Figure S1, related to . Conditional deletion of Erk1/2 and Mek1/2, but not Erk5, in the PNS results in DRG degeneration.
A-B. Compared to E10.5 Wnt1:Cre Rosa26lacZ controls (A), whole mount LacZ staining of Erk1/2CKO(Wnt1) Rosa26lacZ (B) embryos exhibit significant defects in craniofacial neural crest derived tissues, particularly the branchial arches and frontonasal mass (arrows), while the DRG (arrowheads) appears to be intact.
C-D. ERK2 immunofluorescent labeling of E10.5 control (C) and mutant (D) spinal cords reveal a significant loss of ERK2 expression in the Wnt1:Cre expression domain including the DRG (arrow) and dorsal spinal cord (arrowhead).
E. Western blots of DRG whole protein lysates derived from E12.5 Erk1/2CKO(Wnt1) embryos show absence of ERK1 and significantly reduced expression of ERK2 as well as a reduction in phospho-RSK3, a downstream ERK1/2 substrate, when compared to wld-type or Erk1-/- control samples. Quantification of densitometry results from four independent trials resulted in an 89.23±3.2% (p<.001) reduction in ERK2 expression. Similar results were obtained when E12.5 Mek1/2CKO(Wnt1) and Mek2-/- DRG samples were compared.
F-J. H&E stained brachial spinal cord cross sections derived from E17 wild-type (F), Erk1/2CKO(Wnt1) (G), and Mek1/2CKO(Wnt1) (H) embryos revealed an almost complete absence of the DRG in mutant embryos. Cresyl violet stained L4 spinal cord sections derived from the E17.5 wild-type (I) and Erk1/2CKO(Wnt1) (J) show significant degeneration as well, albeit, to a lesser degree than in brachial DRGs at the same stage.
K. A restriction map of the Erk5 gene including extragenic regions and the targeting vector design. Exons are indicated as solid black rectangles. The vector expresses a neomycin phosphotransferase (neo) and a diphtheria toxin A gene (DT). The two FRT sites anchored the two selection genes that were deleted by Flpe recombination in ES cell. Two loxP sites were inserted around Exon 3 of Erk5.
L. Restriction enzymes used to prepare fragments for Southern analysis of genomic DNA from four ES colonies are shown. The genomic probe target is indicated. Southern blotting identified targeted ES cell colonies (first three lanes) expressing both a 10kb wildtype and 7.3 kb modified Erk5 allele.
M. Erk5CKO(Wnt1) mice are noticeably smaller than control littermates and exhibit craniofacial malformations, including mandibular shortening and external ear defects.
N. ERK5 expression in E12.5 DRG lysates is significantly reduced in Erk5CKO(Wnt1) samples.
Figure S2, related to . Glial progenitors require ERK1/2 signaling to colonize the peripheral nerve.
A-B. Analysis of the peripheral nerve of E11.5 control (A) and Erk1/2CKO(Wnt1) (B) embryos demonstrated a decrease in the number of SCPs.
C-D. Compared to E12.5 controls (C), Erk1/2CKO(Wnt1) (D) embryos exhibit BFABP labeled glial progenitors within the DRG, but not in the developing peripheral nerve. In the distal peripheral nerve of control embryos, BFABP positive cells are clearly associated with neurofilament labeled axons (arrows, inset in C). In mutants, defasciculated axons are apparent, but no BFABP staining is seen (arrows, inset in D).
E-F. Generic labeling of all neural crest derivatives was achieved by whole mount staining E12.5 Wnt1:Cre Rosa26LacZ control (E) and mutant (F) forelimbs. LacZ staining was essentially absent in the distal peripheral nerve of mutant embryos (arrows), further demonstrating that neural crest derived progenitors fail to populate the developing peripheral nerve. (Scale bar=50μM)
G. Analysis of E12.5 Erk1/2CKO(Wnt1) DRG samples did not reveal a significant relative decrease in Nrg1 and ErbB2 gene expression as assessed by RT-PCR.
Figure S3, related to . Prior to the effects of SCP loss, ERK1/2 signaling does not alter sensory neuron number or the extent of outgrowth.
