Peptidoglycan recognition in Drosophila is mediated by LysMD3/4.

Microbial recognition is a key step in regulating the immune signaling pathways of multicellular organisms. Peptidoglycan, a component of the bacterial cell wall, exhibits immune stimulating activity in both plants and animals. Lysin motif domain (LysMD) family proteins are ancient peptidoglycan receptors that function in bacteriophage and plants. This report focuses on defining the role of LysMD-containing proteins in animals. Here, we characterize a novel transmembrane LysMD family protein. Loss-of-function mutations at the lysMD3/4 locus in Drosophila are associated with systemic innate immune activation following challenge, so we refer to this gene as immune active (ima). We show that Ima selectively binds peptidoglycan, is enriched in cell membranes, and is necessary to regulate terminal innate immune effectors through an NF-kB-dependent pathway. Hence, Ima fulfills the key criteria of a peptidoglycan pattern recognition receptor. The human Ima ortholog, hLysMD3, exhibits similar biochemical properties. Together, these findings establish LysMD3/4 as the founding member of a novel family of animal peptidoglycan recognition proteins.

Histologic and Functional Outcomes of Conduit Wrapping for Peripheral Nerve Repair: Early Results in a Rat Model.

BACKGROUND: The concept of utilizing a nerve conduit for augmentation of a primary nerve repair has been advocated as a method to prevent neural scarring and decrease adhesions. Despite clinical use, little is known about the effects of a nerve conduit wrapped around a primary repair. To better understand this, we investigated the histologic and functional effects of use of a nerve conduit wrapped around a rat sciatic nerve repair without tension. METHODS: Twenty Lewis' rats were divided into two groups of 10 rats each. In each group, unilateral sciatic nerve transection and repair were performed, with the opposite limb utilized as a matched control. In the first group, direct repair alone was performed; in the second group, this repair was augmented with a porcine submucosa conduit wrapped around the repair site. Sciatic functional index (SFI) was measured at 6 weeks with walking track analysis in both groups. Nonsurvival surgeries were then performed in all animals to harvest both the experimental and control nerves to measure histomorphometric parameters of recovery. Histomorphometric parameters assessed included total number of neurons, nerve fiber density, nerve fiber width, G-ratio, and percentage of debris. Unpaired t-test was used to compare outcomes between the two groups. RESULTS: All nerves healed uneventfully but compared with direct repair; conduit usage was associated with greater histologic debris, decreased axonal density, worse G-ratio, and worse SFI. No significant differences were found in total axon count or gastrocnemius weight. CONCLUSION: In the absence of segmental defects, conduit wrapping primary nerve repairs seem to be associated with worse functional and mixed histologic outcomes at 6 weeks, possibly due to debris from conduit resorption. While clinical implications are unclear, more basic science and clinical studies should be performed prior to widespread adoption of this practice.

Early Expression of MMP-9 Predicts Recovery of Tibialis Anterior after Sciatic Nerve Crush Injury.

Background: The purpose of this study was to assess the expression of molecular markers and epineural blood flow after differing degrees of nerve injury to identify potential tools to predict nerve recovery in a rat sciatic nerve model. Methods: A total of 72 rats were divided into nine groups. Each group was subjected to one of three crush injuries, created by applying one of three vascular clamps for 30 seconds. Vascularity was assessed with laser Doppler flowmetry before and after crush, and at nonsurvival surgery. Nonsurvival surgeries were performed 6 hours, 2 weeks, or 6 weeks later with nerve conduction studies and muscle strength testing. Expression of matrix metalloproteinase 9 (MMP-9) and matrix metalloproteinase 2 (MMP-2) in each nerve was quantified using with enzyme linked immunosorbent analysis. Results: Persistent hyperemia was noted in the zone of injury compared with baseline at 2 weeks and 6 weeks in the groups that displayed incomplete recovery. Expression of MMP-9 at 6 hours increased with increasing severity of crush and was inversely related to tibialis anterior muscle force recovery. The ratio of MMP-9:MMP-2 expression correlated well with recovery of compound nerve action potential amplitude at 6 weeks. Conclusions: Resolution of nerve hyperemia may correlate with nerve recovery from trauma, but early measures of nerve blood flow after injury are not prognostic of recovery. Ratio of MMP-9:MMP-2 expression 6 hours after injury correlates with recovery of compound nerve action potential at 6 weeks, while MMP-9 expression alone predicts tibialis anterior recovery. These findings together suggest that increased MMP-9 expression is a potentially useful marker of more severe nerve injury.

