ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube For Chemical Industry chemical component ,Deficiency of SPECC1L leads to increased stability of spliced ​​joints and reduced shedding of cranial neural crest cells.

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The cranial neural crest cells (CNCC) slough off the embryonic neural folds and migrate to the pharyngeal arches, which form most of the midface structures. CNCC dysfunction plays an important role in the etiology of orofacial cleft, a common congenital malformation. Heterozygous SPECC1L mutations have been found in patients with atypical and syndromic clefts. Here, we report enhanced staining of canonical adhesive junction (AJ) components, β-catenin and E-cadherin in cultured SPECC1L knockdown cells, and electron micrographs show apical-basal diffusion of AJ. To understand the role of SPECC1L in craniofacial morphogenesis, we created a Specc1l deficient mouse model. Homozygous mutants are embryonic lethal and exhibit impaired neural tube closure and CNCC lamination. AJ protein staining is increased in mutant neural folds. This AJ defect is consistent with a defect in CNCC delamination, requiring AJ dissolution. In addition, Specc11 mutants have reduced PI3K-AKT signaling and increased apoptosis. In vitro, mild inhibition of PI3K-AKT signaling in wild-type cells was sufficient to induce AJ changes. Importantly, AJ changes induced by SPECC1L knockdown can be reversed by activation of the PI3K-AKT pathway. Taken together, these data suggest that SPECC1L, as a novel regulator of PI3K-AKT signaling and AJ biology, is required for neural tube closure and CNCC stratification.
Cranial neural crest cells (CNCCs) localize to the dorsal neuroectoderm and detach from the neuroepithelium of the developing neural folds through a process involving the epithelial-mesenchymal transition (EMT)1,2,3. Premigrating epithelial CNCCs disrupt intercellular junctions and become migrating mesenchymal CNCCs that fill the first and second pharyngeal arches and form most of the craniofacial cartilage. Thus, genes that regulate CNCC function are often disrupted in the etiology of craniofacial congenital anomalies such as orofacial clefts, most commonly affecting 1/800 births in the US alone. One of the congenital deformities8.
Delamination of the CNCC coincides with the closure of the anterior neural tube between 8.5 and 9.5 days of embryonic development in mice. Mutants of a number of mouse orofacial cleft-associated genes also exhibit some form of neural tube defect, including Irf69,10, Ghrl310, Cfl111, and Pdgfrα12. However, the processes of neural tube closure and CNCC stratification can be considered independent, as the Splotch mutant mouse (Pax3) exhibits defects in neural tube closure without any effect on CNCC stratification or migration 13,14. Additional mouse models with defects in CNCC dissection and neural tube closure will help delineate the common molecular basis of these two processes.
Isolation of CNCC from neuroepithelial cells requires the dissolution of adhesive junctions (AJs), which are composed of protein complexes containing, among others, E-cadherin, β-catenin, α-E-catenin, and α-actinin associated with actin filaments 2. Overexpression studies E-cadherin in neural folds showed a reduction or delay in CNCC delamination. Conversely, suppression of E-cadherin results in early stratification15,16. Many of the factors that mediate EMT during CNCC stratification are transcription factors (AP2α, Id2, FOXD3, SNAIL, TWIST, SOX10) and extracellular matrix (ECM) remodeling proteins such as matrix metalloproteinases (MMPs), however CNCCs are direct cytoskeletal AJ regulators are not yet known. The PI3K-AKT pathway is known to antagonize E-cadherin levels, mainly from cancer research17. Recent studies have shown that loss of PDGFα-based PI3K-AKT signaling in mice leads to craniofacial abnormalities, including cleft palate and neural tube defects12. However, the relationship between the PI3K-AKT pathway and AJ stability upon CNCC stratification is unclear.
We previously identified SPECC1L as the first mutant gene in two people with a severe cleft that extends from the mouth to the eye, known as oblique cleft (ObFC) or Tessier IV18 cleft. SPECC1L mutations have been identified in two multigenerational families with the autosomal dominant Opitz G/BBB syndrome (OMIM #145410), in which affected individuals exhibited hyperdistance and cleft lip/palate19, and in one family with Tibi overdistance syndrome ( OMIM #145420)20. more than half of cases of Opitz G/BBB syndrome are X-linked (OMIM #300000) and are caused by mutations in the MID1 gene, which encodes protein 22 of the microtubule-associated cell skeleton. We hypothesize that SPECC1L, also a protein associated with microtubules and the actin cytoskeleton, may mediate signaling required for actin cytoskeleton remodeling during cell adhesion and migration 18 . Through in vitro and in vivo studies, we now describe SPECC1L as a novel regulator of AJ stability through PI3K-AKT signaling. At the cellular level, SPECC1L deficiency resulted in a decrease in the level of the pan-AKT protein and an increase in the apical-basal dispersion of AJ, which was eliminated by chemical activation of the AKT pathway. In vivo, Specc11-deficient embryos show impaired neural tube closure and reduced CNCC dissection. Thus, SPECC1L functions in highly regulated cell adhesion-based signaling required for normal CNCC function during facial morphogenesis.