A-F. The number of sensory neurons in wild-type (A,C) and Erk1/2CKO(Wnt1) (B,D) DRGs was assessed by counting the number of Islet1/2 labeled cells at E12.5 and LacZ labeled cells in embryos crossed with the TauSTOP reporter line at E15.5. E12.5 is the earliest stage that pyknotic nuclei could be detected in Hoechst stained mutant DRGs (inset in B, arrowheads). Quantification of neuronal counts at E12.5 show a non-significant reduction in mutant embryos (p=.081), while counts at E15.5 show significant neuron loss (* = p-value < 0.001, n=3) (E). Western blots of E12.5 DRG lysates show an increase in activated caspase-3 expression when compared to controls (F).
G-H. The TauSTOP reporter line will express mGFP in sensory and sympathetic projections when crossed with the Wnt1:Cre driver. Forelimbs from E13 control (Erk1-/-Erk2fl/wtWnt1:Cre TauSTOP) (G) and Erk1/2CKO(Wnt1) TauSTOP (H) embryos were immunolabeled with GFP and Neurofilament antibodies in whole mount. GFP labeled projections can be detected at the tips of developing nerves as depicted in magnified images of the ulnar nerve.
I-J. The Brn3aTau-LacZ mouse drives the expression of a Tau-LacZ fusion protein specifically in sensory neurons. While whole mount lacZ staining in these mice does not label peripheral nerves to the same extent as immunolabeling methods, sensory neuron outgrowth between E12.5 control (I) and Erk1/2CKO(Wnt1) (J) embryos appears similar, however, defasciculation can still be detected (arrows).
Figure S4, related to . Neuronal specific deletion of MEK/ERK signaling inhibits NGF dependent cutaneous innervation, but not proprioceptive innervation of muscle spindles in vivo.
A. Mek1/2CKO(Nestin) mice exhibit significant reductions in MEK1/2 expression and near complete inhibition of ERK1/2 phosphorylation in both spinal cord and DRG lysates derived from E14.5 mutant embryos.
B-D. Erk1/2CKO(Advillin) mice are smaller relative to controls at P16 (B). Further, mutant mice (D) exhibit a clasping phenotype when raised by the tail.
E-L. Both P3 control (E) and Erk1/2CKO(Advillin) (F) L3 DRGs exhibit a normal pattern of expression of the proprioceptive sensory neuron marker Parvalbumin. Central proprioceptive afferents into the spinal cord also appeared intact (G-H). Proprioceptive peripheral innervation of soleus muscle spindles labeled with the pan-axonal marker, PGP9.5, did not appear significantly different from controls in P3 mutant mice (I-J), but by P18, spindle innervation in mutant mice appeared significantly disrupted (K-L).
Figure S5, related to . Schwann cell specific Erk1/2 inactivation results in hypomyelination.
A. P1 Erk1/2CKO(Dhh) sciatic nerve lysates exhibit decreased expression of ERK2.
B-C. Schwann cells labeled with a Cre dependent reporter line (Z/EG) were detected adjacent to NMJ’s at the very tip of phrenic nerve projections into the diaphragm in P20 control (B) and Erk1/2CKO(Dhh) (C) mice.
Figure S6, related to . Differentially expressed genes in the DRG of control vs. Erk1/2CKO(Wnt1) mice.
The expression level of each gene is given in log2ratio and its percentile within each condition. Differential expression is provided as the difference in log2ratio (control-mutant) and also transformed into fold-increase. Genes are listed in ascending order based on fold difference. The second table lists the functional annotation of differentially expressed genes in mutant DRGs.
Figure S7, related to . ERK1/2 signaling is not required for spinal motor neuron development.
A. E14.5 control (A) and Erk1/2CKO(Olig2) (B) spinal cords were labeled with the motor neuron marker, Islet1/2, and ERK2. Note the lack of ERK2 expression in the progenitor domain from which motor neurons and oligodendrocytes arise (arrows) as well as in the motor neurons within the lateral motor column (outlined).
Figure S8, related to . Oligodendrocyte proliferation and the timing of myelination require Erk1/2.
A-B. Analysis of oligodendrocyte number in the white matter labeled with PDGF-Rα or nuclear markers revealed a decrease in E14.5 Erk1/2CKO(Olig2) (B) spinal cords compared to controls.
C-D. P1 MBP positive oligodendrocytes in the ventral spinal cord of control (C) and mutant (D) mice were co-labeled ERK2. ERK2 expression is indeed significantly reduced in mutant oligodendrocytes expressing MBP.