Influences of Repair Site Tension and Conduit Splinting on Peripheral Nerve Reconstruction.

BACKGROUND: We investigated the use of a conduit splinting technique to mitigate tension at the coaptation site of a rodent nerve defect model to determine the optimal reconstruction method for segmental nerve defects. METHODS: A rat sciatic nerve segmental defect model was created by excising 5mm of the sciatic nerve unilaterally. Four groups of 10 rats were each reconstructed using 1 of 4 techniques: primary repair, repair with conduit splinting, reverse isograft with conduit splinting, and reverse isograft without splinting. Functional outcomes were assessed at 6 weeks by measurement of Sciatic Functional Index (SFI), and sciatic nerves were harvested at the nonsurvival surgery. Histomorphologic measurements were reported as a value normalized to the average measurements of the control side. The primary outcomes were assessment of nerve continuity and the proportion of nerve fibers in the regenerating nerve compared with the uninjured side. RESULTS: The number of repair site rupture rates was lower when a conduit splint was used-less than half of the primary repairs under tension remained intact at 6 weeks. No difference was seen in axon number, size, and density between primary repairs and those augmented by conduit splints, but worse functional outcomes and more debris were present compared with the intact primary repairs. CONCLUSIONS: Nerve conduit splinting reduced rupture rates, particularly for nerve repairs associated with a segmental defect. No significant difference was seen in the number of axons among techniques. Primary nerve repair under tension that did not rupture demonstrated superior SFI.

The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis.

G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina, and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84's importance in lens, cornea, and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino-injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea.

Gene expression profiles of lens regeneration and development in Xenopus laevis.

Seven hundred and thirty-four unique genes were recovered from a cDNA library enriched for genes up-regulated during the process of lens regeneration in the frog Xenopus laevis. The sequences represent transcription factors, proteins involved in RNA synthesis/processing, components of prominent cell signaling pathways, genes involved in protein processing, transport, and degradation (e.g., the ubiquitin/proteasome pathway), matrix metalloproteases (MMPs), as well as many other proteins. The findings implicate specific signal transduction pathways in the process of lens regeneration, including the FGF, TGF-beta, MAPK, Retinoic acid, Wnt, and hedgehog signaling pathways, which are known to play important roles in eye/lens development and regeneration in various systems. In situ hybridization revealed that the majority of genes recovered are expressed during embryogenesis, including in eye tissues. Several novel genes specifically expressed in lenses were identified. The suite of genes was compared to those up-regulated in other regenerating tissues/organisms, and a small degree of overlap was detected.

Beta-catenin is required for the establishment of vegetal embryonic fates in the nemertean, Cerebratulus lacteus.