To characterize the role of SPECC1L at the cellular level, we used the previously described stable osteosarcoma cell line U2OS deficient in SPECC1L18. These stable U2OS cells with SPECC1L (kd) knockdown had a moderate (60–70%) decrease in the levels of SPECC1L transcripts and proteins, along with defects in migration and reorganization of the actin cytoskeleton 18. In contrast, a severe transient decrease in SPECC1L has been shown to lead to mitotic defects 23 . Upon further characterization, we found that our stable SPECC1L-kd cells changed morphology at a very high degree of confluence (Figure 1). Individual control cells and kd cells at low confluence looked similar (Figure 1A,D). 24 hours after fusion, control cells retained their cuboidal shape (Fig. 1B, E), while SPECC1L-kd cells elongated (Fig. 1C, F). The extent of this change in cell shape was captured by in vivo live imaging of control cells and kd cells (movie 1). To determine the role of SPECC1L in confluent cells, we first examined its expression. We found that SPECC1L protein levels increased upon fusion (Figure 1G), whereas SPECC1L transcript levels did not increase (Figure 1H). In addition, as cell density increased, SPECC1L protein accumulated at intercellular boundaries (Fig. 2A-E), with a pattern overlapping with that of membrane-associated β-catenin (Fig. 2A’-E’). Given the association of SPECC1L with the actin cytoskeleton 18,23 we hypothesized that SPECC1L interacts with actin-based adhesive junctions (AJ).
(AF) SPECC1L knockdown (DF) cells elongate at high confluence (F) compared to control U2OS cells (AC). Shown here are three of the six time points (T1, T3, T6) that we selected for different cell densities. (G) Western blot analysis showing that the SPECC1L protein is stabilized at a high degree of confluence compared to a low degree of confluence in control cells. Western blot of SPECC1L shows the expected 120 kDa band and a higher molecular weight band, possibly post-translationally modified (*). Western blot analysis was performed under the same conditions for low and high confluence. Images showing SPECC1L at low and high confluence were taken from the same blot. The same blot was removed and re-examined with β-actin antibody. (H) Quantitative RT-PCR analysis showed no significant changes in SPECC1L transcript levels. Error bars represent SEMs from four independent experiments.
(AE) We chose six time points (T1-T6) representing a range of cell densities to normalize cell shape analysis and AJ changes in U2OS cells with SPECC1L knockdown (kd). The first five of these time points included single cells (T1), 50-70% fusion of small cell clusters (T2), fusion without reshaping kd cells (T3), reshaping kd cells (T4), and 24 hour changes. in the posterior form of kd (T5) cells. The SPECC1L protein was predominantly dispersed in the cytoplasm at T1 (A), but its accumulation was observed at intercellular boundaries at subsequent time points (B–E, arrows). (FJ) β-catenin shows similar accumulation at intercellular boundaries associated with the AJ complex. (A’-E’) SPECC1L and β-catenin show overlapping staining at cell borders at high cell density (arrows). (F’-J’) In SPECC1L-kd cells, β-catenin staining appears normal at low cell density (F’-H’), but expands as cell shape changes (I’, J’; arrows), indicating that AJ have changed. Bars = 10 µm.
We then tried to determine the effect of SPECC1L deficiency on AJ. We used several AJ-associated markers, including the canonical components F-actin, myosin IIb, β-catenin, and E-cadherin24,25,26,27. Actin stress fibers increased in SPECC1L-kd cells as described previously (Fig. 3A,B) 18 . Myosin IIb associated with actin filaments showed a similar increase in SPECC1L-kd cells in vitro (Fig. 3C,D). AJ-associated β-catenin binds to cadherin at the cell membrane, showing a normal “honeycomb” expression pattern in control cubocytes (Fig. 3E,G). Interestingly, in flat images using confocal microscopy, β-catenin (Fig. 3E,F) and E-cadherin (Fig. 3G,H) staining on the cell membrane of confluent SPECC1L-deficient cells showed prominent patterns of extended staining. This expansion of AJ-associated β-catenin staining in kd cells was most pronounced at confluence, but appeared to precede changes in cell shape (Fig. 2F-J, F’-J’). To determine the physical nature of this extended AJ staining, we examined cell borders on the apical-basal surface of SPECC1L-kd U2OS cells by transmission electron microscopy (TEM) (Figure 3I,J). In contrast to control cells (Fig. 3I), which had separate electron dense regions indicative of AJ (arrows), kd cells (Fig. 3J) showed large, contiguous regions of high electron density indicative of AJ along the apicobasal plane. . In addition, on transverse sections, we observed extensive cell membrane folds in kd cells (Fig. S1A,B), which explains the extended pattern of β-catenin and E-cadherin staining bands (Fig. 3F,H). In support of the role of SPECC1L in AJs, β-catenin was co-immunoprecipitated with SPECC1L in lysates of confluent U2OS cells (Fig. 3K). Along with extended immunostaining for AJ markers, TEM analysis was consistent with our hypothesis that SPECC1L deficiency increases AJ apical-basal density and variance.
