Birgit Weinhold

Serum response factor is essential for mesoderm formation during mouse embryogenesis

The transcription factor serum response factor (SRF), a phylogenetically conserved nuclear protein, mediates the rapid transcriptional response to extracellular stimuli, e.g. growth and differentiation signals. DNA–protein complexes containing SRF or its homologues function as nuclear targets of the Ras/MAPK signalling network, thereby directing gene activities associated with processes as diverse as pheromone signalling, cell‐cycle progression (transitions G0–G1 and G2–M), neuronal synaptic transmission and muscle cell differentiation. So far, the activity of mammalian SRF has been studied exclusively in cultured cells. To study SRF function in a multicellular organism we generated an Srf null allele in mice. SRF‐deficient embryos (Srf−/−) have a severe gastrulation defect and do not develop to term. They consist of misfolded ectodermal and endodermal cell layers, do not form a primitive streak or any detectable mesodermal cells and fail to express the developmental marker genes Bra (T), Bmp‐2/4 and Shh. Activation of the SRF‐regulated immediate early genes Egr‐1 and c‐fos, as well as the α‐Actin gene, is severely impaired. Our study identifies SRF as a new and essential regulator of mammalian mesoderm formation. We therefore suggest that in mammals Ras/MAPK signalling contributes to mesoderm induction, as is the case in amphibia.

Genetic Ablation of Polysialic Acid Causes Severe Neurodevelopmental Defects Rescued by Deletion of the Neural Cell Adhesion Molecule

Poly-α2,8-sialic acid (polySia) is a unique modification of the neural cell adhesion molecule, NCAM, tightly associated with neural development and plasticity. However, the vital role attributed to this carbohydrate polymer has been challenged by the mild phenotype of mice lacking polySia due to NCAM-deficiency. To dissect polySia and NCAM functions, we generated polySia-negative but NCAM-positive mice by simultaneous deletion of the two polysialyltransferase genes, St8sia-II and St8sia-IV. Beyond features shared with NCAM-null animals, a severe phenotype with specific brain wiring defects, progressive hydrocephalus, postnatal growth retardation, and precocious death was observed. These drastic defects were selectively rescued by additional deletion of NCAM, demonstrating that they originate from a gain of NCAM functions because of polySia deficiency. The data presented in this study reveal that the essential role of polySia resides in the control and coordination of NCAM interactions during mouse brain development. Moreover, this first demonstration in vivo that a highly specific glycan structure is more important than the glycoconjugate as a whole provides a novel view on the relevance of protein glycosylation for the complex process of building the vertebrate brain.

Polysialylated Neural Cell Adhesion Molecule Is Involved in Induction of Long-Term Potentiation and Memory Acquisition and Consolidation in a Fear-Conditioning Paradigm

Polysialic acid (PSA) regulates functions of the neural cell adhesion molecule (NCAM) during development and in neuroplasticity in the adult; the underlying mechanisms at different phases of learning and memory consolidation are, however, unknown. To investigate the contributions of PSA versus the extracellular domain of the NCAM glycoprotein backbone to synaptic plasticity, we applied NCAM, PSA-NCAM, and PSA to acute slices of the hippocampal CA1 region of NCAM-deficient mice and measured their effects on long-term potentiation (LTP). Remarkably, only PSA and PSA-NCAM, but not NCAM restored normal LTP. Application of these molecules to the dorsal hippocampus of wild-type mice showed that PSA-NCAM and PSA, but not NCAM, injected before fear conditioning, impaired formation of hippocampus-dependent contextual memory. Consolidation of contextual memory was affected by PSA-NCAM only when injected during its late, but not early phases. None of the tested compounds disturbed extrahippocampal-cued memory. Mice lacking the polysialyltransferase (ST8SialV/PST) responsible for attachment of PSA to NCAM in adulthood showed a mild deficit only in hippocampal contextual learning, when compared with NCAM-deficient mice that were disturbed in both contextual and cued memories. Contextual and tone memory in NCAM-deficient mice could be partially restored by injection of PSA-NCAM, but not of NCAM, into the hippocampus, suggesting that the impact of PSA-NCAM in synaptic plasticity and learning is not mediated by modulation of NCAM–NCAM homophilic interactions. In conclusion, our data support the view that polysialylated NCAM is involved in both formation and late consolidation of contextual memory.

