A genetically defined asymmetry underlies the inhibitory control of flexor-extensor locomotor movements.

V1 and V2b interneurons (INs) are essential for the production of an alternating flexor-extensor motor output. Using a tripartite genetic system to selectively ablate either V1 or V2b INs in the caudal spinal cord and assess their specific functions in awake behaving animals, we find that V1 and V2b INs function in an opposing manner to control flexor-extensor-driven movements. Ablation of V1 INs results in limb hyperflexion, suggesting that V1 IN-derived inhibition is needed for proper extension movements of the limb. The loss of V2b INs results in hindlimb hyperextension and a delay in the transition from stance phase to swing phase, demonstrating V2b INs are required for the timely initiation and execution of limb flexion movements. Our findings also reveal a bias in the innervation of flexor- and extensor-related motor neurons by V1 and V2b INs that likely contributes to their differential actions on flexion-extension movements.

Identification of a spinal circuit for light touch and fine motor control.

Sensory circuits in the dorsal spinal cord integrate and transmit multiple cutaneous sensory modalities including the sense of light touch. Here, we identify a population of excitatory interneurons (INs) in the dorsal horn that are important for transmitting innocuous light touch sensation. These neurons express the ROR alpha (RORα) nuclear orphan receptor and are selectively innervated by cutaneous low threshold mechanoreceptors (LTMs). Targeted removal of RORα INs in the dorsal spinal cord leads to a marked reduction in behavioral responsiveness to light touch without affecting responses to noxious and itch stimuli. RORα IN-deficient mice also display a selective deficit in corrective foot movements. This phenotype, together with our demonstration that the RORα INs are innervated by corticospinal and vestibulospinal projection neurons, argues that the RORα INs direct corrective reflex movements by integrating touch information with descending motor commands from the cortex and cerebellum.

Shp2/MAPK signaling controls goblet/paneth cell fate decisions in the intestine.

In the development of the mammalian intestine, Notch and Wnt/ß-catenin signals control stem cell maintenance and their differentiation into absorptive and secretory cells. Mechanisms that regulate differentiation of progenitors into the three secretory lineages, goblet, paneth, or enteroendocrine cells, are not fully understood. Using conditional mutagenesis in mice, we observed that Shp2-mediated MAPK signaling determines the choice between paneth and goblet cell fates and also affects stem cells, which express the leucine-rich repeat-containing receptor 5 (Lgr5). Ablation of the tyrosine phosphatase Shp2 in the intestinal epithelium reduced MAPK signaling and led to a reduction of goblet cells while promoting paneth cell development. Conversely, conditional mitogen-activated protein kinase kinase 1 (Mek1) activation rescued the Shp2 phenotype, promoted goblet cell and inhibited paneth cell generation. The Shp2 mutation also expanded Lgr5+ stem cell niches, which could be restricted by activated Mek1 signaling. Changes of Lgr5+ stem cell quantities were accompanied by alterations of paneth cells, indicating that Shp2/MAPK signaling might affect stem cell niches directly or via paneth cells. Remarkably, inhibition of MAPK signaling in intestinal organoids and cultured cells changed the relative abundance of Tcf4 isoforms and by this, promoted Wnt/ß-catenin activity. The data thus show that Shp2-mediated MAPK signaling controls the choice between goblet and paneth cell fates by regulating Wnt/ß-catenin activity.

Genetic dissection of rhythmic motor networks in mice.

Simple motor behaviors such as locomotion and respiration involve rhythmic and coordinated muscle movements that are generated by central pattern generator (CPG) networks in the spinal cord and hindbrain. These CPG networks produce measurable behavioral outputs and thus represent ideal model systems for studying the operational principles that the nervous system uses to produce specific behaviors. Recent advances in our understanding of the transcriptional code that controls neuronal development have provided an entry point into identifying and targeting distinct neuronal populations that make up locomotor CPG networks in the spinal cord. This has spurred the development of new genetic approaches to dissect and manipulate neuronal networks both in the spinal cord and hindbrain. Here we discuss how the advent of molecular genetics together with anatomical and physiological methods has begun to revolutionize studies of the neuronal networks controlling rhythmic motor behaviors in mice.

The tyrosine phosphatase Shp2 in development and cancer.

Deregulation of signaling pathways, through mutation or other molecular changes, can ultimately result in disease. The tyrosine phosphatase Shp2 has emerged as a major regulator of receptor tyrosine kinase (RTK) and cytokine receptor signaling. In the last decade, germline mutations in the human PTPN11 gene, encoding Shp2, were linked to Noonan (NS) and LEOPARD syndromes, two multisymptomatic developmental disorders that are characterized by short stature, craniofacial defects, cardiac defects, and mental retardation. Somatic Shp2 mutations are also associated with several types of human malignancies, such as the most common juvenile leukemia, juvenile myelomonocytic leukemia (JMML). Whereas NS and JMML are caused by gain-of-function (GOF) mutations of Shp2, loss-of-function (LOF) mutations are thought to be associated with LEOPARD syndrome. Animal models that carry conditional LOF and GOF mutations have allowed a better understanding of the mechanism of Shp2 function in disease, and shed light on the role of Shp2 in signaling pathways that control decisive events during embryonic development or during cellular transformation/tumorigenesis.