Downstream components of the canonical Wnt signaling pathway that result in the nuclear localization of beta-catenin are involved in diverse developmental processes including the formation of the mesendoderm, the regulation of axial properties and asymmetric cell divisions in a wide array of metazoans. The nemertean worm, Cerebratulus lacteus, represents a member of the understudied lophotrochozoan clade that exhibits a highly stereotyped spiral cleavage program in which ectodermal, endodermal, and mesodermal origins are known from intracellular fate mapping studies. Here, the embryonic distribution of beta-catenin protein was studied using injection of synthetic mRNA, encoding GFP-tagged beta-catenin, into fertilized eggs. During the early cleavage stages beta-catenin was destabilized/degraded in animal hemisphere blastomeres and became localized to the nuclei of the four vegetal-most cells at the 64-cell stage, which give rise to definitive larval and adult endoderm. Functional assays indicate that beta-catenin plays a key role in the development of the endoderm. Morpholino knockdown of endogenous beta-catenin, as confirmed by Western analysis, resulted in the failure to gastrulate, absence of the gut and an animalized phenotype in the resulting larvae, including the formation of ectopic (anterior) apical organ tissue with elongated apical tuft cilia and no indications of dorsoventral polarity. Similarly, over-expression of the cytoplasmic domain of cadherin or a beta-catenin-engrailed repressor fusion construct prevented endoderm formation and generated the same animalized phenotype. Injections of mRNA encoding either a stabilized, constitutively activated form of beta-catenin or a dominant negative form of GSK3-beta converted all or nearly all cells into endodermal fates expressing gut-specific esterase. Thus, beta-catenin appears to be both necessary and sufficient to promote endoderm formation in C. lacteus, consistent with its role in endoderm and endomesoderm formation in anthozoan cnidarians, ascidians, and echinoderms. Consistent with the results of other studies, beta-catenin may be viewed as playing a role in the development of posterior/vegetal larval fates (i.e., endoderm) in C. lacteus. However, unlike the case found in polychaete annelid and soil nematode embryos, there is no evidence for a role of beta-catenin in regulating cell fates and asymmetric cell divisions along the entire anterior-posterior axis.

Psf2 plays important roles in normal eye development in Xenopus laevis.

PURPOSE: Psf2 (partner of Sld5 2) represents a member of the GINS (go, ichi, ni, san) heterotetramer [1] and functions in DNA replication as a "sliding clamp." Previous in situ hybridization analyses revealed that Psf2 is expressed during embryonic development in a tissue-specific manner, including the optic cup (retina) and the lens [2]. This article provides an analysis of Psf2 function during eye development in Xenopus laevis. METHODS: A morpholino targeted to Psf2 mRNA was designed to knockdown Psf2 translation and was injected into specific embryonic cells during early cleavage stages in the frog, Xenopus laevis. Injected embryos were assayed for specific defects in morphology, cell proliferation, and apoptosis. Synthetic Psf2 RNA was also co-injected with the morpholino to rescue morpholino-mediated developmental defects. It is well known that reciprocal inductive interactions control the development of the optic cup and lens. Therefore, control- and morpholino-injected embryos were used for reciprocal transplantation experiments to distinguish the intrinsic role of Psf2 in the development of the optic cup (retina) versus the lens. RESULTS: Morpholino-mediated knockdown of Psf2 expression resulted in dosage-dependent phenotypes, which included microphthalmia, incomplete closure of the ventral retinal fissure, and retinal and lens dysgenesis. Defects were also observed in other embryonic tissues that normally express Psf2 including the pharyngeal arches and the otic vesicle, although other tissues that express Psf2 were not found to be grossly defective. Eye defects could be rescued by co-injection of synthetic Psf2 RNA. Examination of cell proliferation via an antibody against phospho-histone H3 S10P revealed no significant differences in the retina and lens following Psf2 knockdown. However, there was a significant increase in the level of apoptosis in retinal as well as forebrain tissues, as revealed by TUNEL (terminal deoxynucleotide transferase dUTP nick end labeling) assay. CONCLUSIONS: The results demonstrate intrinsic roles for Psf2 in both retinal and to a lesser extent, lens tissues. Observed lens defects can mainly be attributed to deficiencies in retinal development and consequently the late phase of lens induction, which involves instructive cues from the optic cup. Developmental defects were not observed in all tissues that express Psf2, which could be related to differences in the translation of Psf2 or redundant effects of related factors such as proliferating cell nuclear antigen (PCNA).