Serum response factor is required for immediate-early gene activation yet is dispensable for proliferation of embryonic stem cells.

ABSTRACT Addition of serum to mitogen-starved cells activates the cellular immediate-early gene (IEG) response. Serum response factor (SRF) contributes to such mitogen-stimulated transcriptional induction of many IEGs during the G0-G1 cell cycle transition. SRF is also believed to be essential for cell cycle progression, as impairment of SRF activity by specific antisera or antisense RNA has previously been shown to block mammalian cell proliferation. In contrast, Srf −/− mouse embryos grow and develop up to E6.0. Using the embryonic stem (ES) cell system, we demonstrate here that wild-type ES cells do not undergo complete cell cycle arrest upon serum withdrawal but that they can mount an efficient IEG response. This IEG response, however, is severely impaired in Srf −/− ES cells, providing the first genetic proof that IEG activation is dependent upon SRF. Also, Srf−/− ES cells display altered cellular morphology, reduced cortical actin expression, and an impaired plating efficiency on gelatin. Yet, despite these defects, the proliferation rates of Srf −/− ES cells are not substantially altered, demonstrating that SRF function is not required for ES cell cycle progression.

Synaptic cell adhesion molecule SynCAM 1 is a target for polysialylation in postnatal mouse brain

Among the large set of cell surface glycan structures, the carbohydrate polymer polysialic acid (polySia) plays an important role in vertebrate brain development and synaptic plasticity. The main carrier of polySia in the nervous system is the neural cell adhesion molecule NCAM. As polySia with chain lengths of more than 40 sialic acid residues was still observed in brain of newborn Ncam−/− mice, we performed a glycoproteomics approach to identify the underlying protein scaffolds. Affinity purification of polysialylated molecules from Ncam−/− brain followed by peptide mass fingerprinting led to the identification of the synaptic cell adhesion molecule SynCAM 1 as a so far unknown polySia carrier. SynCAM 1 belongs to the Ig superfamily and is a powerful inducer of synapse formation. Importantly, the appearance of polysialylated SynCAM 1 was not restricted to the Ncam−/− background but was found to the same extent in perinatal brain of WT mice. PolySia was located on N-glycans of the first Ig domain, which is known to be involved in homo- and heterophilic SynCAM 1 interactions. Both polysialyltransferases, ST8SiaII and ST8SiaIV, were able to polysialylate SynCAM 1 in vitro, and polysialylation of SynCAM 1 completely abolished homophilic binding. Analysis of serial sections of perinatal Ncam−/− brain revealed that polySia-SynCAM 1 is expressed exclusively by NG2 cells, a multifunctional glia population that can receive glutamatergic input via unique neuron-NG2 cell synapses. Our findings sug-gest that polySia may act as a dynamic modulator of SynCAM 1 functions during integration of NG2 cells into neural networks.

Dissecting polysialic acid and NCAM functions in brain development

The unique modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is tightly associated with nervous system development and plasticity. The prevailing view that this large carbohydrate polymer acts as an anti‐adhesive factor seems straightforward at first sight. However, during almost 25 years of polySia research it became increasingly clear that the impact of polySia on cell surface interactions can not be explained by one unifying mechanism. Recent progress in the generation of mouse models, which partially or completely lack polySia due to ablation of one or both of the two polySia synthesizing enzymes, provides novel insights into the function of this unique post‐translational modification. The present review is focused on a phenotype comparison between the newly established mouse strains which combine polySia‐deficiency with normal NCAM expression and the well‐characterized NCAM negative mouse model. Analysis of shared and individual phenotypes allows a clear distinction between NCAM and polySia functions and revealed that polySia plays a vital role as a specific control element of NCAM‐mediated interactions.

