

Loc Dinh Nguyen et al. iScience. .

Abstract

Although the unfolded protein response (UPR) is activated during brain development, its roles remain unclear. Here, we report that deletion of activating transcription factor 6 (ATF6), consisting of ATF6α and ATF6β, in the developing brain caused microcephaly and neonatal death in mice. Analysis of Atf6a/Atf6b double conditional knockout (dcKO) brains revealed diverse neuronal phenotypes, such as reduced neurogenesis, increased cell death, impaired cortical layer formation, and axon projection defects. Furthermore, hypervasculature, glial defects, and neuroinflammation were observed in dcKO brains. Notably, hypervasculature was detected at E14.5, when endoplasmic reticulum (ER) stress was morphologically unclear, but the UPR was activated to a greater extent in dcKO brains. Expression profiles revealed reduced levels of molecular chaperones in the ER and enhanced levels of PERK- and IRE1-downstream molecules, including VEGFA, in dcKO brains. Administration of a chemical chaperone 4-phenylbutyric acid partially rescued dcKO mice, suggesting roles of ATF6 for improving proteostasis and for coordinating the UPR.

Keywords: Cell biology; Developmental biology; Neuroscience.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Expression of UPR genes in developing brain (A) Expression of UPR genes in wild-type cerebral cortex at different stages of development (n = 4 brains per time point), data are shown as mean ± SEM. E12.5, E14.5, E16.5, and E18.5 denote embryonic 12.5, 14.5, 16.5, and 18.5 days after gestation, respectively. P0.5 and P56 denote postnatal day 0.5 and 56, respectively. (B) Crossing scheme for parental mating, embryos or neonatal mice were used for experiments after genotyping. (C and D) Expression of Atf6a and Atf6b mRNA (C) (n = 6–7 brains per group) and protein (D) (n = 3 brains per group) in E16.5 cerebral cortices from control and dcKO embryos. Data are shown as mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 by Mann-Whitney U test. Arrowhead: full length ATF6α and ATF6β, arrow: N-terminal fragment, asterisk: non-specific band.
Figure 2
Figure 2
Behavior and brain size of the control and dcKO mice (A) Newborn mice at P0.5 in the same littermate. Arrow indicates a milk spot. (B) Illustration of suckling behavior test. Nipple searching time is shown in the graph as mean ± SEM (n = 8 for control mice including 4 Atf6afl/flAtf6bfl/fl mice and 4 Atf6afl/flAtf6bfl/+ mice, n = 5 for dcKO mice). All dcKO mice failed to find and attach to the nipple (arrowhead) after 120 s and were considered negative in this test. (C and D) Neonatal mice after caesarean delivery were monitored in warm-humidified chamber either without feeding (n = 8 for control mice, n = 3 for dcKO mice, p = 0.5093 by Mantel-Cox test) (C) or with administration of the artificial milk directly into the stomach (n = 6 for control mice, n = 4 for dcKO mice, p = 0.6822 by Mantel-Cox test). (E) Brains from E16.5 embryos and P0.5 mice. Brain and body weights were measured at both E16.5 (n = 4–8 embryos for each group) and P0.5 (n = 9–12 mice for each group). Data are represented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by Mann-Whitney U test. Scale bars: 5 mm (A), 2 mm (E).
Figure 3
Figure 3
Impaired neurogenesis and increased cell death in dcKO (A, B, and D) Immunohistochemistry of the brain section from E14.5 control or dcKO embryos for the indicated molecules. Cortical thickness was also measured by the thickness of DAPI positive area (A) (n = 4–7 brains for each group). Dashed lines indicate brain surface. (C) EdU incorporation was conducted at E14.5 for 1 h to see proliferating cells in S-phase (EdU) (4 control brains including 2 Atf6afl/flAtf6bfl/fl embryos and 2 Atf6afl/flAtf6bfl/+ embryos, and 4 dcKO brains). (E) Apoptosis was evaluated by TUNEL staining using sections prepared for immunohistochemistry as aforementioned (n = 4 brains for each group). Dashed lines indicate brain surface and lateral ventricle. Data are represented as mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01 by Mann-Whitney U test. Scale bars: 100 μm (A–D), 500 μm (E).
Figure 4
Figure 4
Hypervasculature in dcKO mice (A–C) Brain sections from E14.5 (A and B) and E16.5 (C) embryos were subjected to immunohistochemistry for the indicated molecules. Nuclei were visualized with DAPI (blue). (B) Higher magnification in the cortical cortex (Cx) and caudate putamen (CPu) in (A). The area of CD31 (+) cells and the number of ERG (+) cells were measured using ImageJ, as described in the text (n = 3 embryos per group). (D) RT-qPCR. Brain samples from E14.5 brains were subjected to RT-qPCR using specific primers for the indicated genes (n = 3–4 per group). Data are represented as mean ± SEM. ∗p < 0.05 by Mann-Whitney U test. Scale bars: 500 μm (A and C), 50 μm (B).
Figure 5
Figure 5