(AH) Increased F-actin staining in kd cells at 48 hours post-fusion (T6; A, B). Altered staining of myosin IIb associated with F-actin (C, D). The smooth pattern of β-catenin and E-cadherin membrane staining in control cells (E, G) was enhanced in SPECC1L-kd (F, H) cells. Bars = 10 µm. (I–J) Electron micrographs observing the apical-basal intercellular junction. Control cells show distinct electron-dense regions indicating sticky junctions (I, arrows). In contrast, the entire apical-basal junction in SPECC1L-kd cells appeared electron dense (J, arrows), indicating increased density and dispersion of adhesive junctions. (K) β-catenin was co-immunoprecipitated with SPECC1L in confluent U2OS cell lysates. Image taken from one spot representing one of four independent experiments.
To understand the role of SPECC1L in craniofacial morphogenesis, we created a Specc1l deficient mouse model using two independent ES trap cell lines, DTM096 and RRH048 (BayGenomics, CA), which represent intron 1 and Specc1l transcripts were captured at 15 ( Fig. 1). 4A, figure S2). The genomic location of the decoy vector insert was determined by whole genome sequencing and confirmed by PCR (Fig. S2). Both gene trap designs also allowed in-frame fusion of the Specc11-lacZ reporters upon capture. Therefore, lacZ expression determined by X-gal staining was used as an indicator of Specc11 expression. Both alleles showed similar lacZ expression patterns, with the DTM096 gene trap in intron 1 showing stronger expression than RRH048 in intron 15 (not shown). However, Specc1l is widely expressed, with particularly strong expression in the neural folds at E8.5 (Figure 4B), in the neural tube and facial processes at E9.5 and E10.5 (Figure 4C,D), and in developing limbs at E10. 5 and eyes (Figure 4D). We previously reported that SPECC1L expression in the first pharyngeal arch at E10.5 was present in the epithelium and underlying mesenchyme18, consistent with the CNCC lineage. To test SPECC1L expression in CNCC, we performed E8.5 neural folds (Figure 4E-J) and E9.5 skull sections (Figure 4K-). At E8.5, SPECC1L stained neural folds intensely (Fig. 4E, H), including cells stained with NCC markers (Fig. 4G, J). At E9.5, SPECC1L (Fig. 4K, N) strongly stained migrating CNCC co-stained with AP2A (Fig. 4L, M) or SOX10 (Fig. 4O, P).
(A) Schematic representation of the mouse Specc11 gene showing decoy vector insertion in ES DTM096 (intron 1) and RRH048 (intron 15) cell clones. (BD) lacZ staining of heterozygous Specc1lDTM096 embryos representing Specc1l expression from E8.5 to E10.5. NE = neuroectoderm, NF = neural fold, PA1 = first pharyngeal arch. (EP) SPECC1L immunostaining with NCC markers AP2A and SOX10 in E8.5 (NF; EJ) neural folds and E9.5 (KP) skull sections. SPECC1L staining was widely observed in neural folds E8.5 (E, H; arrowheads), including cells labeled with AP2A (F, G; arrowheads) and SOX10 (I, J; arrowheads). At E9.5, SPECC1L strongly stained migrating CNCCs (K, N; arrows) labeled AP2A (L, M; arrows) and SOX10 (O, P; arrows).
Crossing between heterozygous Specc1lDTM096/+ and Specc1lRRH048/+ mice shows that the two gene trap alleles are not complementary and that compound heterozygotes and embryonic homozygotes for either gene trap allele are embryonic lethal (Table S1). Mendelian ratios indicated a decrease in the survival rate of heterozygotes at birth (expected 1.34 vs. 2.0). We noted low perinatal mortality among heterozygotes, some had craniofacial anomalies (Fig. S3). However, the low penetrance of these perinatal craniofacial phenotypes makes it difficult to study their underlying pathophysiological mechanisms. Therefore, we focused on the embryonic lethal phenotype of homozygous Specc11 mutants.
