Richard P. Tucker

The roles of microtubule-associated proteins in brain morphogenesis: a review

Microtubule-associated proteins (MAPs) are a diverse family of cytoskeletal proteins that copurify with tubulin in vitro. Recently a number of novel approaches have been used to learn more about the functions of MAPs during brain development, including: localization of MAPs and their mRNA in the developing brain, comparisons of MAPs between species to learn potential fundamental characteristics, biochemical analysis of changes in MAPs in process-bearing cell lines, and sequence analysis of MAP cDNAs and cDNA transfection studies. Taken together, these data allow us to assign roles to MAPs which are abundant in the developing brain, and to develop models for future studies. Four MAPs are particularly abundant in the developing brain: MAP1B, the high and low-molecular weight forms of MAP2, and juvenile tau. MAP1B is the only MAP to be found consistently in extending processes in both the developing and adult brain, making it a likely regulator of neurite outgrowth. High-molecular weight MAP2 and tau crosslink microtubules in dendrites and axons, respectively. Low-molecular weight MAP2 may be able to regulate MAP2-mediated crosslinking to make processes more labile during development and in adult brain regions where synaptogenesis is active. Tau-mediated crosslinking may be regulated by temporal regulation of the expression of tau forms with different binding affinities to tubulin. High-molecular weight MAP2 is sequestered into dendrites by the selective transport of its mRNA. This allows rapid and local regulation of MAP2 synthesis.

The sequential expression of tenascin mRNA in epithelium and mesenchyme during feather morphogenesis

SummaryI have studied the distribution of tenascin and its transcript during feather germ morphogenesis using immunohistochemistry and in situ hybridization. Anti-tenascin staining is intense in the periphery of dermal core condensations in both the feather rudiment and bud. There is faint anti-tenascin immunoreactivity in the overlying epithelium, but the apex of the bud is unstained. The appearance of tenascin in the developing feather is transient, as no significant anti-tenascin staining can be detected in the feather shaft or follicle at later stages of development. In situ hybridization with a tenascin cDNA probe reveals tenascin mRNA in the epithelial placode of the feather rudiment and early bud. In contrast, tenascin mRNA is concentrated in the dermis in the late feather bud. Therefore, at the time when inductive events are taking place, the expression of tenascin flips from the epithelium overlying tenascin-rich mesenchyme to the mesenchyme itself.

Differential localization of the high- and low-molecular weight variants of MAP2 in the developing retina

Microtubule-associated protein 2 (MAP2) occurs in developing mammalian neuronal tissue as both high (280 kDa)- and low (70 kDa)-molecular weight forms with temporally regulated expression. We have studied the developing avian retina with a monoclonal antibody that recognizes both the high- and low-molecular weight forms of MAP2 and a second monoclonal antibody that recognizes only high-molecular weight MAP2. The developmentally regulated, low-molecular weight protein, MAP2c, has a more widespread distribution in the embryonic avian retina than high-molecular weight MAP2. Our results suggest that MAP2c is the first form of MAP2 to appear in differentiated embryonic retinal neurons, and that the high-molecular weight isoforms of MAP2 appear only later when they may confer stability to neuronal processes.

The human carotid atherosclerotic plaque stimulates angiogenesis on the chick chorioallantoic membrane

The chick chorioallantoic membrane was used to determine whether the carotid atherosclerotic plaque stimulates angiogenesis. Carotid endarterectomy specimens (1 mm3) with fibromuscular plaque (n = 8) and complicated plaque (n = 11) were implanted on the membrane on day nine of incubation and the response evaluated on day 11. Following fixation in situ with 10% formalin the angiogenic response was evaluated by: (1) examining whole membrane mounts, (2) quantitatively from a vascular density index and (3) from a histological study. Unmanipulated chorioallantoic membrane (n = 11) and plaque boiled prior to implantation (n = 6) served as controls. The vascularity of whole mounts of both fibromuscular and complicated plaque was greater than the controls. Vessel density of the membrane was estimated by counting the number of vessels intersecting four concentric circles (144.5 mm total circumference) placed on the formalin fixed membrane. The vascular density index due to the fibromuscular plaque (390.6 +/- 8.3) and complicated plaque (391.0 +/- 14.9) were similar (P greater than 0.9) but were significantly greater (P less than 0.001) than the unmanipulated membrane (327.9 +/- 5.6) or after treatment with the boiled plaque (283.8 +/- 15.6). Transforming growth factor beta 1 confirmed the validity of the experimental model to study angiogenesis. The histology of the chorioallantoic membrane due to either type of plaque was similar. Numerous vessels surrounded the plaque, and intraplaque vessels containing nucleated chick erythrocytes were observed. Although scattered vessels surrounded the boiled plaque, intraplaque vessels were not observed. This study demonstrates that the atherosclerotic plaque has angiogenic properties that may account for the increase in vasa vasorum that is associated with the plaque.

Immunohistochemical localization of a tenascin-like extracellular matrix protein in sea urchin embryos

SummaryWe have used polyclonal antisera raised against vertebrate tenascin to identify and localize tenascin-like proteins in the developing sea urchin. These antisera recognize high-molecular weight proteins on immunoblots of sea urchin embryo homogenates that are similar in size and appearance to tenascin from vertebrates. These proteins appear as a doublet with an apparent molecular weight of 150 kDa and a larger, broad band with an apparent molecular weight of 350 kDa. Whole mounts of sea urchin embryos and larvae were stained with one of these antisera. The anti-tenascin stained the surface of primary mesenchyme cells during their phase of active migration. This staining was sensitive to detergent, suggesting that the protein recognized by the antiserum was associated with the cell surface. During later stages of development, the bulk of the antitenascin staining was found dispersed throughout the blastocoel matrix, and was no longer sensitive to detergent. We conclude that sea urchins express tenascin-like proteins during early stages of development, and that these proteins may play a role associated with primary mesenchyme cell morphogenesis.

Microtubule-Associated Proteins in Cerebellar Morphogenesis

In his studies of the cerebellum, Ramon y Cajal (1888,1960) noted, with characteristic clarity, the major features of morphogenesis in the various cell types. Many of his observations are now so familiar as to be commonplace, whereas others are less frequently cited, perhaps because they deal with cellular phenomena whose regulation is still not properly understood. For example, Ramon y Cajal was the first to report that the growth of axons and dendrites involves several distinct steps. He observed that the neuroblast first extends short processes of rather irregular outline, as, for example, the granule cell does during its initial horizontal, bipolar phase. Only later do these initial protrusions narrow and lengthen to form a proper “axis cylinder.” Dendrites appear later still. Thus, Purkinje cells have already produced extensive, well formed axons long before dendrites appear. At this early stage the Purkinje cell body is decorated with multiple short protoplasmic extensions that are subsequently resorbed to be replaced by the growth of the mature dendritic tree. Neurogenesis thus involves an initial outgrowth of short, irregular processes followed by the formation of a distinct axon and finally by the gradual development of the dendrites.