Impaired layer formation and axon projection in dcKO (A and B) Brain sections from P0.5 control or dcKO mice were subjected to immunohistochemistry (A) or Hematoxylin and eosin (H&E) staining (B). (A) CUX1 (a layer II-IV marker)(+) cells were distributed tightly in upper layer in control, but scattered and expressed deeply to lower layer in dcKO mice (n = 4 brains per group). Solid line indicates layer II-IV, and dashed line indicates layer V-VI. Data are presented as mean ± SEM. ∗p < 0.05 by Mann-Whitney U test. (B) H&E staining showing abnormal corpus callosum (CC) in dcKO brains. Arrows indicate thicken CC (middle) and Probst bundles (right). (C and D) Immunohistochemistry of brain sections from E16.5 control and dcKO embryos for the indicated molecules. Axon direction is parallel in control, but disorganized and surrounded by IBA1(+) macrophages/microglia in dcKO brains (n = 4 brains per group). Scale bars: 100 μm (A and D), 500 μm (B), 200 μm (C).
Figure 6
Figure 6
Expression of UPR genes in control and dcKO brains (A) Total RNA isolated from E16.5 control or dcKO cerebral cortex was subjected to RNA-sequencing (n = 3 brains per group). Heatmaps show gene expressions in each GO term or in ATF6 branch including molecular chaperones in the ER. Color scale bars indicate Z score by row. (B) RT-qPCR. Brains samples from E16.5 control or dcKO cerebral cortex were subjected to RT-qPCR with specific primers for the indicated genes (n = 6–7 brains per group). Expressions levels of molecular chaperones in the ER are downregulated, while those of IRE1-and PERK-downstream genes are upregulated in dcKO brains. (C) Western blotting. Protein was extracted from cerebral cortices of E16.5, and subjected to western blotting with indicated antibodies (n = 3–7 brains per group). Expression of REELIN was reduced in dcKO brain. (D and E) Immunohistochemistry of brain section from E16.5 control and dcKO embryos for p-PERK and SOX2. p-PERK(+) cells are distributed around ventricular zone (D) and majority of them are co-localized with SOX2(+) cells (E) (n = 4 brains per group). Cx, cortex; CPu, caudate putamen. Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 by Mann-Whitney U test. Scale bars: 500 μm (D), 50 μm (E).
Figure 7
Figure 7
Expression of genes related to brain development in control and dcKO brains (A) Total RNA isolated from E16.5 control or dcKO cerebral cortex was subjected to RNA-sequencing (n = 3 brains per group). Heatmaps show gene expressions in each GO term. Color scale bars indicate Z score by row. (B) Western blotting. Protein was extracted from cerebral cortex of P0.5 control or dcKO mice, and subjected to western blotting with indicated antibodies (n = 4–7 brains per group). (C and D) Immunohistochemistry of brain section from P0.5 control and dcKO mice for GFAP (C) and PDGFRα (D). GFAP(+) Glial wedge (boxed area) are observed in control mice, but not clear in dcKO mice. By contrast, indusium griseum glia (arrow), and midline zipper glia (arrowhead) are observed in both genotypes. GFAP(+) areas in the glial wedge were measured (n = 4 brains per group). (D) Oligodendrocyte progenitor cells (PDGFRα(+) cells) are less observed in dcKO mice. The number of PDGFRα(+) cells was counted in each area (n = 4 brains per group). CC, corpus callosum; Cx, cortex; IZ, intermediate zone; CPu, caudate putamen. Data are represented as mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01 by Mann-Whitney U test. Scale bars: 200 μm (C, upper row), 50 μm (C, lower row, D).
Figure 8
Figure 8
Rescue experiment using 4-PBA. Pregnant mice were administered with 4-PBA from E10.5 to E15.5 (A) Brains from E16.5 embryos (n = 4–8 brains per group). (B) Cortical thickness was measured by the thickness of DAPI positive area from immunohistochemistry brain section (n = 4–6 brain per group). (C and D) Immunohistochemistry of brain section from E16.5 control and dcKO embryos for the indicated molecules. (C) Note that the number of proliferating cells in dcKO brains increases by 4-PBA both in the apical and basal areas (n = 4 brains per group).(D) Axon direction in dcKO brains is also improved to the parallel pattern by 4-PBA, but is still surrounded by IBA1(+) macrophages/microglia (n = 4 brains per group). (E) RT-qPCR. Expression of IRE1-downstream gene (sXbp1) in dcKO brains decreases by 4-PBA, while that of PERK-downstream genes (Ddit3 and Atf3) does not change (n = 3–4 brains per group). Data are represented as mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01 by Mann-Whitney U test. None: non-treatment group, 4-PBA: treatment group. Scale bars: 2 mm (A), 50 μm (C), 100 μm (D).
Figure 9
Figure 9
Scheme for the roles of ATF6α and ATF6β in the developing brain Deletion of Atf6a and Atf6b genes causes reduced levels of expression of molecular chaperones in the ER, leading to hyperactivation of two other UPR branches and impairment of proteostasis. These changes disrupt the microenvironment in the developing brain, resulting in microcephaly and possibly neonatal death.
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