The embryonic patterning gene Dbx1 governs the survival of the auditory midbrain via Tcf7l2-Ap2δ transcriptional cascade

Abstract

At the top of the midbrain is the inferior colliculus (IC), which functions as the major hub for processing auditory information. Despite the functional significance of neurons in the IC, our understanding of their formation is limited. In this study, we identify the embryonic patterning gene Dbx1 as a key molecular player that governs genetic programs for IC survival. We find that Dbx1 plays a critical role in preventing apoptotic cell death in postnatal IC by transcriptionally repressing c-Jun and pro-apoptotic BH3 only factors. Furthermore, by employing combined approaches, we uncover that Tcf7l2 functions downstream of Dbx1. Loss of Tcf7l2 function causes IC phenotypes with striking similarity to those of Dbx1 mutant mice, which include defective embryonic maturation and postnatal deletion of the IC. Finally, we demonstrate that the Dbx1-Tcf7l2 cascade functions upstream of Ap-2δ, which is essential for IC development and survival. Together, these results unravel a novel molecular mechanism for IC maintenance, which is indispensable for normal brain development.

Fig. 1. Differential expression profiling identifies Dbx1 target genes in the IC.

A Volcano plot representing transcriptional changes in Dbx1^del IC versus wildtype IC at E18.5. Orange dots correspond to upregulated genes whereas blue dots correspond to downregulated genes. B Gene ontology (GO) analysis of differentially expressed genes in the IC of Dbx1^del embryos at E18.5 compared with wildtype embryos. Bar charts showing 10 GO terms for biological process, molecular function and cellular components, ranked by fold enrichment. C, F, H Clustering heatmaps represent expression patterns of the genes with overrepresented GO terms: transcription factors (C), neuron projection and adhesion (F), and synaptic transmission and excitability (H). Each column represents an independent biological replicate. The log₂ values of expression in relation to the median of the row are shown as a blue-red color scale. Localization of each gene expression in the brain was estimated according to the Allen Brain Atlas, and is indicated by boxes. IC-enriched, expression enriched in the IC; IC-depleted, expressed broadly in the brain except for the IC; Ubiquitous, expressed throughout the brain; Low, low overall expression in the brain; N/A, not available in the Allen Brain Atlas. D, E, G, I In situ hybridization on E18.5 sagittal brain sections. Scale bars, 200 μm.

Fig. 2. Loss of Dbx1 leads to increased levels of c-Jun and BH3 only factors, and to cell death in the IC.

A Differential expression of BH3 only (Bid, Bad, Bim, Puma, Hrk) factors and c-Jun in E18.5 Dbx1^del compared with control based on RNA-seq (pink bars; ****FDR q ≤ 0.0001) and qRT-PCR (light blue bars; Student’s t-test; ***p ≤ 0.001); error bars indicate standard error for log2 fold change). B In situ hybridization with Bim, Hrk, and c-Jun riboprobes in E18.5 sagittal brain sections. C–E Immunohistochemistry using anti-cleaved caspase 3 (Cas3) was performed on E18.5 coronal sections (C) and E12.5 (D) and E15.5 (E) sagittal sections to detect apoptosis. E18.5, but not E12.5 or E15.5 Dbx1^del mutants exhibited increased numbers of apoptotic cells in the IC. For E12.5, Cas3⁺ cells were counted from 5 sections from each embryo (n = 5). For counting Cas3⁺ cells at E15.5 and E18.5, 10–20 counting sites (counting grid for SC, 200 μm × 200 μm; for IC, 100 μm × 100 μm) were evaluated in one section. The values were calculated from 5 sections from each embryo (n = 5). *P < 0.01 (Student’s t-test). Scale bars, 200 μm.

Fig. 3. Absence of the inferior colliculus in Dbx1^cko mutant mice.

A A close-up dorsal view of P3 control and Dbx1^cko mutant brains. Dashed lines indicate the position of the IC. Compared with controls, Dbx1^cko mutant brains showed a reduction in IC size. B In situ hybridization on P3 sagittal sections of control and Dbx1^cko brains. C Immunohistochemistry with anti-Cas3 on P3 sagittal sections of control and Dbx1^cko brains. Cas3⁺ cells were counted from five sections from three embryos using 20 counting sites (counting grid 100 μm × 100 μm) in one section. *P < 0.05; **P < 0.001 (Student’s t-test). D The P20 brain phenotype of Dbx1^cko mutants. The entire brain (dorsal view) and histological sections of the midbrain. Section ISH on sagittal sections indicates an almost complete lack of the IC with the SC fused to the cerebellum in Dbx1^cko mutants. Scale bars, 200 μm.

Fig. 4. Tcf7l2 acts downstream of Dbx1 to orchestrate a genetic program of morphological maturation in the developing IC.

