Silvia Arber

Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase

Cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells depend directly on the regulated dynamics of the actin cytoskeleton,. To understand the mechanisms of these basic cellular processes, the signalling pathways that link external signals to the regulation of the actin cytoskeleton need to be characterized,. Here we identify a pathway for the regulation of cofilin, a ubiquitous actin-binding protein that is essential for effective depolymerization of actin filaments. LIM-kinase 1, also known as KIZ, is a protein kinase with two amino-terminal LIM motifs that induces stabilization of F-actin structures in transfected cells. Dominant-negative LIM-kinase1 inhibits the accumulation of the F-actin. Phosphorylation experiments in vivo and in vitro provide evidence that cofilin is a physiological substrate of LIM-kinase 1. Phosphorylation by LIM-kinase 1 inactivates cofilin, leading to accumulation of actin filaments. Constitutively active Rac augmented cofilin phosphorylation and LIM-kinase 1 autophosphorylation whereas phorbol ester inhibited these processes. Our results define a mechanism for the regulation of cofilin and hence of actin dynamics in vivo. By modulating the stability of actin cytoskeletal structures, this pathway should play a central role in regulating cell motility and morphogenesis.

MLP-Deficient Mice Exhibit a Disruption of Cardiac Cytoarchitectural Organization, Dilated Cardiomyopathy, and Heart Failure

MLP is a LIM-only protein of terminally differentiated striated muscle cells, where it accumulates at actin-based structures involved in cytoarchitecture organization. To assess its role in muscle differentiation, we disrupted the MLP gene in mice. MLP (-/-) mice developed dilated cardiomyopathy with hypertrophy and heart failure after birth. Ultrastructural analysis revealed dramatic disruption of cardiomyocyte cytoarchitecture. At birth, these hearts were not hypertrophic, but already abnormally soft, with cell-autonomous and MLP-sensitive alterations in cytoarchitecture. Thus, MLP promotes proper cardiomyocyte cytoarchitecture, whose perturbation can lead to dilated cardiomyopathy. In vivo analysis revealed that MLP-deficient mice reproduce the morphological and clinical picture of dilated cardiomyopathy and heart failure in humans, providing the first model for this condition in a genetically manipulatable organism.

Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice

Regulation of neurite outgrowth and structural plasticity may involve the expression of intrinsic determinants controlling growth competence. We have tested this concept by targeting constitutive expression of the growth-associated protein GAP-43 to the neurons of adult transgenic mice. Such mice showed striking spontaneous nerve sprouting at the neuromuscular junction and in the terminal field of hippocampal mossy fibers. In control mice, these nerve fibers did not express GAP-43, and did not sprout spontaneously. Lesion-induced nerve sprouting and terminal arborization during reinnervation were greatly potentiated in GAP-43-overexpressing mice. A mutant GAP-43 that cannot be phosphorylated by PKC had reduced sprout-promoting activity. The results establish GAP-43 as an intrinsic presynaptic determinant for neurite outgrowth and plasticity.

A developmental switch in the response of DRG neurons to ETS transcription factor signaling

Two ETS transcription factors of the Pea3 subfamily are induced in subpopulations of dorsal root ganglion (DRG) sensory and spinal motor neurons by target-derived factors. Their expression controls late aspects of neuronal differentiation such as target invasion and branching. Here, we show that the late onset of ETS gene expression is an essential requirement for normal sensory neuron differentiation. We provide genetic evidence in the mouse that precocious ETS expression in DRG sensory neurons perturbs axonal projections, the acquisition of terminal differentiation markers, and their dependence on neurotrophic support. Together, our findings indicate that DRG sensory neurons exhibit a temporal developmental switch that can be revealed by distinct responses to ETS transcription factor signaling at sequential steps of neuronal maturation.

Requirement for the Homeobox Gene Hb9 in the Consolidation of Motor Neuron Identity

The homeobox gene Hb9, like its close relative MNR2, is expressed selectively by motor neurons (MNs) in the developing vertebrate CNS. In embryonic chick spinal cord, the ectopic expression of MNR2 or Hb9 is sufficient to trigger MN differentiation and to repress the differentiation of an adjacent population of V2 interneurons. Here, we provide genetic evidence that Hb9 has an essential role in MN differentiation. In mice lacking Hb9 function, MNs are generated on schedule and in normal numbers but transiently acquire molecular features of V2 interneurons. The aberrant specification of MN identity is associated with defects in the migration of MNs, the emergence of the subtype identities of MNs, and the projection of motor axons. These findings show that HB9 has an essential function in consolidating the identity of postmitotic MNs.

Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation

Muscle LIM protein (MLP) is a novel positive regulator of myogenesis. Its expression and that of its Drosophila homolog DMLP1 are enriched in striated muscle and coincide with myogenic differentiation. In the absence of MLP, induced C2 cells express myogenin but fail to exit from the cell cycle and to differentiate. Over-expression of MLP in C2 myoblasts potentiates myogenic differentiation and reduces its sensitivity to TGF beta. Like MLP, single LIM domain deletion mutants of MLP and nonmuscle LIM-only proteins promote myogenic differentiation. In 3T3 fibroblasts, the same LIM proteins prevent phorbol ester-induced inhibition of DNA replication. These results establish MLP as an essential promoter of myogenesis and suggest that LIM-only proteins act via similar mechanisms to regulate aspects of cell differentiation.

ETS Gene Er81 Controls the Formation of Functional Connections between Group Ia Sensory Afferents and Motor Neurons

The connections formed between sensory and motor neurons (MNs) play a critical role in the control of motor behavior. During development, the axons of proprioceptive sensory neurons project into the spinal cord and form both direct and indirect connections with MNs. Two ETS transcription factors, ER81 and PEA3, are expressed by developing proprioceptive neurons and MNs, raising the possibility that these genes are involved in the formation of sensory-motor connections. Er81 mutant mice exhibit a severe motor discoordination, yet the specification of MNs and induction of muscle spindles occurs normally. The motor defect in Er81 mutants results from a failure of group Ia proprioceptive afferents to form a discrete termination zone in the ventral spinal cord. As a consequence there is a dramatic reduction in the formation of direct connections between proprioceptive afferents and MNs. ER81 therefore controls a late step in the establishment of functional sensory-motor circuitry in the developing spinal cord.

Patterning of Muscle Acetylcholine Receptor Gene Expression in the Absence of Motor Innervation

The patterning of skeletal muscle is thought to depend upon signals provided by motor neurons. We show that AChR gene expression and AChR clusters are concentrated in the central region of embryonic skeletal muscle in the absence of innervation. Neurally derived Agrin is dispensable for this early phase of AChR expression, but MuSK, a receptor tyrosine kinase activated by Agrin, is required to establish this AChR prepattern. The zone of AChR expression in muscle lacking motor axons is wider than normal, indicating that neural signals refine this muscle-autonomous prepattern. Neuronal Neuregulin-1, however, is not involved in this refinement process, nor indeed in synapse-specific AChR gene expression. Our results demonstrate that AChR expression is patterned in the absence of innervation, raising the possibility that similarly prepatterned muscle-derived cues restrict axon growth and initiate synapse formation.

Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9.

The initial stages of pancreatic development occur early during mammalian embryogenesis, but the genes governing this process remain largely unknown. The homeodomain protein Pdx1 is expressed in the developing pancreatic anlagen from the approximately 10-somite stage, and mutations in the gene Pdx1 prevent the development of the pancreas. The initial stages of pancreatic development, however, still occur in Pdx1-deficient mice. Hlxb9 (encoding Hb9; ref. 6) is a homeobox gene that in humans has been linked to dominant inherited sacral agenesis and we show here that Hb9 is expressed at early stages of mouse pancreatic development and later in differentiated β-cells. Hlxb9 has an essential function in the initial stages of pancreatic development. In absence of Hlxb9 expression, the dorsal region of the gut epithelium fails to initiate a pancreatic differentiation program. In contrast, the ventral pancreatic endoderm develops but exhibits a later and more subtle perturbation in β-cell differentiation and in islet cell organization. Thus, dorsally Hlxb9 is required for specifying the gut epithelium to a pancreatic fate and ventrally for ensuring proper endocrine cell differentiation.

Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload

Iron homeostasis plays a critical role in many physiological processes, notably synthesis of heme proteins. Dietary iron sensing and inflammation converge in the control of iron absorption and retention by regulating the expression of hepcidin, a regulator of the iron exporter ferroportin. Human mutations in the glycosylphosphatidylinositol-anchored protein hemojuvelin (HJV; also known as RGMc and HFE2) cause juvenile hemochromatosis, a severe iron overload disease, but the way in which HJV intersects with the iron regulatory network has been unclear. Here we show that, within the liver, mouse Hjv is selectively expressed by periportal hepatocytes and also that Hjv-mutant mice exhibit iron overload as well as a dramatic decrease in hepcidin expression. Our findings define a key role for Hjv in dietary iron sensing and also reveal that cytokine-induced inflammation regulates hepcidin expression through an Hjv-independent pathway.