Allan M. Sheppard

E‐cadherin‐mediated adhesion inhibits ligand‐dependent activation of diverse receptor tyrosine kinases

E‐cadherin is an essential adhesion protein as well as a tumor suppressor that is silenced in many cancers. Its adhesion‐dependent regulation of signaling has not been elucidated. We report that E‐cadherin can negatively regulate, in an adhesion‐dependent manner, the ligand‐dependent activation of divergent classes of receptor tyrosine kinases (RTKs), by inhibiting their ligand‐dependent activation in association with decreases in receptor mobility and in ligand‐binding affinity. E‐cadherin did not regulate a constitutively active mutant RTK (Neu*) or the ligand‐dependent activation of LPA receptors or muscarinic receptors, which are two classes of G protein‐coupled receptors. EGFR regulation by E‐cadherin was associated with complex formation between EGFR and E‐cadherin that depended on the extracellular domain of E‐cadherin but was independent of β‐catenin binding or p120‐catenin binding. Transfection of E‐cadherin conferred negative RTK regulation to human melanoma and breast cancer lines with downregulated endogenous E‐cadherin. Abrogation of E‐cadherin regulation may contribute to the frequent ligand‐dependent activation of RTK in tumors.

Abnormal reorganization of preplate neurons and their associated extracellular matrix: An early manifestation of altered neocortical development in the reeler mutant mouse

The formation of the distinct layers of the cerebral cortex begins when cortical plate neurons take up positions within the extracellular matrix (ECM)‐rich preplate, dividing it into the marginal zone above and the subplate below. We have analyzed this process in the reeler mutant mouse, in which cortical lamination is severely disrupted. The recent observation that the product of the reeler gene is an ECM‐like protein that is expressed by cells of the marginal zone indicates a critical role for ECM in cortical lamination. We have found that preplate cells in normal cortex that are tagged during their terminal division with bromodeoxyuridine (BrdU) are closely associated with chondroitin sulfate proteoglycans (CSPGs), which were identified by immunolabeling; this association is maintained in the marginal zone and subplate after the preplate is divided by cortical plate formation. Cortical plate cells do not aggregate within the preplate in reeler; instead, preplate cells remain as an undivided superficial layer containing abundant CSPGs, and cortical plate neurons accumulate below them. These findings indicate that preplate cells are responsible for the formation of a localized ECM, because the association of CSPGs with preplate cells is maintained even when these cells are in abnormal positions. The failure of cortical plate neurons to aggregate within the framework of the preplate and its associated ECM and to divide it is one of the earliest structural abnormalities detectable in reeler cortex, suggesting that this step is important for the subsequent formation of cortical layers. J. Comp. Neurol. 378:173–179, 1997.

Expanding Roles for α4 Integrin and its Ligands in Development

Interaction of alpha 4 integrins with vascular cell adhesion molecule-1 (VCAM-1) is classically important for immune function. However, we found recently that these receptors have a second role, in embryogenesis, where they mediate cell-cell interactions that are important for skeletal muscle differentiation. Here, we present evidence of an expanding role for these receptors in murine development. alpha 4 and VCAM-1 were found at embryonic sites of hematopoiesis, suggesting a role for these receptors during embryogenesis that parallels their hematopoietic function in adult bone marrow. During angiogenesis in the lung, alpha 4 and VCAM-1 were found on mesenchyme that gives rise to vascular endothelium and smooth muscle. alpha 4 persisted on the smooth muscle and the endothelium of newly forming vessels where it colocalized with its extracellular matrix ligand, fibronectin (FN). These patterns suggest several roles for alpha 4 integrins and their ligands in angiogenesis. alpha 4 was also found on neural crest derivatives where it colocalized with FN. alpha 4 was expressed selectively on cells in the dorsal root ganglia: it was apparent along ventral projections, but absent from dorsal projections, suggesting that alpha 4 integrins could be involved in defining neuronal fates. Although VCAM-1 was not expressed on most neural crest derivatives, it was found in the neural crest-derived outflow tract of the embryonic heart, where it colocalized with alpha 4. These results imply that alpha 4 integrins and their ligands could be important for migration or differentiation of neural crest. alpha 4 was also expressed on embryonic retina and FN was found on inductive mesenchyme surrounding the eye, suggesting a role for these proteins in eye development. Finally, based on their patterns of expression, we conclude that VCAM-1 only participates in a subset of interactions involving alpha 4 integrins, whereas FN appears to be the more general ligand.