Most compound heterozygous or homozygous Specc1lDTM096/RRH048 mutant embryos did not develop after E9.5–10.5 (Figs. 5A–D), and the neural tube did not close anteriorly (Figs. 5B, D) and sometimes closed posteriorly (not shown). . This cranial neural tube closure defect was associated with the majority of CNCC marked DLX2 remaining in the neural folds at E10.5, indicating no dissection (Figure 5A’-D’). To determine if the overall size of CNCC was also reduced, we tagged CNCC lines with GFP in our gene trap lines with Wnt1-Cre and ROSAmTmG. We stream sorted GFP+ NCC and GFP- (RFP+) non-NCC from whole embryos. At E9.5, the proportion of flow-sorted GFP-labeled CNCCs did not change significantly between WT and mutant embryos (not shown), indicating normal CNCC specification. Therefore, we hypothesized that residual Wnt1-Cre and DLX2 staining in exposed neural folds (Figure 5B’) was due to defective CNCC layering, possibly due to increased density or dispersion of AJ cells, as seen in SPECC1L-kd cells. We used the NCC markers SOX10, AP2A, and DLX2 to confirm the presence of CNCC in the neural fold (Figure 5E-R). At E8.5, neural fold staining for all three NCC markers was observed in sections of WT (Fig. 5E, G, I) and Specc1l mutant (Fig. 5F, H, J). At E9.5, while NCC markers stained migrating NCC in WT sections (Fig. 5M, O, Q), residual NCC staining was observed in exposed neural folds of Specc1l mutant embryos (Fig. 5N, P, R). Because SOX10 and DLX2 mark migrating CNCCs, this result suggests that SPECC1L-deficient CNCCs achieve post-migratory specification but fail to migrate from neural folds.
Specc11 deficiency leads to defective neural tube closure, delamination of cranial neural crest cells and AJs.
(A, B’) E9.5 WT (A) Embryo carrying migrating cranial neural crest cells (CNCC) labeled with Wnt1-Cre (A’). In contrast, Specc11 mutant embryos show open neural folds (B), arrowheads) and CNCCs that have not migrated (B’, arrowheads). (C, D’) Bright field images (C, D’) and immunostaining (C’, D’) of the CNCC marker DLX2 of E10.5 WT embryos (C, C’) and Specc1l (D, D’). In WT E10.5 embryos, DLX2-positive CNCC colonize the gill arches (C’, arrows), while in mutants, conspicuous staining persists in the open neural folds (D’, arrows) and in the first pharyngeal arches (D’, arrows). ) with some staining (arrows) indicating poor delamination and migration of CNCC. ER) Sections of WT and Specc1l mutant embryos at stages E8.5 (E–L) and E9.5 (M–R) were labeled with NCC markers SOX10 (E, F, M, N), AP2A (G, H, O, P ) and DLX2 (I, J, Q, R). At E8.5, NCC staining was observed in wild-type neural fold (NF) and mutant sections. Co-staining of SOX10 and β-catenin in E8.5 WT (K) and mutant (L) revealed increased β-catenin staining at cell boundaries in the neural folds. At E9.5, wild-type staining of migrating CNCCs (M, O, Q) was observed, while in mutants, unstratified CNCCs stained open neural folds (N, P, R). (S–Z) In vivo AJ labeling analysis in coronal sections of WT and Specc11DTM096/RRH048 embryos with the E9.5 mutation. An approximate sectional plane is shown in the upper right corner. In sections of mutant tissues, increased staining of F-actin (S, T) and myosin IIb (U, V) was observed. Similar to the in vitro results in Fig. 3, in mutant embryos, enhanced membrane staining for β-catenin (W, X) and E-cadherin (Y, Z) was observed. (AA-BB) An electron micrograph of a section of a wild-type embryo looking beyond the border of the apical-basal cell shows a distinct electron-dense region indicative of adhesive junctions (AA, arrows). In contrast, in sections of Specc11 mutant embryos (BB, arrows), the entire apicobasal junction is electron dense, indicating an increased density and dispersion of adhesive junctions.
To test our hypothesis that reduced layering is due to altered AJ, we examined AJ labeling in exposed neural folds of Specc1l mutant embryos (Fig. 5S-Z). We observed an increase in actin stress fibers (Fig. 5S, T) and a concomitant increased localization of myosin IIB staining on actin fibers (Fig. 5U, V). Importantly, we observed increased staining of β-catenin (Fig. 5W,X) and E-cadherin (Fig. 5Y,Z) at intercellular boundaries. We also examined β-catenin staining of NCC in the neural folds of E8.5 embryos (Fig. 5K, L). β-catenin staining appeared to be stronger in Specc1l mutant neural folds (Fig. 5L and K), suggesting that AJ changes had begun. In electron micrographs of skull sections of E9.5 embryos, we again observed increased diffuse electron-dense staining in Specc1l mutant embryos compared to WT (Fig. 5AA, BB and S1E-H). Taken together, these results support our in vitro results in SPECC1L-kd U2OS cells and suggest that aberrant AJ staining precedes CNCC stratification in our mutant embryos.
