SrGAP3 interacts with lamellipodin at the cell membrane and regulates Rac-dependent cellular protrusions.

SrGAP3/MEGAP is a member of the Slit-Robo GAP (srGAP) family and is implicated in repulsive axon guidance and neuronal migration through Slit-Robo-mediated signal transduction. Here we describe an inhibitory role of srGAP3 on actin dynamics, specifically on lamellipodia formation. We show that the F-BAR domain localizes srGAP3 to the leading edge of cellular protrusions whereas the SH3 domain is important for focal adhesion targeting. We report on a novel srGAP3 interaction partner, lamellipodin, which localizes with srGAP3 at the leading edge. Live-cell analyses revealed that srGAP3 influences lamellipodin-evoked lamellipodial dynamics. Furthermore, we show that mouse embryonic fibroblasts derived from homozygous srGAP3-knockout embryos display an increased cell area and lamellipodia formation that can be blocked by shRNA-mediated knockdown of lamellipodin.

Islet1 is a direct transcriptional target of the homeodomain transcription factor Shox2 and rescues the Shox2-mediated bradycardia.

The heart's rhythm is initiated and regulated by a group of specialized cells in the sinoatrial node (SAN), the primary pacemaker of the heart. Abnormalities in the development of the SAN can result in irregular heart rates (arrhythmias). Although several of the critical genes important for SAN formation have been identified, our understanding of the transcriptional network controlling SAN development remains at a relatively early stage. The homeodomain transcription factor Shox2 is involved in the specification and patterning of the SAN. While the Shox2 knockout in mice results in embryonic lethality due to severe cardiac defects including improper SAN development, Shox2 knockdown in zebrafish causes a reduced heart rate (bradycardia). In order to gain deeper insight into molecular pathways involving Shox2, we compared gene expression levels in right atria of wildtype and Shox2 (-/-) hearts using microarray experiments and identified the LIM homeodomain transcription factor Islet1 (Isl1) as one of its putative target genes. The downregulation of Isl1 expression in Shox2 (-/-) hearts was confirmed and the affected region narrowed down to the SAN by whole-mount in situ hybridization. Using luciferase reporter assays and EMSA studies, we identified two specific SHOX2 binding sites within intron 2 of the ISL1 locus. We also provide functional evidence for Isl1 as a transcriptional target of Shox2 by rescuing the Shox2-mediated bradycardia phenotype with Isl1 using zebrafish as a model system. Our findings demonstrate a novel epistatic relationship between Shox2 and Isl1 in the heart with important developmental consequences for SAN formation and heart beat.

Srgap3⁻/⁻ mice present a neurodevelopmental disorder with schizophrenia-related intermediate phenotypes.

Mutations in the SRGAP3 gene residing on chromosome 3p25 have previously been associated with intellectual disability. Genome-wide association studies have also revealed SRGAP3, together with genes from the same cellular network, as risk genes for schizophrenia. SRGAP3 regulates cytoskeletal dynamics through the RHO protein RAC1. RHO proteins are known to be involved in cytoskeletal reorganization during brain development to control processes such as synaptic plasticity. To elucidate the importance of SRGAP3 in brain development, we generated Srgap3-knockout mice. Ten percent of these mice developed a hydrocephalus and died before adulthood. Surviving mice showed various neuroanatomical changes, including enlarged lateral ventricles, white matter tracts, and dendritic spines together with molecular changes, including an increased basal activity of RAC1. Srgap3(-/-) mice additionally exhibited a complex behavioral phenotype. Behavioral studies revealed an impaired spontaneous alternation and social behavior, while long-term memory was unchanged. The animals also had tics. Lower locomotor activity was observed in male Srgap3(-/-) only. Srgap3(-/-) mice showed increased methylphenidate stimulation in males and an impaired prepulse inhibition in females. Together, the results show neurodevelopmental aberration in Srgap3(-/-) mice, with many of the observed phenotypes matching several schizophrenia-related intermediate phenotypes. Mutations of SRGAP3 may thus contribute to various neurodevelopmental disorders.

The distinct and overlapping phenotypic spectra of FOXP1 and FOXP2 in cognitive disorders.

Rare disruptions of FOXP2 have been strongly implicated in deficits in language development. Research over the past decade has suggested a role in the formation of underlying neural circuits required for speech. Until recently no evidence existed to suggest that the closely related FOXP1 gene played a role in neurodevelopmental processes. However, in the last few years, novel rare disruptions in FOXP1 have been reported in multiple cases of cognitive dysfunction, including intellectual disability and autism spectrum disorder, together with language impairment. As FOXP1 and FOXP2 form heterodimers for transcriptional regulation, one may assume that they co-operate in common neurodevelopmental pathways through the co-regulation of common targets. Here we compare the phenotypic consequences of FOXP1 and FOXP2 impairment, drawing on well-known studies from the past as well as recent exciting findings and consider what these tell us regarding the functions of these two genes in neural development.

Recurrent craniofacial dermatofibrosarcoma protuberans: long-term prognosis after close surgical removal.