c-Myb and Ets proteins synergize to overcome transcriptional repression by ZEB

The Zfh family of zinc finger/homeodomain proteins was first identified in Drosophila where it is required for differentiation of tissues such as the central nervous system and muscle. ZEB, a vertebrate homolog of Zfh‐1, binds a subset of E boxes and blocks myogenesis through transcriptional repression of muscle genes. We present evidence here that ZEB also has an important role in controlling hematopoietic gene transcription. Two families of transcription factors that are required for normal hematopoiesis are c‐Myb and Ets. These factors act synergistically to activate transcription, and this synergy is required for transcription of at least several important hematopoietic genes. ZEB blocks the activity of c‐Myb and Ets individually, but together the factors synergize to resist this repression. Such repression imposes a requirement for both c‐Myb and Ets for transcriptional activity, providing one explanation for why synergy between these factors is important. The balance between repression by ZEB and transcriptional activation by c‐Myb/Ets provides a flexible regulatory mechanism for controlling gene expression in hematopoietic cells. We demonstrate that one target of this positive/negative regulation in vivo is the α4 integrin, which play a key role in normal hematopoiesis and function of mature leukocytes.

Developmental expression of keratan sulfate-like immunoreactivity distinguishes thalamic nuclei and cortical domains

Proteoglycans influence axonal outgrowth in several experimental paradigms, and their distribution during development suggests a role in axon guidance. We have used a monoclonal antibody, 5D4, that recognizes an epitope on sulfated keratans (KS), to define the distribution of keratan sulfate proteoglycans (KSPGs) in the developing thalamus and cortex of the rat. During development, 5D4 immunolabeling is present on thalamic axons as they grow through the internal capsule and subplate but is not present in the adjacent pathway for cortical efferent axons. Individual thalamic nuclei differ markedly in their expression of KSPGs; these distinctions persist throughout the period of developmentally regulated expression. Major cortical domains also differ in their expression of KSPGs, which are expressed throughout medial (cingulate and retrosplenial) cortex well before neocortex. Immunolabeling for KSPGs diminishes 2 weeks after birth; in the adult it is associated with small glia. The 5D4 epitope is present on several KSPGs (320, 220, and 160 kD) on Western blots during development but only in a broad 200‐kD band in adult brain. Immunolabeling is degraded on sections and Western blots by keratanase II but not by keratanase I or chondroitinase ABC, confirming that the antibody recognizes KS. Bands identified by 5D4 on Western blots differ from those identified by antibodies to known KSPGs (aggrecan, claustrin, SV2, ABAKAN, phosphacan‐KS), indicating that 5D4 is labeling KSPGs not previously described in the brain. The selective expression of KSPGs during development suggests that they may be a part of the molecular identity of thalamic nuclei and cortical domains that defines their connectivity. J. Comp. Neurol. 380:533–552, 1997.

Chapter 9 Extracellular matrix in early cortical development

Publisher Summary This chapter focuses on the recent findings and those of other laboratories on extracellular matrix in developing cortex and provides a brief review of recent observations on the distribution of receptors for extracellular matrix (ECM). The studies of the distribution and production of ECM components during development of the cerebral cortex have suggested several hypotheses regarding their functional role. In the earliest stages of cortical development, fibronectin is produced by cells in the ventricular zone throughout the telencephalic vesicle, where it may serve as a part of the local environment that supports cell division and determines cell fate. Fibronectin is also distributed along radial glial processes. Axons leaving the cortical plate cross the chondroitin sulfate proteoglycan (CSPG)-rich subplate and then turn to follow a path containing much less CSPG. In contrast, the cortical trajectory of thalamic axons is centered on the subplate, indicating that CSPGs in the subplate are not a barrier to axon outgrowth and may instead be serving as guidance cues that distinguish afferent from efferent pathways. Neuro can, a central nervous system (CNS)-specific CSPG with many molecular features that indicate roles in cell-cell and cell-substrate interactions, is the only CSPG defined to date whose distribution supports a role in distinguishing afferent from efferent pathways.

Erratum: B. Miller, A.M. Sheppard, and A.L. Pearlman (1997) Developmental expression of keratan sulfate-like immunoreactivity distinguishes thalamic nuclei and cortical domains. J.Comp. Neurol.380:533-552

During the production and printing process, the contrast of the original figure was altered, and several control lanes in Figure 9, page 547, were inadvertently blocked out and therefore appear white instead of the same grey as the background in other lanes. The affected lanes were: A, Cortex: E16; C, P4: K2, Adult: K2; TED15: K1, and K1 + ABC. The corrected figure and legend are reprinted on the following page.