Given the known antagonistic relationship between AKT activity and E-cadherin stability,17,28 we hypothesized the involvement of PI3K-AKT signaling. In addition, we observed subepidermal blistering in some of our mutant embryos that escaped lethality (<5%) at E9.5-10.5 and instead settled at around E13.5 (Fig. S3). Subepidermal vesicles are a hallmark of reduced PI3K-AKT signaling based on PDGFRα12. Fantauzzo et al. (2014) reported that disruption of PDGFRα-based PI3K activation in PdgfraPI3K/PI3K mutant embryos results in subepidermal vesicles, neural tube defects, and cleft palate phenotypes. Indeed, levels of pan-AKT and active phosphorylated Ser473-AKT were reduced in vivo in Specc1l mutant tissues to E9.5 embryonic arrest (Fig. 6A-D). The decrease in levels of phosphorylated Ser473-AKT may be entirely due to the decrease in levels of pan-AKT in vivo (FIG. 6E) and in vitro (FIG. 6F). An in vitro decrease was observed only when U2OS cells were strongly confluent with changes in cell shape and AJ density (Figure 6D). Thus, our data suggest that SPECC1L is a novel positive regulator of PI3K-AKT signaling in craniofacial morphogenesis.
(A–E) E8.5 (A,B) and E9.5 (C,D) skull sections or E9.5 lysates from Specc1l mutant embryos (E) showing levels of active phosphorylated S473-AKT and pan-AKT Protein reduction , compared to control WT. Western blotting was performed on wild-type lysates and mutant lysates under the same conditions. The images shown for SPECC1L were taken from one blot. The same blot was removed and re-examined with anti-pan-ACT and β-actin antibodies. Pan-AKT levels in E8.5 neural folds (A, B) and levels of phosphorylated S473-AKT in E9.5 skull sections were significantly reduced. (F) Pan-AKT levels were similarly reduced in lysates of SPECC1L-kd U2OS cells harvested at high confluence. Error bars represent SEMs from three independent Western blot quantifications. (GJ) Sections of WT embryos at E9.5 stained with KI67 and cleaved caspase 3, respectively, showing cell proliferation (G, G’) and little apoptotic activity (H, H’). Specc11 mutant embryos show comparable cell proliferation (I), but the number of cells undergoing apoptosis is significantly increased (J).
We then examined markers of proliferation and apoptosis. We did not observe any difference in the proliferation of E9.5 embryos (Fig. 6E, G compared to I) with a proliferation index of 82.5% for WT mutants and 86.5% for Specc1l mutants measured by KI67 staining (p <0.56, Fisher’s exact test). Similarly, we did not observe any difference in apoptosis measured by staining for cleaved caspase 3 in neural folds at E8.5 until embryo arrest (not shown) (not shown). In contrast, apoptosis was significantly increased in all E9.5 mutant embryos (Fig. 6F, H and J). This overall increase in apoptosis is consistent with reduced PI3K-AKT signaling and early embryonic lethality29,30,31.
Next, to confirm a causal role for PI3K-AKT signaling in AJ changes in our kd cells, we chemically altered the pathway in control and kd cells (Figure 7A-F). We used as a marker the cell shape change phenotype observed in confluent SPECC1L-kd cells, which we quantified using the ratio of the longest dimension (length) to the corresponding vertical dimension (width). A ratio of 1 is expected for relatively round or cuboidal cells (Figure 7G). In addition to cell shape, we also confirmed the effect on AJ by β-catenin staining (Fig. 7A’-F’). Inhibition of the PI3K-AKT pathway using wortmannin was sufficient to change cell shape in control cells (Figure 7A,C) and AJ (Figure 7A’). PI3K-AKT activator SC-79 did not affect cell shape (FIG. 7A, E) or AJ expansion (FIG. 7A’) in control cells. In SPECC1L-kd cells, further suppression of the PI3K-AKT pathway resulted in increased apoptosis (Fig. 7B,D) and a marked increase in β-catenin staining (Fig. 7B’), consistent with our in vivo heavy mutants. Importantly, activation of the PI3K-AKT pathway significantly improved cell shape (Figure 7B,F) and AJ phenotypes (Figure 7B”). Changes in cell shape were quantified as cell roundness ratio (CCR) and compared for significance as described above (FIG. 7G). Indeed, in control cells (Fig. 7G, CCR = 1.56), wortmannin treatment was sufficient to significantly alter the cell shape (Fig. 7G, CCR = 3.61, p < 2.4 × 10-9) to the extent similar to the one observed in SPECC1L. -kd cells (Fig. 7G, CCR = 3.46). Wortmannin treatment of SPECC1L-kd cells (Fig. 7G, CCR = 3.60, negligible) was no more significant than untreated kd cells (Fig. 7G, CCR = 3.46, negligible) or wortmannin-treated control cells (Fig. 7G). , CCR = 3.46, negligible) additionally affects cell elongation (7G, CCR = 3.61, negligible). Most importantly, SC-79 AKT activator restored the elongated phenotype of SPECC1L-kd cells (Fig. 7G, CCR = 1.74, p < 6.2 × 10-12). These results confirm that SPECC1L regulates PI3K-AKT signaling and suggest that a moderate decrease in SPECC1L affects cell adhesion, while a strong decrease leads to apoptosis (Fig. 8).
