Phenotypes observed in Eml3 null mice

Lethality in Eml3 null mice

Eml3−/− embryos are smaller than control littermates and are developmentally delayed.

At each gestational age between E8.5 and E18.5, the Eml3−/− embryos (A) and their placentas (B) (E10.5-E18.5) are significantly smaller than their Eml3+/+ and Eml3+/− control littermates. The size was measured either by area (E7.5-E10.5 embryos; E10.5-E18.5 placentas) or weight (E11.5-E18.5 embryos); for each litter analyzed, the size of each embryo or placenta was measured relative to the average of the control embryos (Eml3+/+ and Eml3+/−) and then normalized to the average of the Eml3+/+ embryos. The average percent size difference is shown below each image (n = 5-32). Dunn’s pairwise comparison, * = p<0.05, ns = not significant. Scale bars, 100µm for E7.5, 500µm for E8.5, and 1mm for E9.5-E18.5. (C) At each gestational age between E8.5 and E12.5, the Eml3−/− embryos have significantly fewer somite pairs than Eml3+/+ and Eml3+/− control littermates; average difference in number of somite pairs ± SD shown (n = 20 - 50). (D) Weight of embryos was plotted against the number of somite pairs counted for Eml3−/− and control embryos. Linear regression was used to fit the datapoints; Eml3−/− embryos are significantly smaller (p<0.0001) than control embryos with the same number of somite pairs.

Focal neuronal ectopias (FNEs) in Eml3−/− embryos.

(A) Nissl (cresyl-violet) staining of coronal sections of forebrains from E18.5 Eml3+/+ and Eml3−/− embryos. The Eml3+/+ sections in the top panels show normal brain architecture with a close-up view in the panel on the right. An FNE is present in the section of the Eml3−/− cortex shown in the bottom panels. The close-up in the bottom-right panel shows over-migrating neurons (dark cresyl-violet staining) extending past the marginal zone and into the subarachnoid space. Ca, calvarium; CP, cortical plate; MZ, marginal zone; SAS, subarachnoid space; SVZ, subventricular zone; V, ventricle; VZ, ventricular zone. Scale bars, 100µm. (B) Immunolabelling of an E18.5 Eml3−/− mouse brain section for laminin (green) a major component of the extracellular matrix of the pial basement membrane, and for neuron-specific tubulin beta-3 (TUBB3, red). The pial basement membrane is disrupted at the FNEs, with neurons (TUBB3+) over-migrating into the subarachnoid space. Laminin speckles are observed (arrow). CP, cortical plate; PBM, pial basement membrane; SAS, subarachnoid space. Scale bar, 50µm. (C) Heads of E15.5 Eml3+/+ and Eml3−/− embryos were immuno-stained and cleared before imaging with a fluorescence stereomicroscope (a-c) or with a confocal microscope (’a-c”). Immunostaining was performed with antibodies against the ECM protein laminin (green in a-c and a’-c’, white in ”a-c”) and a marker of terminally differentiated neurons, tubulin beta-3 (TUBB3, red). The asterisks point to the same area in the high magnification confocal microscope images as in the low magnification stereomicroscope images. Scale bars, 1mm (a-c) and 0.1mm (’a-c”).

Eml3−/− embryos are developmentally delayed vs. littermate controls but the onset and progression of neurogenesis is equal in somite-matched Eml3−/−and control forebrains; additionally, neurons of all cortical plate layers are present in Eml3−/− FNEs.

(A) The onset and progression of neurogenesis is delayed in Eml3−/− vs. littermate control forebrains. Forebrains from embryos collected at E11.5 were cryosectioned and processed for indirect immunofluorescence with markers for cell populations whose relative abundances indicate the onset and progression of neurogenesis. The percentage of TBR2-positive intermediate progenitors was determined. Eml3+/+ and Eml3+/− data are pooled as controls (Ctrls) vs. Eml3−/− embryos. The total number of cells in the embryonic brain section was determined with the nuclear stain Hoechst. Each dot on the graph represents the mean percentage of cells positive for TBR2 from analysis of 2-4 sections per embryo. Error bars represent SEM. (B) The onset and progression of neurogenesis is equal in somite-matched Eml3−/− and control forebrains. Forebrains from embryos collected at E10.5 and E11.5 were cryosectioned and processed for indirect immunofluorescence with markers for cell populations whose relative abundances indicate the onset and progression of neurogenesis. The percentage of TBR2-positive intermediate progenitors was determined and is plotted against the number of somite pairs counted in each embryo. The total number of cells in the embryonic brain section was determined with the nuclear stain Hoechst. Each dot on the graph represents the mean percentage of cells positive for TBR2 in an individual embryo. Error bars represent SD from analysis of 2-4 sections per embryo. Eml3+/+ and Eml3+/− data are pooled as controls (Ctrls) vs. Eml3−/− embryos. Shown below the graphs is the Theiler stage of development that corresponds to the number of somite pairs counted. Also indicated is the age of the embryos in days post-coitum. (C) Neurons of all cortical plate layers are present in Eml3−/− FNEs at E18.5. (a, b) Nissl (cresyl-violet) staining of serial coronal sections through a single FNE. (a’, b’) Immunolabeling of adjacent sections of the same FNE with markers of cortical plate layers. (a’) CUX1 immunostaining of late-born upper layer neurons (II-IV) and CTIP2 immunostaining of intermediate layer neurons (V). (b’) TLE4 immunostaining of early-born deeper layer neurons (VI) and CTIP2 immunostaining. Scale bars, 500µm (a, b) and 100µm (a’, b’).

Onset of neuronal over-migration and structural integrity of pial basement membrane ECM.