A Volcano plot of differential gene expression measured by RNA-seq of wildtype and Tcf7l2^del IC. Upregulated or downregulated genes are indicated by orange and blue dots, respectively. B GO term enrichment for gene sets that are differentially regulated in Tcf7l2^del embryos. Bar charts showing 10 GO terms for biological process, molecular function, and cellular components, ranked by fold enrichment. C Intersections of differentially expressed genes in Dbx1^del and Tcf7l2^del IC identify commonly upregulated and downregulated gene sets. D Correlation between the significantly differentially expressed genes (n = 342) observed in Dbx1^del and Tcf7l2^del mutants. Thresholds used to determine differential expression are indicated by dashed lines (log₂FC ≥ 0.4 and ≤ −0.4; P_adj ≤ 0.05). The Pearson correlation coefficient is indicated in the lower right corner. E, G, I Clustering heatmap indicating relative expression of the genes with the overrepresented GO terms: transcription factors (E), neuron projection and adhesion (G), and synaptic transmission and excitability (I). Each column represents an independent biological replicate. The log₂ values of expression in relation to the median of the row are shown as a blue-red color scale. F, H, J In situ hybridization on E18.5 sagittal brain sections. Scale bars, 200 μm.

Fig. 5. Tcf7l2 inactivation leads to apoptosis and lack of the inferior colliculus.

A Differential expression of BH3-only apoptotic factors and Jun in E18.5 Tcf7l2^del mutants compared with controls based on RNA-seq (pink bars, **FDR q ≤ 0.005; ****FDR q ≤ 0.0001) and qRT-PCR (light blue bars, Student’s t-test; ***p ≤ 0.001; error bars indicate standard error for log2 fold change). B ISH with Bim, Hrk and c-Jun probe in E18.5 sagittal brain sections. C Immunohistochemistry using anti-Cas3 was performed on E15.5 and E18.5 sagittal sections to detect apoptosis. For counting of Cas3⁺ cells at E15.5 and E18.5, 10–20 counting sites (counting grid 100 μm x 100 μm) were evaluated in one section. The values were calculated for five sections from six embryos for each genotype. *P < 0.001 (Student’s t-test). D Immunohistochemistry for phospho-histone H3 (PH) on sagittal sections of E15.5 midbrain. The number of PH3-positive cells was counted for three sections from three embryos for each genotype. E Section ISH on coronal sections of E12.5 control and Tcf7l2^cko embryos with a riboprobe derived from exon 10 of Tcf7l2. F The dorsal view of control and Tcf7l2^cko mutant P20 brains. Section ISH on sagittal sections indicates large deletion of the IC, whereas the SC abutted on the cerebellum in Tcf7l2^cko mutants. Scale bars, 200 μm.

Fig. 6. Ap-2δ is co-regulated by Dbx1 and Tcf7l2 in the IC.

A Differential expression of Ap-2δ in Dbx1^del and Tcf7l2^del mouse embryos compared with controls based on RNA-seq (****FDR q ≤ 0.0001) and qRT-PCR (Student’s t-test; ****p ≤ 0.0001). Error bars indicate standard error for log2 fold change). B Representative midbrain sections of wild-type, Dbx1^del, Dbx1^cko, and Tcf7l2^del mouse embryos hybridized with Ap-2δ at E18.5. Scale bars, 200 μm.

Fig. 7. Restored expression of Tcf7l2 and/or Ap-2δ attenuates apoptosis in Dbx1-deficient IC cells.

A Isolation and primary cultures of IC cells from E15.5 control and Dbx1^del mutant embryonic brains. IC cells were cultured on coverslips coated with L-ornithine and laminin in 24-well plates. IC cell identity was verified by immunocytochemistry of 5 days in vitro (DIV5) cells using antibodies against Nrgn and Tcf7l2. Bright-field (BF) images display cellular processes including neurite outgrowth. Consistent with gene expression profiles in embryos, the majority of primary cultured Dbx1-deficient IC cells are Tcf7l2⁻, Nrgn⁻. B Caspase 3 (Cas3) immunostaining were performed at DIV7 cells to identify quantity of apoptosis. Co-immunostaining revealed that most of Cas3⁺ cells are Tcf7l2⁻ (n = 5 biological replicates; Student’s t-test; *p ≤ 0.001). C Primary IC cells were established from E15.5 embryos and kept in culture for 21 days. Compared to control IC cells with round cell bodies and a complex network of neurites, most of Dbx1^del mutant cell bodies were smaller, shrunken, and the neurites were fragmented and lost. D Calcein AM staining of viable cells. The number of viable cells was determined by Calcein fluorescence at DIV14, DIV21, and DIV28. Six counting sites were evaluated in one coverslip (n = 5 biological replicates). *P < 0.01; **P < 0.001 (Student’s t-test). E, F IC cells isolated from Dbx1^del mutant E15.5 brains were cultured for 2 DIV and transfected with Tcf7l2 (p043mTcf-4B), Ap-2δ (pCMV-Tfap2d) expression constructs or both DNAs. Nuclear GFP expression construct (CAG-NLS-GFP) was co-transfected to identify the transfected cells. E Quantification of apoptosis was achieved from IC cells cultured for 7 additional DIV by counting the proportion of Cas3⁺ cells among GFP⁺ cells. Approximately 3000 GFP+ cells were considered for each experiments. Histograms represent the means normalized to 1 in the proportion of mock control (empty vector). Experiments were performed in 4 biological replicates. Error bars correspond to standard deviation. Means were compared using Student’s t-test; *p ≤ 0.01). F Quantification of survival was achieved from IC cells cultured for 7, 14 and 21 additional DIV following transfection. The number of viable cells was determined by Calcein fluorescence for 6 counting sites in each coverslip (n = 4 biological replicates). (Student’s t-test; *p ≤ 0.05; **p ≤ 0.01).