(A–F’) Control (A, C, E) and SPECC1L-kd (B, D, F) cells treated with PI3K-AKT pathway inhibitor wortmannin (C, D) or SC-79 activator (E, F) Treatment .Untreated control cells are cuboidal (A) with normal β-cat cellular staining (A’), while kd cells are elongated (B) with increased β-cat staining (B’). After suppression of the PI3K-AKT pathway, control cells elongated (C) with β-cat expansion (C’), while kd cells began to undergo apoptosis (D), similar to our highly mutated embryos and showing extremely enhanced β-cat. staining (D’). After activation of the PI3K-AKT pathway, control cells remained cuboidal (E) and had normal β-cat (E’) staining, while kd cells showed significantly improved cell shape (F) and β-cat (F’) staining, indicating (G) The degree of cell shape change in (AF) was quantified using the cell roundness ratio (CCR) of the longest dimension (length) and the corresponding vertical dimension (width) using MetaMorph software. Untreated (NT) SPECC1L-kd cells (CCR = 3.46) were significantly longer than control cells (CCR = 1.56, p < 6.1 × 10–13). Wort’s inhibition of the PI3K-AKT pathway in control cells was sufficient to cause a similar elongation in cell shape (CCR=3.61, p<2.4×10-9). Similarly, AKT activation by SC-79 in SPECC1L-kd cells restored cell elongation to control levels (CCR = 1.74, p < 6.2 × 10–12). Wortmannin treatment of SPECC1L-kd cells resulted in increased apoptosis but no further increase in cell shape change (CCR=3.60) compared to untreated kd (CCR=3.46, ns) or wortmannin-treated control cells (3.61) observed in . ns = doesn’t matter. +/- SEM measurements for 50 cells are shown. Statistical differences were calculated using Student’s t-test.
(A) Schematic representation of inhibition and activation of the PI3K-AKT pathway resulting in AJ changes and rescue, respectively. (B) Proposed model for AKT protein stabilization by SPECC1L.
Premigratory CNCCs require AJ lysis to separate from anterior neural fold neuroepithelial cells1,15,32. Increased staining of AJ components and loss of the apical-basal AJ asymmetric distribution in SPECC1L-deficient cells both in vitro and in vivo, combined with the physical proximity of SPECC1L to β-catenin, suggest that SPECC1L functions to properly maintain AJ local stability for organization muscles. actin cytoskeleton. The association of SPECC1L with the actin cytoskeleton and β-catenin and the increase in the number of condensed actin filaments in the absence of SPECC1L is consistent with the observed increase in AJ density. Another possibility is that an increased number of actin fibers in SPECC1L-deficient cells leads to a change in intercellular tension. Because cellular stress affects AJ 33 dynamics, voltage changes can result in more diffuse AJ 34 . So any changes will affect the CNCC layers.
Wnt1 is expressed in the early neural folds that give rise to neural crest cells. Thus, Wnt1-cre lineage tracing marks both pre- and migrating NCC35. However, Wnt1 also marks dorsal brain tissue clones also derived from early neural folds 35,36 , making it likely that our staining of E9.5 mutants for Wnt1 markers in open neural folds is not CNCC. Our positive staining for the NCC markers AP2A and SOX10 confirmed that the exposed neural folds of Specc11 mutant embryos did indeed contain CNCC. In addition, since AP2A and SOX10 are markers of early migrating NCC, positive staining indicated that these cells are post-migratory CNCC that cannot be stratified by E9.5.
Our data suggest that molecular regulation of AJ by SPECC1L is mediated by PI3K-AKT signaling. AKT signaling is reduced in SPECC1L deficient cells and tissues. Findings by Fantauzzo et al. support a direct role for PI3K-AKT signaling in craniofacial morphogenesis. (2014) showed that the lack of activation of PDGFRα-based PI3K-AKT signaling leads to a cleft palate phenotype. We also show that inhibition of the PI3K-AKT pathway is sufficient to change AJ and cell shape in U2OS cells. Consistent with our findings, Cain et al. 37 showed that downregulation of the PI3K α110 subunit in endothelial cells results in a similar increase in pericellular β-catenin staining, referred to as an increase in the “connectivity index”. However, in endothelial cells whose actin filaments are already highly organized, suppression of the PI3K-AKT pathway results in a loose cell shape. In contrast, SPECC1L-kd U2OS cells showed an elongated cell shape. This difference may be cell type specific. While suppression of PI3K-AKT signaling permanently affects the actin cytoskeleton, the effect on cell shape is determined by changes in tension caused by changes in the density and organization of central actin fibers. In U2OS cells, we used only cell shape changes as a marker of SPECC1L-deficient AJ change and recovery. In conclusion, we hypothesize that inhibition of the AKT pathway in SPECC1L deficiency increases AJ stability and reduces delamination in CNCC.
