Hic-5 remodeling of the stromal matrix promotes breast tumor progression.

The remodeling of the stromal extracellular matrix (ECM) has a crucial, but incompletely understood role during tumor progression and metastasis. Hic-5, a focal adhesion scaffold protein, has previously been implicated in tumor cell invasion, proliferation and metastasis. To investigate the role of Hic-5 in breast tumor progression in vivo, Hic-5-/- mice were generated and crossed with the Mouse Mammary Tumor Virus-Polyoma Middle T-Antigen mouse. Tumors from the Hic-5-/-;PyMT mice exhibited increased latency and reduced growth, with fewer lung metastases, as compared with Hic-5+/-;PyMT mice. Immunohistochemical analysis showed that Hic-5 is primarily expressed in the cancer-associated fibroblasts (CAFs). Further analysis revealed that the Hic-5-/-;PyMT tumor stroma contains fewer CAFs and exhibits reduced ECM deposition. The remodeling of the stromal matrix by CAFs has been shown to increase tumor rigidity to indirectly regulate FAK Y397 phosphorylation in tumor cells to promote their growth and invasion. Accordingly, the Hic-5-/-;PyMT tumor cells exhibited a reduction in FAK Y397 phosphorylation. Isolated Hic-5-/-;PyMT CAFs were defective in stress fiber organization and exhibited reduced contractility. These cells also failed to efficiently deposit and organize the ECM in two and three dimensions. This, in turn, impacted three-dimensional MDA-MB-231 tumor cell migration behavior. Thus, using a new knockout mouse model, we have identified Hic-5 expression in CAFs as a key requirement for deposition and remodeling of the stromal ECM to promote non-cell autonomous breast tumor progression.

Reelin binds alpha3beta1 integrin and inhibits neuronal migration.

Mice that are mutant for Reelin or Dab1, or doubly mutant for the VLDL receptor (VLDLR) and ApoE receptor 2 (ApoER2), show disorders of cerebral cortical lamination. How Reelin and its receptors regulate laminar organization of cerebral cortex is unknown. We show that Reelin inhibits migration of cortical neurons and enables detachment of neurons from radial glia. Recombinant and native Reelin associate with alpha3beta1 integrin, which regulates neuron-glia interactions and is required to achieve proper laminar organization. The effect of Reelin on cortical neuronal migration in vitro and in vivo depends on interactions between Reelin and alpha3beta1 integrin. Absence of alpha3beta1 leads to a reduction of Dab1, a signaling protein acting downstream of Reelin. Thus, Reelin may arrest neuronal migration and promote normal cortical lamination by binding alpha3beta1 integrin and modulating integrin-mediated cellular adhesion.

LIS1 regulates CNS lamination by interacting with mNudE, a central component of the centrosome.

LIS1, a microtubule-associated protein, is required for neuronal migration, but the precise mechanism of LIS1 function is unknown. We identified a LIS1 interacting protein encoded by a mouse homolog of NUDE, a nuclear distribution gene in A. nidulans and a multicopy suppressor of the LIS1 homolog, NUDF. mNudE is located in the centrosome or microtubule organizing center (MTOC), and interacts with six different centrosomal proteins. Overexpression of mNudE dissociates gamma-tubulin from the centrosome and disrupts microtubule organization. Missense mutations that disrupt LIS1 function block LIS1-mNudE binding. Moreover, misexpression of the LIS1 binding domain of mNudE in Xenopus embryos disrupts the architecture and lamination of the CNS. Thus, LIS1-mNudE interactions may regulate neuronal migration through dynamic reorganization of the MTOC.

Properties of ectopic neurons induced by Xenopus neurogenin1 misexpression.

