An image-based RNAi screen identifies SH3BP1 as a key effector of Semaphorin 3E–PlexinD1 signaling

Extracellular signals have to be precisely interpreted intracellularly and translated into diverse cellular behaviors often mediated by cytoskeletal changes. Semaphorins are one of the largest families of guidance cues and play a critical role in many systems. However, how different cell types translate extracellular semaphorin binding into intracellular signaling remains unclear. Here we developed and performed a novel image-based genome-wide functional RNAi screen for downstream signaling molecules that convert the interaction between Semaphorin 3E (Sema3E) and PlexinD1 into cellular behaviors. One of the genes identified in this screen is a RhoGAP protein, SH3-domain binding protein 1 (SH3BP1). We demonstrate that SH3BP1 mediates Sema3E-induced cell collapse through interaction with PlexinD1 and regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1) activity. The identification and characterization of SH3BP1 as a novel downstream effector of Sema3E-PlexinD1 provides an explanation for how extracellular signals are translated into cytoskeletal changes and unique cell behavior, but also lays the foundation for characterizing other genes identified from our screen to obtain a more complete picture of plexin signaling

Nano-scale architecture of blood-brain barrier tight-junctions

Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust physical barrier, whose damage underlies BBB dysfunctions related to several neurodegenerative diseases. What makes these highly specialized BBB-TJs extremely restrictive remains unknown. Here, we use super-resolution microscopy (dSTORM) to uncover new structural and functional properties of BBB TJs. Focusing on three major components, Nano-scale resolution revealed sparse (occludin) vs. clustered (ZO1/claudin-5) molecular architecture. In mouse development, permeable TJs become first restrictive to large molecules, and only later to small molecules, with claudin-5 proteins arrangement compacting during this maturation process. Mechanistically, we reveal that ZO1 clustering is independent of claudin-5 in vivo. In contrast to accepted knowledge, we found that in the developmental context, total levels of claudin-5 inversely correlate with TJ functionality. Our super-resolution studies provide a unique perspective of BBB TJs and open new directions for understanding TJ functionality in biological barriers, ultimately enabling restoration in disease or modulation for drug delivery

Fgd5 identifies hematopoietic stem cells in the murine bone marrow

Hematopoietic stem cells (HSCs) are the best-characterized tissue-specific stem cells, yet experimental study of HSCs remains challenging, as they are exceedingly rare and methods to purify them are cumbersome. Moreover, genetic tools for specifically investigating HSC biology are lacking. To address this we sought to identify genes uniquely expressed in HSCs within the hematopoietic system and to develop a reporter strain that specifically labels them. Using microarray profiling we identified several genes with HSC-restricted expression. Generation of mice with targeted reporter knock-in/knock-out alleles of one such gene, Fgd5, revealed that though Fgd5 was required for embryonic development, it was not required for definitive hematopoiesis or HSC function. Fgd5 reporter expression near exclusively labeled cells that expressed markers consistent with HSCs. Bone marrow cells isolated based solely on Fgd5 reporter signal showed potent HSC activity that was comparable to stringently purified HSCs. The labeled fraction of the Fgd5 reporter mice contained all HSC activity, and HSC-specific labeling was retained after transplantation. Derivation of next generation mice bearing an Fgd5-CreERT2 allele allowed tamoxifen-inducible deletion of a conditional allele specifically in HSCs. In summary, reporter expression from the Fgd5 locus permits identification and purification of HSCs based on single-color fluorescence

Elimination of aberrant DRG circuitries in Sema3A mutant mice leads to extensive neuronal deficits.

Axon guidance molecules determine the pattern of neuronal circuits. Accuracy of the process is ensured by unknown mechanisms that correct early guidance errors. Since the time frame of error correction in Sema3A null mice partly overlaps with the period of naturally occurring cell death in dorsal root ganglia (DRG) development, we tested the hypothesis that apoptosis of misguided neurons enables error correction. We crossed BAX null mice, in which DRG apoptosis is blocked, with Sema3A null mice to induce errors. Analyses of these double-null mouse embryos showed that the elimination of abnormal projections is not blocked in the absence of BAX. Surprisingly however, there are fewer surviving neurons in Sema3A null or Sema3A/BAX double-null newborn mice than in wild-type mice. These results suggest that guidance errors are corrected by a BAX-independent cell death mechanism. Thus, aberrant axonal guidance may lead to reductions in neuronal numbers to suboptimal levels, perhaps increasing the likelihood of neuropathological consequences later in life

Abnormal axon projection elimination is not blocked in the absence of BAX.