Interestingly, pan-AKT levels were reduced in vitro and in vivo in addition to phosphorylated 473-AKT levels in the absence of SPECC1L, suggesting regulation of PI3K-AKT signaling at the level of AKT protein stability or turnover. The SPECC1L and MID1 genes, both associated with Opitz/GBBB syndrome, encode proteins that stabilize microtubules 18,22 . The mechanism by which SPECC1L and MID1 mediate microtubule stabilization is not fully understood. In the case of SPECC1L, this stabilization includes enhanced acetylation of a subset of microtubules 18 . It is possible that SPECC1L uses a similar mechanism to stabilize other proteins such as AKT. It has been shown that acetylation of lysine residues in the AKT protein leads to a decrease in membrane localization and phosphorylation38. In addition, ubiquitination of the K63 chain at the same lysine residue on AKT is required for its membrane localization and activation39,40. Among several factors interacting with SPECC1L proteins identified in various high throughput yeast two-hybrid screens, four – CCDC841, ECM2942, APC and UBE2I43 – have been implicated in protein turnover or stability via ubiquitination or sumoylation. SPECC1L may be involved in post-translational modification of AKT lysine residues, affecting AKT stability. However, the critical role of SPECC1L in the localization and stability of the AKT protein remains to be elucidated.
Severe defects in SPECC1L expression in vivo resulted in increased AJ marker staining and defective CNCC overlay, as well as increased apoptosis and early embryonic lethality. Previous reports have shown that mouse mutants with increased levels of apoptosis are associated with neural tube defects 44,45,46,47 and craniofacial defects48. It has been suggested that excessive cell death in the neural folds or pharyngeal arches may result in an insufficient number of cells required for proper morphogenetic movement 48,49,50. In contrast, our SPECC1L deficient cell lines with moderately reduced SPECC1L expression showed only AJ changes without evidence of increased cell death. However, chemical inhibition of the PI3K-AKT pathway in these Kd cells did result in increased apoptosis. Thus, a moderate decrease in SPECC1L expression or function ensures cell survival. This is consistent with the observation that rare Specc11 mutant embryos that escape arrest at st. E9.5—perhaps due to reduced gene capture efficiency—are able to close their neural tubes and stop later in development, often with craniofacial defects (Fig. S3). Also consistent with this is the rare occurrence of heterozygous Specc1l embryos with craniofacial abnormalities—probably due to increased gene capture efficiency—as well as the finding in zebrafish in which one of the two SPECC1L orthologues (specc1lb) causes late embryonic phenotypes, including loss of lower jaws and bilateral clefts51. Thus, heterozygous SPECC1L loss-of-function mutations identified in human patients may cause small impairments in SPECC1L function during craniofacial morphogenesis, sufficient to explain their orofacial clefts. SPECC1L-based regulation of intercellular contacts may also play a role in palatogenesis and fusion of the pharyngeal arches. Further studies of SPECC1L function will help elucidate the role of temporary intercellular contacts in CNCC during neural tube closure in neuroepithelial cell motility and craniofacial morphogenesis.
U2OS osteosarcoma control and SPECC1L-kd cells have been described previously (Saadi et al., 2011). Antibodies against SPECC1L have also been characterized previously (Saadi et al., 2011). Anti-β-catenin antibodies (rabbit; 1:1000; Santa Cruz, Dallas, TX) (mouse; 1:1000; Cell Signaling Technology, Danvers, MA), myosin IIb (1:1000; Sigma-Aldrich, St. Louis) , MO) ), E-cadherin (1:1000; Abkam, Cambridge, MA), AP2A (1:1000; Novus Biologicals, Littleton, Colo.), SOX10 (1:1000; 1000; Aviva Systems Biology, San Diego, California), DLX2 (1:1000; Abcam, Cambridge, MA), phospho-Ser473-AKT (1:1000; Cell Signaling Technology, Danvers, MA), pan-AKT (1:1000; ThermoFisher Scientific, Waltham, MA) , KI67 (1:1000; Cell Signaling Technology, Danvers, MA), cleaved caspase 3 (1:1000; Cell Signaling Technology, Danvers, MA) and β-actin (1:2500; Sigma-Aldrich, St. Louis, MO ) was used as described. . Actin filaments were stained with Acti-stain rhodamine phalloidin (Cytoskeleton, Denver, Colorado).