We have examined cells cultured from ectoderm-misexpressing Neurogenin1 (Ngn1) to describe better the extent to which this gene can control aspects of neuronal phenotype including motility, morphology, excitability, and synaptic properties. Like primary spinal neurons which normally express Ngn1, cells in Ngn1-misexpressing cultures exhibit a motility-correlated behavior called circus movements prior to neuritogenesis. Misexpression of NeuroD also causes circus movements and later neuronal differentiation. GSK3beta, which inhibits NeuroD function in vivo, blocks both Ngn1-induced and NeuroD-induced neuronal differentiation, while Notch signaling inhibits only Ngn1-induced neuronal differentiation, confirming that NeuroD is downstream of Ngn1 and insensitive to Notch inhibition. While interfering with NeuroD function in ventral ectoderm inhibits both circus movements and neuronal differentiation, such inhibition in the neural plate inhibits only neuronal differentiation, suggesting that additional factors regulate circus movements in the neural ectoderm. Ngn1-misexpressing cells extend N-tubulin-positive neurites and exhibit tetrodotoxin-sensitive action potentials. Unlike the majority of cultured spinal neurons, however, Ngn1-misexpressing cells do not respond to glutamate and do not form functional synapses with myocytes, suggesting that these cells are either like Rohon-Beard sensory neurons or are not fully differentiated.

Mitochondrial dysfunction is a primary event in glutamate neurotoxicity.

Excitotoxic neuronal death, associated with neurodegenerative disorders and hypoxic insults, results from excessive exposure to excitatory neurotransmitters. Glutamate neurotoxicity is triggered primarily by massive Ca2+ influx arising from overstimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondria are primary targets in excitotoxicity by confocal imaging of intracellular Ca2+ ([Ca2+]i) and mitochondrial membrane potential (delta psi) on cultured rat hippocampal neurons. Sustained activation of NMDA receptors (20 min) elicits reversible elevation of [Ca2+]i. Longer activation (50 min) renders elevation of [Ca2+]i irreversible (Ca2+ overload). Susceptibility to NMDA-induced Ca2+ overload is increased when the 20 min stimuli are applied to neurons pretreated with electron transport chain inhibitors, thereby implicating mitochondria in [Ca2+]i homeostasis during excitotoxic challenges. Remarkably, delta psi exhibits prominent and persistent depolarization in response to NMDA, which closely parallels the incidence of neuronal death. Blockade of the mitochondrial permeability transition pore by cyclosporin A allows complete recovery of delta psi and prevents cell death. These results suggest that early mitochondrial damage plays a key role in induction of glutamate neurotoxicity.

Onset of electrical excitability during a period of circus plasma membrane movements in differentiating Xenopus neurons.

Living neurons are usually first identifiable in primary cultures at the time of neurite initiation, and studies of excitability have been restricted largely to the subsequent period. A morphological early marker is described that identifies neurons for whole-cell voltage-clamp recordings before neurite initiation. Video time-lapse recordings of cultured cells dissociated from neurectoderm of Xenopus neural plate stage embryos reveal cells demonstrating circus movements, in which blebs of plasma membrane propagate around the cell circumference within a period of several minutes. All neurons demonstrate circus movements before morphological differentiation; the fraction of cells exhibiting circus movements that differentiate morphologically depends on the substrate on which they are cultured. Blockade of circus activity with cytochalasin B does not prevent neuronal differentiation. Circus movements are not neurectoderm-specific because they similarly predict differentiation of myocytes developing in mesodermal cultures. Initially inexcitable, neurons develop voltage-dependent K+, Na+, and Ca2+ currents during the period of several hours in which they exhibit circus movements. The early development of depolarization-induced elevations of [Ca2+]i several hours before morphological differentiation corresponds to the previously described onset of functionally significant spontaneous elevations of [Ca2+]i in these neurons and demonstrates a role for early expression of voltage-dependent ion channels.

Spontaneous neuronal calcium spikes and waves during early differentiation.