Dermatofibrosarcoma protuberans (DFSP) is a low-grade malignant neoplasm of the dermis that rarely manifests in the craniofacial area. In this retrospective analysis, we investigated the long-term survival of 7 patients with recurrent craniofacial DFSP. This study includes all patients in our department with recurrences of DFSP between 1989 and 2006. All patients were treated by radical surgery with 1-cm free safety margin in every direction and remained in routine long-term follow-up for tumor patients. Two of the 7 patients showed a local recurrence, which was again successfully treated surgically with the same technique. Advanced reconstruction with free full-thickness skin transfers, regional flaps, and forearm flaps, respectively, was required in 5 of the 7 patients. The other 2 patients were reconstructed locally. The long-term prognosis of craniofacial DFSP can be assessed optimistically even if the tumor already reoccurred. All 7 patients included in this study are still alive and so far not suffering from local recurrence. Advanced reconstructive techniques are often required in the management of reoccurring craniofacial DFSP. Late recurrences have been reported; therefore, a long-term follow-up for these patients should be considered.

Assessment of the cytocompatibility of different coated titanium surfaces to fibroblasts and osteoblasts.

Over the years, many metal and polymer implants have been developed for internal fracture fixation. However, there are always some problems associated with their application, such as implant loosening or infection. This study describes how the morphology and adhesion of both fibroblasts and osteoblasts to two manufactured commercially pure, medical implant-quality anodized titanium surfaces (TS and TSS), and five modified titanium surfaces (TLF, low friction gray anodized titanium; TIG, nitrogen ion implanted TSS; THY, TSS grafted with sodium hyaluronate; TAST, TSS coated with hydrophilic hydrogel; and TT, tiodized TS) were used to obtain an indication of their relative cytocompatibility and to assess which modified surface could potentially be used in vivo. Small variations were observed both qualitatively and quantitatively in the spreading and adhesion of fibroblasts and osteoblasts to the studied surfaces. Overall, fibroblast spreading and adhesion were greatest on the TIG and TLF surfaces. Osteoblast spreading and adhesion were greatest on TS, TIG, and TAST surfaces. No fibroblasts or osteoblasts were found attached to the THY-coated surfaces. Coating medical implant-quality anodized titanium surfaces (TS and TSS) with a TLF, TAST, or more specifically TIG could probably improve soft tissue adhesion and/or osseointegration of bone in vivo. However, it seemed that a hyaluronic acid coating (THY) has potential as a coating in areas where cell adhesion is undesirable, such as orbital fractures, where muscles should avoid adhesion to the implant, and distal radius fractures, where tendons should freely glide over the implant.

Facial basal cell carcinoma with successive metastases to the neck, thyroid gland and lung.

Basal cell carcinoma of the skin is the most common malignancy in the head and neck area. Regional and distant metastases rarely occur with this type of tumour. We report an uncommon case of a sclerodermiform basal cell carcinoma of the facial skin in which metastases developed several years after the primary tumour. The metastases occurred in the soft tissue of the neck, the thyroid gland and the lung. This is the first case of BCC with triple metastases which were histologically confirmed.

The cellular function of srGAP3 and its role in neuronal morphogenesis.

The Slit-Robo GTPase activating protein 3 (srGAP3) dynamically regulates cytoskeletal reorganisation through inhibition of the Rho GTPase Rac1 and interaction with actin remodelling proteins. SrGAP3-mediated reorganisation of the actin cytoskeleton is crucial for the normal development of dendritic spines and loss of srGAP3 leads to abnormal synaptic activity and impaired cognitive behaviours in mice, which is reminiscent of an association between disrupted srGAP3 and intellectual disability in humans. Additionally, srGAP3 has been implicated to act downstream of Slit-Robo signalling in commissural axons of the spinal cord. Thus, srGAP3-mediated cytoskeletal reorganisation has an important influence on a variety of neurodevelopmental processes, which may be required for normal cognitive function.

Cranio-maxillofacial non-Hodgkin's lymphoma: clinical and histological presentation.

Non-Hodgkin's lymphoma represents about 5% of all malignant lesions of the head and neck. In this study we retrospectively evaluated clinical presentation, histological subtype and long-term prognosis of 42 patients with non-Hodgkin's lymphoma involving the craniofacial area. The mean age at diagnosis was 64 years. More than half of the patients presented with disseminated disease at multiple sites (55%, n=23). In 62% (n=26) the first manifestation was extranodal. The most common affected region was the oral cavity (65%, n=17). Treatment consisted of local therapy, including surgical resection and radiation, as well as chemotherapy with or without local therapy. Recurrence occurred in 31% (n=13) of the treated patients. Mean survival after first diagnosis varied from 17 months in patients presenting with diffuse large B-cell lymphoma (DLBCL), to 8.5 years in patients with follicular lymphoma. The most common histological subtype is DLBCL. Standard treatment for DLBCL consists of chemotherapy combined with CD 20 monoclonal antibody, even after total resection of the tumour. There is high risk of systemic disease in patients presenting with non-Hodgkin's lymphoma and high risk of post therapy recurrence.

