The Mediator Subunit, Med23 Is Required for Embryonic Survival and Regulation of Canonical WNT Signaling During Cranial Ganglia Development

Abstract

Development of the vertebrate head is a complex and dynamic process, which requires integration of all three germ layers and their derivatives. Of special importance are ectoderm-derived cells that form the cranial placodes, which then differentiate into the cranial ganglia and sensory organs. Critical to a fully functioning head, defects in cranial placode and sensory organ development can result in congenital craniofacial anomalies. In a forward genetic screen aimed at identifying novel regulators of craniofacial development, we discovered an embryonically lethal mouse mutant, snouty, which exhibits malformation of the facial prominences, cranial nerves and vasculature. The snouty mutation was mapped to a single nucleotide change in a ubiquitously expressed gene, Med23, which encodes a subunit of the global transcription co-factor complex, Mediator. Phenotypic analyses revealed that the craniofacial anomalies, particularly of the cranial ganglia, were caused by a failure in the proper specification of cranial placode neuronal precursors. Molecular analyses determined that defects in cranial placode neuronal differentiation in Med23 ^sn/sn mutants were associated with elevated WNT/β-catenin signaling, which can be partially rescued through combined Lrp6 and Wise loss-of-function. Our work therefore reveals a surprisingly tissue specific role for the ubiquitously expressed mediator complex protein Med23 in placode differentiation during cranial ganglia development. This highlights the importance of coupling general transcription to the regulation of WNT signaling during embryogenesis.

Keywords: MED23; Wnt signaling; cranial ganglia; cranial placodes; neural crest cells.

FIGURE 1

snouty embryos exhibit small body size and craniofacial defects at E9.5. (A–D) Control and snouty mouse embryo littermates at E9.0 and E9.5 were stained with DAPI and imaged with a confocal microscope demonstrating the smaller body size and shortened frontonasal prominences (arrows) in snouty embryos. (E–F′) Neuronal marker TuJ1 staining of control and snouty embryos at E9.5 suggests that snouty embryos exhibit disrupted cranial ganglia. (E′,F′) Are higher magnification images of (E,F). (G,H) PECAM1 immunostaining of E9.5 control and snouty embryos reveal abnormal development of the vascular tree like network in the head and epibranchial regions of snouty mutants. Intersomitic vessel formation (arrow, G,H) is not affected.opv, optic vesicle; ov, otic capsules; BA, branchial arch; DRG, dorsal root ganglia. Scale bars for (A–D) is 300 um, (E,F) is 350 um, (G) is 300 um, (H) is 200 um.

FIGURE 2

snouty mutant mice have a mutation in the Med23 gene. (A) The snouty mouse mutant phenotype was determined to be caused by a single base pair mutation in Exon 22 of the Med23 gene, which leads to a valine to aspartic acid amino acid change in the protein. Chromosome position of the mutation is noted based on mm10 mouse assembly. (B) Western blot against Med23 shows a drastic reduction of Med23 protein levels in snouty mutant embryos. (C) Ubiquitous expression of LacZ throughout E7.5 to E10.5 embryos is indicative of the Med23 expression. Scale bar is 250 um.

FIGURE 3

Med23^sn/sn mutants display neural crest cell and placodal defects. (A–D) Sox10 expression in E8.5 and E9.5 control and Med23^sn/sn embryos shows reduced Sox10 + cells entering the cranial ganglia region near the 1^st branchial arch and around the otic vesicle in Med23^sn/sn embryos. (E,F) Crabp1 ISH reveals proper neural crest cell migration in the cranial and trunk regions of E9.5 control and Med23^sn/sn embryos. (G,H) Eya2 expression in control and Med23^sn/sn embryos shows proper formation of anterior pre-placodal area. However, very few Eya2⁺ cells are observed in the trigeminal and epibranchial regions of Med23^sn/sn embryos (arrow, H). (I,J) Pax2⁺ cells are present in the optic, otic, and isthmus of both control and Med23^sn/sn embryos. (K,L) Pax8⁺ cells are present but loosely organized in the epibranchial region, otic placode and isthmus of Med23^sn/sn embryos in comparison to controls. Scale bar for (A,B) is 120 um, (C–F,I–L) is 300 um, and (G–H) is 150 um.

FIGURE 4

Med23^sn/sn mutants display defects in neuronal differentiation. (A,B) Ngn1 is expressed in trigeminal (arrow) and epibranchial cells in control embryos, however is downregulated or lost in Med23^sn/sn embryos. (C,D) Ngn2⁺ cells are observed in the epibranchial ganglia of control embryos (arrows, C). Ngn2⁺ cell number is drastically reduced in the epibranchial ganglia of Med23^sn/sn embryos (arrows, D). (E,F) Neurod1⁺ cells are considerably reduced in the developing trigeminal and epibranchial ganglia of Med23^sn/sn embryos at E9.5 compared to controls. (G,H) A higher number of cleaved Caspase 3 positive cells are observed in the developing trigeminal and frontonasal prominence of E9.5 Med23^sn/sn embryos compared to controls. (I) qPCR analyses of E9.5 wild-type and Med23^sn/sn littermate embryos revealed downregulation of Ngn1, Ngn2, and NeuroD1 transcripts in the mutants. Statistical analysis was performed using ANOVA and ^∗ denotes a p-value < 0.05. Scale bar for (A–F) is 150 um, (G,H) is 300 um. *p < 0.05.