The tyrosine phosphatase Shp2 (PTPN11) directs Neuregulin-1/ErbB signaling throughout Schwann cell development.

The nonreceptor tyrosine phosphatase Shp2 (PTPN11) has been implicated in tyrosine kinase, cytokine, and integrin receptor signaling. We show here that conditional mutation of Shp2 in neural crest cells and in myelinating Schwann cells resulted in deficits in glial development that are remarkably similar to those observed in mice mutant for Neuregulin-1 (Nrg1) or the Nrg1 receptors, ErbB2 and ErbB3. In cultured Shp2 mutant Schwann cells, Nrg1-evoked cellular responses like proliferation and migration were virtually abolished, and Nrg1-dependent intracellular signaling was altered. Pharmacological inhibition of Src family kinases mimicked all cellular and biochemical effects of the Shp2 mutation, implicating Src as a primary Shp2 target during Nrg1 signaling. Together, our genetic and biochemical analyses demonstrate that Shp2 is an essential component in the transduction of Nrg1/ErbB signals.

Mutant desmocollin-2 causes arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically heterogeneous heart-muscle disorder characterized by progressive fibrofatty replacement of right ventricular myocardium and an increased risk of sudden cardiac death. Mutations in desmosomal proteins that cause ARVC have been previously described; therefore, we investigated 88 unrelated patients with the disorder for mutations in human desmosomal cadherin desmocollin-2 (DSC2). We identified a heterozygous splice-acceptor-site mutation in intron 5 (c.631-2A-->G) of the DSC2 gene, which led to the use of a cryptic splice-acceptor site and the creation of a downstream premature termination codon. Quantitative analysis of cardiac DSC2 expression in patient specimens revealed a marked reduction in the abundance of the mutant transcript. Morpholino knockdown in zebrafish embryos revealed a requirement for dsc2 in the establishment of the normal myocardial structure and function, with reduced desmosomal plaque area, loss of the desmosome extracellular electron-dense midlines, and associated myocardial contractility defects. These data identify DSC2 mutations as a cause of ARVC in humans and demonstrate that physiologic levels of DSC2 are crucial for normal cardiac desmosome formation, early cardiac morphogenesis, and cardiac function.

Mutations in the desmosomal protein plakophilin-2 are common in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is associated with fibrofatty replacement of cardiac myocytes, ventricular tachyarrhythmias and sudden cardiac death. In 32 of 120 unrelated individuals with ARVC, we identified heterozygous mutations in PKP2, which encodes plakophilin-2, an essential armadillo-repeat protein of the cardiac desmosome. In two kindreds with ARVC, disease was incompletely penetrant in most carriers of PKP2 mutations.

Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation.

Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease. We have ablated the plakophilin 2 gene in mice. The resulting mutant mice exhibit lethal alterations in heart morphogenesis and stability at mid-gestation (E10.5-E11), characterized by reduced trabeculation, disarrayed cytoskeleton, ruptures of cardiac walls, and blood leakage into the pericardiac cavity. In the absence of plakophilin 2, the cytoskeletal linker protein desmoplakin dissociates from the plaques of the adhering junctions that connect the cardiomyocytes and forms granular aggregates in the cytoplasm. By contrast, embryonic epithelia show normal junctions. Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

Tyrosine kinase receptor RON functions downstream of the erythropoietin receptor to induce expansion of erythroid progenitors.

Erythropoietin (EPO) is required for cell survival during differentiation and for progenitor expansion during stress erythropoiesis. Although signaling pathways may couple directly to docking sites on the EPO receptor (EpoR), additional docking molecules expand the signaling platform of the receptor. We studied the roles of the docking molecules Grb2-associated binder-1 (Gab1) and Gab2 in EPO-induced signal transduction and erythropoiesis. Inhibitors of phosphatidylinositide 3-kinase and Src kinases suppressed EPO-dependent phosphorylation of Gab2. In contrast, Gab1 activation depends on recruitment and phosphorylation by the tyrosine kinase receptor RON, with which it is constitutively associated. RON activation induces the phosphorylation of Gab1, mitogen-activated protein kinase (MAPK), and protein kinase B (PKB) but not of signal transducer and activator of transcription 5 (Stat5). RON activation was sufficient to replace EPO in progenitor expansion but not in differentiation. In conclusion, we elucidated a novel mechanism specifically involved in the expansion of erythroblasts involving RON as a downstream target of the EpoR.