Craniofacial metastases: a 20-year survey.

In this study we analysed the long-term prognosis of 52 patients with distant metastases to the craniofacial area. All patients were treated in our department between 1989 and 2009. Possible predictive factors for the overall survival prognosis like age, gender, histopathological type of the metastasis, location and tissue structure of the area in the head and neck region, time between primary tumour and metastasis and the therapy were evaluated. 62% of the patients with distant metastases in the craniofacial area were male (32/52), the average age was 63 years. Adenocarcinoma was the most common histological type (20/52) and lung (12/52), malignant melanoma of the skin (9/52) and breast (8/52) the most common primary tumour site. In 35% of all patients, the primary tumour was not known at the time of the diagnosis of the craniofacial metastasis, this number reduced to 17% without the patients with a CUP syndrome. Patients survived an average of 14.4 months after manifestation of the metastases and 43.4 months after the manifestation of the primary tumour.

Evidence for a role of srGAP3 in the positioning of commissural axons within the ventrolateral funiculus of the mouse spinal cord.

Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.

Dynamic expression of the Slit-Robo GTPase activating protein genes during development of the murine nervous system.

We investigated the expression of the three known Slit-Robo GTPase activating protein (srGAP) genes in the developing murine nervous system using in situ hybridization. The three genes are expressed during embryonic and early postnatal development in the murine nervous system, showing a distinct pattern of expression in the olfactory system, the eye, forebrain and midbrain structures, the cerebellum, the spinal cord, and dorsal root ganglia, which we discuss in relation to Slit-Robo expression patterns and signaling pathways. We also report srGAP2 expression in zones of neuronal differentiation and srGAP3 in ventricular zones of neurogenesis in many different tissues of the central nervous system (CNS). Compared to srGAP2 and srGAP3, the onset of srGAP1 expression is later in most CNS tissues. We propose that these differences in expression point to functional differences between these three genes in the development of neural tissues.

Interdisciplinary combined treatment of craniofacial osteosarcoma with neoadjuvant and adjuvant chemotherapy and excision of the tumour: a retrospective study.

Osteosarcomas are highly malignant tumours of bone, and are rare in the craniofacial area. They account for only 1% of all head and neck malignancies. In this study we describe the treatment of 12 patients who were diagnosed with osteosarcoma of the mandible or maxilla between 1990 and 2004. These patients were given interdisciplinary treatments, either with a combination of neoadjuvant and adjuvant high-dose chemotherapy following the protocol of the cooperative osteosarcoma study group (COSS) with radical excision of the tumour (n=7), with a combination of radiation and radical excision (n=2), or with radical excision alone of the tumour (n=3). The 5-year survival of the group treated only by excision was 1 of 3, whereas that of the group treated by combination of chemotherapy and surgical removal was 7 of 7. The two patients treated with radiation and excision also lived 5 years after the end of the treatment, but had side-effects of radiation, whereas those treated with chemotherapy had no serious side-effects. We therefore conclude that combined interdisciplinary treatment of radical resection of the tumour with high-dose chemotherapy according to standard protocols is the most effective treatment for craniofacial osteosarcomas.

N-WASP regulates extension of filopodia and processes by oligodendrocyte progenitors, oligodendrocytes, and Schwann cells-implications for axon ensheathment at myelination.

The molecular mechanisms used by oligodendrocyte precursor cells (OPCs), oligodendrocytes (OLs), and Schwann cells (SCs) to advance processes for motility in the developing nervous system and to ensheath axons at myelination are currently not well defined. Here we demonstrate that OPCs, OLs, and SCs express the major proteins involved in actin polymerization-driven protrusion; these key proteins including F-actin, the Arp2/3 complex, neural-Wiskott Aldrich Syndrome protein (N-WASP) and WAVE proteins, and the RhoGTPases Rac and Cdc42 are present at the leading edges of processes being extended by OPCs, OLs, and SCs. We reveal by real-time PCR that OLs and SCs have different dominant WAVE isoforms. Inhibition of the WASP/WAVE protein, N-WASP, with wiskostatin that prevents activation of the Arp2/3 complex, blocks process extension by OPCs and SCs. Inhibition of N-WASP also causes OPC and SC process retraction, which is preceded by retraction of filopodia. This implicates filopodia in OPC and SC process stability and also of N-WASP in OPC and SC process dynamics. We also demonstrate that p34 (a component of the Arp2/3 complex), WASP/WAVE proteins, actin, alpha-tubulin, Rac, Cdc42, vinculin, and focal adhesion kinase are detected in water-shocked myelin purified from brain. Inhibition of N-WASP with wiskostatin decreases the number of axons undergoing initial ensheathment in intact optic nerve samples and reduces the Po content of dorsal root ganglia:SC co-cultures. Our findings indicate that OPCs, OLs, and SCs extend processes using actin polymerization-driven protrusion dependent on N-WASP. We hypothesize that inner mesaxons of OLs and SCs use the same mechanism to ensheath axons at myelination.