<p><b>A:</b> Illustrated cross section of the lumbar spinal cord at E15.5 used to analyze abnormal axon projections. Spinal cord in the middle (red circle) with DRG on both sides (red star) is surrounded by pre-cartilage primordial (red rhombus). The left side portrays an abnormal circuit – pseudo-uni-bipolar neurons within the DRG (blue) send branches and enter the spinal cord at the dorsal root entry (green arrow). A second branch crosses abnormally through the pre-cartilage primordial (purple arrow) instead of using the correct path through the ventral root (green arrow – same marking shapes are used hereafter). The right side portrays a normal circuit (note that the ventral root contains the ventral nerve of the DRG and the motor nerve exiting the spinal cord ventrally). <b>B:</b> Experimental design. Mouse genetics used to test the role of apoptosis in the elimination of aberrant circuits. C<b>:</b> Abnormal axon projections in three adjacent cross-sections of DRG from a Sema3A^−/− embryo (E15.5). Note the normal dorsal and ventral roots (short green arrows, upper right image). Abnormal projections were scored only when actual exit from the DRG was noticeable (arrows in upper and lower left images). Serial sections were used to identify <i>bona fide</i> errors. Low magnification in upper panel and high magnification of a different error in lower panel; note that single axon errors could be detected at this magnification (lower right image, purple arrowhead). All scale bars 50 µm. <b>D:</b> Similar axon errors are detected in Sema3A null mice and Sema3A:BAX double null mice. Abnormal axon projections in Sema3A^−/−:BAX^+/+ mice (left) or Sema3A^−/−:BAX^−/− mutant mice (right). The abnormalities are similar in both cases. A representative image of lumbar DRG cross section is shown (low magnification, upper panel) and (higher magnification, lower panel) of the same abnormal projections (anti-neurofilament immunostaining). At E15.5, these DRGs of Sema3A^−/−:BAX^+/+ and Sema3A^−/−: BAX^−/− embryos exhibit abnormal axon bundles (purple arrow). Spinal cord is marked with a red circle, the pre-cartilage primordial marked with a red rhombus, and the DRG is marked with a red star. All scale bars are 50 µm. <b>E:</b> Analysis of temporal error occurrence. Error rate over time in Sema3A^−/: BAX^+/+ embryos compared to error rate in the compound Sema3A^−/−:BAX^−/− embryos. Absence of BAX results in accumulation of errors between E13.5 and E15.5. Massive error elimination is apparent in both genetic backgrounds at E17.5 (n = 30 DRG per genotype in each of the three embryonic ages, E13.5 P = 0.037, E15.5 P = 0.05, and E17.5 P = 0.0042). <b>Fa,b:</b> Representative images of E13.5 lumbar DRG of a Sema3A^−/− embryo cross section are shown (anti-neurofilament immunostaining). a, example of DRG that exhibits three abnormal axon bundles. b, example of DRG that exhibits single axon error. <b>G:</b> Quantification of abnormal axon bundles – Absence of BAX results in accumulation of errors between E13.5 and E15.5. Massive error elimination is apparent in both genetic backgrounds at E17.5 (E13.5 P = 0.0348, E15.5 P = 0.0069, and E17.5 P = 0.0404). <b>H:</b> Quantification of single axon errors – No accumulation of single axon errors is observed between E13.5 and E15.5 in the Sema3A^−/−:BAX^−/− genotype. Single axon errors are fully eliminated by E17.5 in the Sema3A^−/− embryos, while Sema3A^−/−:BAX^−/− embryos still exhibit low but detectable levels of errors (n = 30 DRG per genotype at each of the three embryonic ages, E13.5 P>0.05, E15.5 P>0.05, and E17.5 P<0.05 ).</p

Increased levels of axon guidance errors in BAX null and heterozygote Sema3A:BAX null mice.

<p><b>A:</b> Representative image of errors observed in DRG from Sema3A^+/+:BAX^−/− mice. Three adjacent cross sections of E13.5 DRG are shown. Most of the observed errors in the indicated genotypes were single axons or small bundles. Note the tortuous path an abnormal axon takes in and out of the plane of view. Scale bar 50 µm. <b>B:</b> Analysis of error occurrence in the indicated genotypes at E13.5. Increase in error frequency is detected in BAX null background, indicating cell-death involvement in both wild-type and Sem3A^+/− mice (n = 30 DRG per genotype (Wild type versus Sema3A^+/+:BAX^−/− P = 0.0416, Sema3A^+/− :BAX^+/+ versus Sema3A^+/−:BAX^−/− P = 0.0006). <b>C:</b> DRG neurons from BAX^−/− and BAX^+/+ mice are equally responsive to Sema3A-induced growth cone collapse. DRG explants from E12.5 embryos (BAX^+/+ and BAX^−/− littermates) were cultured in the presence of 10 ng/ml NGF for 20 h, at which time neurons were treated with 0, 6, 7.5, 10, 15 or 30 pM Sema3A. After an additional incubation period of 40 min with or without Sema3A, the explants were fixed and stained with rhodamine phalloidin. Quantification of the growth cone collapse results is shown. Results represent the mean +/− S.E.M. of three independent experiments. None of the Sema3A concentrations induced statistically significant difference in collapse levels between BAX^+/+ and BAX^−/− groups, P>0.05. <b>D:</b> Activation of caspase-3 is not significantly changed in Sema3A null mice. For each embryo proteins were extracted from E15.5 (upper panel) and E16.5 (lower panel) DRGs from lumbar and thoracic levels (at least three embryos of each genotype were used). Relative changes in caspase-3 activation levels were measured by western blot analysis using an activated caspase-3-specific antibody (Cell Signaling Technology). To determine protein levels each membrane was re-blotted for actin. Quantification of band intensity was obtained using scanning densitometry (Quantity One, BioRad) of three blots representing three different experiments. Results were normalized to actin. The average normalized result of the wild-type embryos at each age was defined as 1. At E15.5 the difference between Sema3A null mice and wild-type littermates is not statistically significant (P>0.05).</p