MEGF8 is a modifier of BMP signaling in trigeminal sensory neurons
- Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, United States
- University of Southern California Keck School of Medicine, United States
- University of Toyama, Japan
Figures
Figure 1 with 1 supplement
Disruption of Megf8 causes defasciculation of the TG ophthalmic nerve.
(A) Whole-mount neurofilament staining of E11.5 control and Line 687 mutant littermates, showing the trigeminal ganglia (TG) and its three main projections: the ophthalmic (OP), maxillary (MX), and …
Figure 1—figure supplement 1
Complementation analysis of Megf8Trap and Megf8L1775P alleles.
(A) Schematic diagram of gene trap allele (Megf8Trap). German Gene Trap Consortium cells G037A09 have an intron 2–3 insertion of a secretory trap vector such that the start ATG and signal peptide of …
Figure 2 with 1 supplement
Megf8 is expressed widely during development.
(A–C) Whole-mount in situ hybridization (ISH) for Megf8 at E8.5, E9.5, and E10.5. (D) ISH for Megf8 on E11.5 transverse cryosection shows expression in the DRG. (E) ISH for Megf8 on E11.5 transverse …
Figure 2—figure supplement 1
Megf8 is expressed throughout the developing nervous system.
(A) In situ hybridization on coronal cryosections from E11.5-P0 shows strong Megf8 expression at all time points in the DRG and TG. (B–D) In situ hybridization on coronal cryosections at P0 shows …
Figure 3 with 3 supplements
Megf8 is required for development of the trigeminal ganglia, limb, skeleton, heart, and left-right asymmetry.
(A) Whole-mount neurofilament staining of E11.5 Megf8+/+ and Megf8−/− littermates. The Megf8−/− null mutant phenocopies the point mutant Megf8L1775P/L1775P. (B) Whole-mount images of Megf8+/+ and Meg…
Figure 3—figure supplement 1
Generation of a conditional knock-out mouse line (Megf8Flox).
(A) Schematic diagram of Megf8 gene targeting strategy. This strategy targeted the final exon of Megf8, which is 3.1 kb and comprises the transmembrane domain, intracellular C-terminus, and 3’′ UTR. …
Figure 3—figure supplement 2
Megf8L1775P/L1775P mutants show defects in limb and skeletal development.
(A) Alcian blue staining on E16.5 limbs shows that Megf8L1775P/L1775P mutants have digit duplication as well as duplication of bones in the hand. (B) Alizarin red staining on E16.5 limbs shows that M…
Figure 3—figure supplement 3
Heart development in Megf8L1775P/L1775P mutant embryos.
Sections from control (A–C) and Megf8L1775P/L1775P mutant (D–F) embryos. (A and E) Hematoxylin and eosin (H and E) staining of E15.5 control and mutant sections. The arrowhead in the control E15.5 …
Figure 4 with 1 supplement
Megf8 is required for development of the PNS.
(A and D) Whole-mount neurofilament staining of E11.5 Megf8+/+ and Megf8−/− littermates showing the DRG spinal nerves, which are undergrown in the Megf8−/− (arrow). (B and E) Whole-mount peripherin …
Figure 4—figure supplement 1
Conditional deletion of Megf8 from DRG neurons does not disrupt formation of the radial/ulnar nerves.
Whole-mount peripherin staining of E13.5 forelimbs from Wnt1-Cre;Megf8Flox/+ and Wnt1-Cre;Megf8Flox/Flox littermates. Limbs are outlined with dotted lines. Scale bar represents 500 µm.
Figure 5 with 2 supplements
BMP signaling is required for proper extension of the TG ophthalmic nerve.
(A) Whole-mount β-galactosidase and neurofilament (NFM) co-staining on Bmp4lacZ/+ embryos at E10.5 and E11.5, showing the relationship between Bmp4 expression and the developing TG nerve. (B) …
Figure 5—figure supplement 1
Bmp4 expression in Megf8−/− embryo compared to wild-type littermate.
Whole-mount β-galactosidase and neurofilament co-staining on E11.5 embryos: Bmp4lacZ/+;Megf8+/+ (left) and Bmp4lacZ/+;Megf8−/− (right) littermates. Bmp4lacZ expression is lost at the location where …
Figure 5—figure supplement 2
Bmpr2 and Megf8 are expressed throughout the developing TG.
In situ hybridization on serial transverse cryosections at E12.5 shows strong expression of Bmpr2 (A–C) and Megf8 (D–F) throughout the developing TG. Representative images are shown for ophthalmic (A…
Figure 6
Megf8 mediates the inhibition of TG axon growth by BMP4.
(A) E12.5 wild-type TG explants cultured in 0–100 ng/ml BMP4. (B) E12.5 TG explants from Megf8+/− and Megf8−/− littermates cultured side by side in 0–50 ng/ml BMP4. (C) Quantification of (A): axonal …
Tables
Table 1
Summary of Megf8−/− phenotypes and implicated BMPs
| Phenotype | Megf8-/- | BMP implicated | |
|---|---|---|---|
| Trigeminal nerve (V1) defasciculation | 100% (10/10) | n.d. | |
| Trigeminal patterning | n.d. | BMP4 | (Hodge et al., 2007) |
| Polydactyly | 100% (9/9) | BMP4, BMP7 | (Dudley et al., 1995; Dunn et al., 1997) |
| Reversed heart looping | 33% (7/21) | BMP4 | (Fujiwara et al., 2002) |
| Reversed embryonic turning (E11.5) | 33% (5/15) | BMP4 | (Fujiwara et al., 2002) |
| Edema (E13.5+) | 100% (6/6) | ||
| DRG spinal nerves undergrown (E11.5) | 100% (10/10) | BMP4 | (Guha et al., 2004) |
| Radial/ulnar nerves undergrown (E13.5) | 100% (2/2) | BMP4 | (Guha et al., 2004) |
| Vagus defasciculation | 90% (9/10) | n.d. | |
| Exencephaly | 36% (16/45) | ||
| Disrupted BMP4 expression around V1 | 100% (4/4) | ||
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Phenotypes, stages of observation and penetrances of Megf8−/− mutants are listed. V1 refers to the ophthalmic branch of the trigeminal nerve. Bmp loss-of-function lines known to display similar phenotypes are noted.