(A) Double immunostaining of laminin (green) and tubulin beta-3 (TUBB3, red) at 39 somite pair (sp) embryonic stage (TS17; collected at E11.5). Scale bars, 20µm. (a) In a heterozygous brain the PBM is continuous and migrating neuroblasts are observed underneath, whereas in an Eml3−/− embryo (b) a gap in the ECM and ectopic neuroblasts (arrowhead) were detected. (B, C) Coronal head sections of control and Eml3−/− embryos at different developmental stages were stained for laminin and tubulin beta-3. The length of gaps in the ECM was measured and the presence of over-migrating neuroblasts (NBs) was noted. The total length of PBM evaluated was measured for normalization. The green arrow points to the Eml3−/− embryo that had an FNE at the earliest developmental stage i.e., 39 somite pairs. (B) For each embryo, the total length of gaps per mm of PBM analyzed was plotted against the number of somite pairs counted. The Eml3−/− embryos with and without FNEs were plotted separately. (C) For each embryo, the length of the largest gap observed was plotted against the number of somite pairs counted. For the Eml3−/− embryos, the largest gaps with and without over-migrating NBs through the gap were plotted separately. No over-migrating NBs were ever observed in gaps of control embryos. (D) Transmission electron microscopy was performed on sections of Eml3−/− and control embryos (Ctrls; Eml3+/+ and Eml3+/−) matched by developmental stage. Three stages were analyzed (31-33, 39-41, and 46-47 somite pairs; sp). The embryo ages in days post-coitum are indicated. The total length of PBM was measured in each TEM micrograph. The ECM length was then subdivided into regions with different structural integrity. Normal, dense: sharp, straight, electron-dense ECM. Slightly altered: thicker, diffuse, less dense ECM. Severely altered: delaminating ECM, with electron dense material shedding off a diffuse PBM. Absent: regions with no ECM overlaying the NEP basal end-feet. Representative micrographs shown. At the 31-33sp stage, control ECM was 8.6±7.8% normal (dense), 85.4±8.0% slightly altered, 4.3±3.6% severely altered, and 1.6±1.4% absent, whereas Eml3−/− ECM was 8.5±7.0% normal (dense), 81.4±2.9% slightly altered, 9.3±8.1% severely altered, and 0.7±1.1% absent. At the 39-41sp stage, control ECM was 66.8±6.3% normal, 19.3±9.0% slightly altered and 13.9±15.2% severely altered, whereas Eml3−/− ECM was 14.4±6.7% normal, 55.1±23.8% slightly altered, 29.3±30.2% severely altered and 1.2±0.4% absent. At the 46-47sp stage, control ECM was 56.8±9.1% normal, 28.2±4.0% slightly altered and 15.0±5.1% severely altered, whereas Eml3−/− ECM was 13.9±5.9% normal, 34.1±7.2% slightly altered, 50.8±11.6% severely altered and 1.2±1.6% absent.

EML3 is expressed in the tissues that form and maintain the PBM.

(A) EML3 protein expression (green) as determined by indirect immunofluorescence on a coronal cryosection of an E10.5 Eml3+/+ embryo head. This developmental timepoint, 37 somite pairs, immediately precedes appearance of the first FNE in Eml3−/− embryos (at 39 somite pairs). An Eml3−/− littermate with 35 somite pairs was included as control for antibody staining specificity. Ctx, cerebral cortex; Me, mesenchyme; PP, preplate; SVZ, subventricular zone; VZ, ventricular zone; V, ventricle. Hoechst nuclear stain (blue). Scale bars, 100µm. (B) EML3 protein expression (green) during forebrain development as determined by indirect immunofluorescence on E9.5-E18.5 Eml3+/+ embryo cryosections. In the bottom panels, the same EML3-stained section is shown with additional immunostaining for markers of neural progenitor cells (SOX1, white) and of post-mitotic neuronal cells (TUBB3, red), as well as Hoechst nuclear stain (blue). The dotted line indicates the location of the PBM which separates mesenchyme (Me) and cerebral cortex whose cell composition changes during development. At E9.5 the cortex is spanned by neuroepithelial cells (NE). At E10.5 and E11.5, the neuroepithelial progenitors are differentiating into radial glial cells and the cortex can now be divided into ventricular zone (VZ), subventricular zone (SVZ), and the post-mitotic neurons of the preplate (PP). At E12.5-E18.5, radially migrating neurons are populating the region below the PBM and form the developing cortical plate (CP). V, ventricle. Scale bars, 25µm.

Verification of EML3 protein interactions in co-transfected cells.

HEK293T cells were transfected with plasmids encoding for the indicated full-length proteins. A fraction of the cell lysates was immunoblotted for verification of protein expression (INPUT lanes, 2% or 4% of the total lysate) and the remainder was used for immunoprecipitations with EML3 rabbit polyclonal antibody (EML3 IP lanes). HEK293T cells express low amounts of endogenous EML3 protein. (A) EML3 interacts with DYNLL1 and YWHAE proteins in co-transfection experiments. DYNLL1 co-immunoprecipitates with co-expressed M 3 protein in lane A’. YWHA co-immunoprecipitates with co-expressed M 3 protein in lane B’. ROM1 does not co-immunoprecipitate with co-expressed M 3 protein in lane C’. DYN 1 and YWHA are not immunoprecipitated by the EML3 antibody when no EML3-expressing construct is co-transfected into the cells in lane D’. (B) Mutating the TQT86 motif of the EML3 protein to AAA abolishes binding to DYNLL1. DYNLL1 co-immunoprecipitates with co-expressed M 3 protein in lane A’. DYN 1 does not co-immunoprecipitate with co-expressed EML3TQT86AAA mutant protein in lane B’.

EML3 protein coIP-MS data from three different tissues