Calcium ions play critical roles in neuronal development, but the factors that govern spontaneous fluctuations in intracellular calcium are not well understood. Transient, repeated elevations of calcium in embryonic Xenopus spinal neurons have been recorded over periods of 1 hr in vitro and in vivo, confocally imaging fluo-3-loaded cells at 5 sec intervals. Calcium spikes and calcium waves are found both in neurons in culture and in the intact spinal cord. Spikes rise rapidly to approximately 400% of baseline fluorescence and have a characteristic double exponential decay, while waves rise slowly to approximately 200% of baseline fluorescence and decay slowly as well. Imaging of fura-2-loaded neurons indicates that intracellular calcium increases from 50 to 500 nM during spikes. Both spikes and waves are abolished by removal of extracellular calcium. Developmentally, the incidence and frequency of spikes decrease while the incidence and frequency of waves are constant. Spikes are generated by spontaneous calcium-dependent action potentials that can be triggered by low-threshold, T-type calcium current and are eliminated by agents that block voltage-dependent calcium channels. They can be elicited by depolarization, are generated in an all-or-none manner, and are rapidly and bidirectionally propagated. Spikes also utilize intracellular calcium stores, since blocking release from stores substantially reduces their amplitude. Waves are not elicited by depolarization nor by activation of glutamate receptors, and are propagated at a rate consistent with diffusion of calcium. Waves are blocked by Ni2+ at a higher concentration than required to block classical voltage-dependent calcium channels. Previous work now suggests that spikes are required for expression of the transmitter GABA and for potassium channel modulation. The present study indicates that waves in growth cones are likely to regulate neurite extension.

The problem of missing links: today and yesterday.

The problems of "missing links" over the years since Darwin's On the Origin of Species is analyzed in the perspective of our increased knowledge of the fossil record and our changing concepts of evolution, geology, and biology. The history of application of evolutionary theory to the fossil record and the consequences of the conflict of categorical, essential classification and evolutionary continuity are examined, as they relate to the "naive" idea of "missing links". Transitions between taxonomic categories are treated at two levels: the infraspecies-species level, and the supraspecies level. In the latter category case analyses of transitions within and between the major groups of metazoans and metaphytes are made, with special attention to the evidence of origins of groups in the fossil record. Throughout, the impacts on evolutionary theory of changes in knowledge and interpretations of the record of the history of life are considered. The problem of the existence of linkages and phylogenies at the species and generic levels has been much reduced during the last one hundred and twenty years. How this reduction supports or denies Darwin's concepts of phyletic gradualism is still a matter of interpretation of the evidence. At familial and higher levels, the establishment of linkages between categories has been much less successful, and decreasingly so at each successive higher level. Under the very best circumstances, however, morphological and stratigraphically graded transitions between classes and subclasses have been found. At the level of phyla and higher categories, any information on transitions as far as the fossil record is concerned is essentially non-existent. Fairly standard patterns of transitions between high categories can be established on the basis of the optimal cases, and these point up the continuing problems in evolutionary theory as being the interrelationships and integration of micro-evolutionary and macro-evolutionary processes.

Enhancement of mast cell differentiation in vitro by T cell factor(s).

Mast cell colonies were obtained when lymph node cells of horse serum-immunized Balb/c mice were cultured in a horse serum-containing medium on embryonic fibroblast monolayer. In order to characterized precursors of mast cells, mesenteric lymph node cells from the immunized mice were fractionated to obtain nonadherent cells, a B cell-depleted fraction and a T cell-depleted fraction; and each fraction was cultured on fibroblast monolayer. Mast cell colonies developed from nonadherent cells and from the B cell-depleted fraction but not from the T cell-depleted fraction. However, cultures of the same T cell-depleted fraction developed mast cell colonies if cell-free supernatant obtained from culture of horse serum-primed T cells was added. Soluble factors promoting mast cell growth were not obtained when the same T cells were incubated in horse serum-free medium. It appears that the majority of mast cell precursors in the lymph nodes are nonadherent cells and bear neither immunoglobulin nor Thy 1 antigen. The results also suggested that soluble factor(s) released from antigen-stimulated T cells enhanced the differentiation of the precursors to mature mast cells.