FIGURE 5

WNT signaling is upregulated and ectopically expressed in the lateral nasal processes of Med23^sn/sn embryos. (A) qPCR analysis of cDNA obtained from E9.5 wild-type and Med23^sn/sn littermate embryos shows a reduction in the levels of Med23, Dkk1, and Ccnd1. (B–E′) Lateral and frontal images of control BATGal and Med23^sn/sn;BATGal embryos reveal an increase in WNT signaling in the frontonasal processes at E9.5. The lateral nasal processes, which are usually devoid of active WNT signaling (*, asterisk, control) exhibit positive LacZ staining (WNT activity) in Med23^sn/sn embryos (*, asterisk, Med23^sn/sn). (B′–E′) are high magnification images of (B–E). Scale bar for (B–E) is 450 um, (B′–E′) is 100 um.

FIGURE 6

Rescue of the Med23^sn/sn phenotype by modulating WNT signaling. DAPI stained E9.5 (A) wild-type, (B) Med23^sn/sn, (C) Med23^sn/sn; Lrp6^+/–, (D) Med23^sn/sn; Wise^+/– and (E) Med23^sn/sn; Lrp6^+/–; Wise^+/– embryos. TuJ1 immunostained (green) E9.5 (F) wild-type (G) Med23^sn/sn (H) Med23^sn/sn; Lrp6^+/–, (I) Med23^sn/sn; Wise^+/– and (J) Med23^sn/sn; Lrp6^+/–; Wise^+/–, embryos. Combinatorial loss of Lrp6 and Wise restores neuronal differentiation in the epibranchial region of Med23^sn/sn embryos. (F′–J′) are higher magnification images of (F–J) focused on the trigeminal ganglia. (K) Quantification of the area of Tuj1 staining in the trigeminal ganglia is shown as a ratio of the length of the embryo for wild-type, Med23^sn/sn, Med23^sn/sn; Lrp6^+/–, Med23^sn/sn; Wise^+/– and Med23^sn/sn; Lrp6^+/–; Wise^+/– (L) qPCR analysis of cDNA obtained from E9.5 wild-type, Med23^sn/sn, Med23^sn/sn; Lrp6^+/–, Med23^sn/sn; Wise^+/–, and Med23^sn/sn; Lrp6^+/–; Wise^+/– embryos indicates a significant increase in Ccnd1 and Dkk1 transcripts in Med23^sn/sn; Lrp6^+/–; Wise^+/– embryos compared to WT and Med23^sn/sn. Statistical analysis was performed using Students t-test with Med23^sn/sn as one of the nominal variables. Scale bars for (A–E) is 300 um, (F–J) is 200 um, (F′–J′) is 50 um. *p < 0.05.

FIGURE 7

Conditional deletion of Med23 in neural crest cells. (A,B) Brightfield images of P0 Med23^fx/+;Wnt1-Cre and Med23^fx/fx;Wnt1-Cre embryos indicate that the mutants exhibit micrognathia. (C,D) TuJ1 staining of E10.5 Med23^fx/+;Wnt1-Cre and Med23^fx/fx;Wnt1-Cre embryos however revealed proper formation of the cranial ganglia in mutant embryos. Scale bars for (A,B) is 450 um, (C,D) is 375 um.

FIGURE 8

Conditional deletion of Med23 in endothelial cells. (A,B) Brightfield images of E16.5 Med23^fx/+;Tek-Cre and Med23^fx/fx;Tek-Cre embryos indicate that the mutant embryos exhibit severe hemorrhaging and edema, probably resulting in lethality. (C,D) Immunostaining for endothelial cells using PECAM1 suggests development in Med23^fx/fx;Tek-Cre embryos is normal. Scale bars for (A,B) is 450 um, (C,D) is 300 um.

FIGURE 9

Temporal systemic deletion of Med23 at E6.5 phenocopies Med23^sn/sn embryos. (A,B) Med2^fx/fx;Cre-ER^T2 embryos treated with tamoxifen at E5.5 are small in size compared to Med2^fx/+;Cre-ER^T2 embryos. These embryos also display a shortened frontonasal prominence similar to Med23^sn/sn embryos. (C,D) TuJ1 staining revealed severely hypoplastic cranial as well as vagal ganglia in Med2^fx/fx;Cre-ER^T2 embryos. (E,F) PECAM1 staining revealed that endothelial cells are disorganized in the Med2^fx/fx;Cre-ER^T2 embryos compared to control Med2^fx/+;Cre-ER^T2 embryos. Scale bars for (A,B) is 250 um, (C,D) is 375 um, (E,F) is 300 um.

FIGURE 10

Model for Med23-mediated regulation of cranial placode development. In wild-type embryos, placode precursors express Six and Eya genes. Their subsequent expression of different Pax genes defines them as placode specific progenitor cells. Some of these cells then express Ngn1 and Ngn2 thus becoming neural progenitor cells. Ngn1 and Ngn2 regulate the expression of NeuroD1 which then defines these cells as neural precursors. However, in Med23^sn/sn embryos, placode precursors exhibit reduced Eya2 expression, and although Pax genes are similarly expressed in Med23^sn/sn embryos as they are in wild-type embryos, Ngn1, Ngn2, and NeuroD1 are downregulated. Collectively, this reduces the number of neural precursors resulting in defects in cranial ganglia development in Med23^sn/sn embryos.