Srf–/– ES cells display non-cell-autonomous impairment in mesodermal differentiation

The serum response factor (SRF) transcription factor is essential for murine embryogenesis. Srf−/− embryos stop developing at the onset of gastrulation, lacking detectable mesoderm. This developmental defect may reflect cell‐autonomous impairment of Srf−/− embryonic cells in mesoderm formation. Alternatively, it may be caused by a non‐cell‐autonomous defect superimposed upon inappropriate provision of mesoderm‐inducing signals to primitive ectodermal cells. We demonstrate that the ability of Srf−/− embryonic stem (ES) cells to differentiate in vitro into mesodermal cells is indeed impaired. However, this impairment can be modulated by external, cell‐independent factors. Retinoic acid, but not dimethylsulfoxide, permitted activation of the mesodermal marker gene T(Bra), which was also activated when SRF was expressed in Srf−/− ES cells. Embryoid bodies from Srf−/− ES cell aggregates also activated mesodermal marker genes, but displayed unusual morphologies and impairment in cavitation. Finally, in nude mice, Srf−/− ES cells readily differentiated into mesodermal cells of Srf−/− genotype, including cartilage, bone or muscle cells. We demonstrate that SRF contributes to mesodermal gene expression of ES cells and that Srf−/− ES cells display a non‐cell‐autonomous defect in differentiation towards mesoderm.

In vivo synaptic plasticity in the dentate gyrus of mice deficient in the neural cell adhesion molecule NCAM or its polysialic acid

The neural cell adhesion molecule NCAM and its associated polysialic acid (PSA) play important roles in synaptic plasticity in the CA1 and/or CA3 regions of the hippocampus in vitro. Here, we address the question of whether NCAM and PSA are involved in regulation of synaptic transmission and plasticity also in vivo at synapses formed by entorhinal cortex axons in the dentate gyrus of mice anaesthetized with urethane. We show that basal synaptic transmission, measured as the slope of field excitatory postsynaptic potentials, was reduced strongly in mice lacking ST8SiaII/STX, the enzyme involved in polysialylation of NCAM in stem cell‐derived immature granule cells, but not in mice deficient either in the NCAM glycoprotein or the enzyme ST8SiaIV/PST involved in polysialylation of NCAM in mature neurons. Strikingly, only mice deficient in NCAM, but not in PST or STX, were impaired in long‐term potentiation (LTP) induced by theta‐burst stimulation, suggesting that LTP in the dentate gyrus depends on the NCAM glycoprotein alone rather than on its associated PSA. As also patterns of synaptic activity during and immediately after induction of LTP were impaired in NCAM‐deficient mice, it is likely that induction of LTP requires NCAM. These data are the first to describe that NCAM is necessary for induction of synaptic plasticity in identified synapses in vivo and suggest that polysialylation of NCAM expressed by immature granule cells in the dentate gyrus supports development of basal excitatory synaptic transmission in this region.

Polysialic Acid Profiles of Mice Expressing Variant Allelic Combinations of the Polysialyltransferases ST8SiaII and ST8SiaIV

The post-translational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) represents a remarkable example of dynamic modulation of homo- and heterophilic cell interactions by glycosylation. The synthesis of this unique carbohydrate polymer depends on the polysialyltransferases ST8SiaII and ST8SiaIV. Aiming to understand in more detail the contributions of ST8SiaII and ST8SiaIV to polySia biosynthesis in vivo, we used mutant mouse lines that differ in the number of functional polysialyltransferase alleles. The 1,2-diamino-4,5-methylenedioxybenzene method was used to qualitatively and quantitatively assess the polySia patterns. Similar to the wild-type genotype, long polySia chains (>50 residues) were detected in all genotypes expressing at least one functional polysialyltransferase allele. However, variant allelic combinations resulted in distinct alterations in the total amount of poly-Sia; the relative abundance of long, medium, and short polymers; and the ratio of polysialylated to non-polysialylated NCAM. In ST8SiaII-null mice, 45% of the brain NCAM was non-polysialylated, whereas a single functional allele of ST8SiaII was sufficient to polysialylate ∼90% of the NCAM pool. Our data reveal a complex polysialylation pattern and show that, under in vivo conditions, the coordinated action of ST8SiaII and ST8SiaIV is crucial to fine-tune the amount and structure of polySia on NCAM.

Polysialic Acid, a Glycan with Highly Restricted Expression, Is Found on Human and Murine Leukocytes and Modulates Immune Responses

Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV−/− mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.