U2OS control cells and SPECC1L-kd cells were cultured in standard high glucose DMEM supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA). For AJ changes, 2 x 105 cells were seeded on glass treated with 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, MO) and observed for changes in cell shape. Cells were collected at different indicated time points: 4 hours after seeding (t = 1), 24 hours after seeding (t = 2), confluence without change in cell shape (t = 3), change in cell shape (t = 4), 24 h after cell shape change (t = 5) and 48 h after cell shape change (t = 6) (Fig. 1, 2, 3). To modulate the PI3K-AKT pathway, cells were cultured at the indicated concentrations with the PI3K-AKT inhibitor wortmannin (TOCRIS Biosciences, Minneapolis, Minnesota) or SC-79 activator (TOCRIS Biosciences, Minneapolis Adams, Minnesota). The medium containing the chemicals was changed daily.
Frame-by-frame recordings were made on live control and KD cells under normal culture conditions, and phase contrast images were collected every 10 minutes for 7 days. Images were acquired using a computer-controlled Leica DM IRB inverted microscope equipped with a mechanical stage and a 10 × N-PLAN objective connected to a QImaging Retiga-SRV camera. During imaging, cell cultures were maintained at 37°C in a humid atmosphere with 5% CO2.
Two gene trap ES cell lines DTM096 and RRH048 from the Regional Mutant Mouse Resource Center (UC Davis, CA) were used to generate Specc11 deficient mouse lines, designated Specc1lgtDTM096 and Specc1lgtRRH046. Briefly, 129/REJ ES cells were injected into C57BL6 blastocysts. The resulting chimeric male mice were bred with female C57BL6 mice to identify offspring with agouti coat coloration. The presence of gene trap vector inserts was used to identify heterozygotes. Mice were kept on a mixed background of 129/REJ;C57BL6. The location of the insertion site of the genetic trap vector was confirmed by RT-PCR, genome sequencing, and genetic complementation (Supplementary Figure 1). To trace the CNCC lineage of double heterozygous Specc1lGT mice, ROSAmTmG (#007576) and Wnt1-Cre (#003829) mice (Jackson Laboratory, Bar Harbor, ME) were crossed to produce the ROSAmTmG and Wnt1-Cre allele in Specc1l mutant embryos. All experiments in mice were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center.
Embryos were fixed in (1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 0.02% NP-40, 5 mM EGTA) for 60 min at room temperature. After fixation in X-gal staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 1 mg/ml X-gal) Stain development was performed at 37°C. °C within 1-6 hours. Embryos were post-fixed in 4% PFA and visualized.
For Western blotting, cells were lysed in passive lysis buffer (Promega, Fitchburg, WI) supplemented with a mixture of HALT protease inhibitors (Sigma-Aldrich, St. Louis, MO). Lysates were processed on 12% polyacrylamide Mini-PROTEAN TGX ready-made gels (Bio-Rad, Hercules, CA) and transferred to Immobilon PVDF membranes (EMD Millipore, Billerica, MA). The membranes were blocked in 5% milk in PBS containing 0.1% Tween. Antibodies were incubated overnight at 4°C or for one hour at room temperature. Femto SuperSignal West ECL reagent (Thermo Scientific, Waltham, MA) was used for signal generation. For immunostaining, embryos were fixed overnight in 4% PFA/PBS and cryopreserved. Tissue cryosections were blocked in PBS containing 1% normal goat serum (Thermo Scientific, Waltham, MA) and 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) and then incubated at 4°C in an incubator during the night. with anti-antibody and fluorescent secondary antibody (1:1000) for 1 hour at 4°C. Stained sections were placed in ProLong gold medium (Thermo Scientific, Waltham MA) and flat images were obtained using a Leica TCS SPE confocal microscope. Each immunostaining was performed as three independent experiments on cirossections of at least two mutant embryos. A representative experiment is shown.
Cells were incubated in modified RIPA buffer (20 mM Tris-HCl, pH 8.0, 1% NP-40, 130 mM NaCl, 10% glycerol, 2 mM EDTA, and HALT protease inhibitor (Sigma-Aldrich, St. Louis, MO) Briefly, lysates were prepurified with protein G magnetic beads (Life Technologies, Carlsbad, CA) and then incubated overnight at 4° C. with anti-SPECC1L or IgG protein G protein beads were used to extract SPECC1L and Western blotting was performed using the anti-β-catenin antibody described above The co-IP experiments shown are representative of four independent experiments.
Fixed cultured cells or mouse embryonic tissues were provided to the electron microscopy center at the University of Kansas Medical Center. Briefly, samples were embedded in EMbed 812 resin (Electron Microscopy Sciences, Fort Washington, PA), polymerized overnight at 60°C, and sectioned at 80 nm using a Leica UC7 ultramicrotome equipped with a diamond blade. Sections were visualized using a JEOL JEM-1400 transmission electron microscope equipped with a 100 kV Lab6 gun.
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