Michael A. Kirby

A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system.

Axon pathfinding relies on the ability of the growth cone to detect and interpret guidance cues and to modulate cytoskeletal changes in response to these signals. We report that the murine POU domain transcription factor Brn-3.2 regulates pathfinding in retinal ganglion cell (RGC) axons at multiple points along their pathways and the establishment of topographic order in the superior colliculus. Using representational difference analysis, we identified Brn-3.2 gene targets likely to act on axon guidance at the levels of transcription, cell-cell interaction, and signal transduction, including the actin-binding LIM domain protein abLIM. We present evidence that abLIM plays a crucial role in RGC axon pathfinding, sharing functional similarity with its C. elegans homolog, UNC-115. Our findings provide insights into a Brn-3.2-directed hierarchical program linking signaling events to cytoskeletal changes required for axon pathfinding.

The Role of Leukocyte Traffic and Activation in Parturition

Objective: This review focuses on the contribution of immune cell trafficking and activities during the initial phase of activation in the process of parturition. Although uterine contractile activity has been the predominant focus for the mechanism that initiates labor, significant cellular and biochemical chanes cause remodeling of the cervix well before term. A convergence of evidence suggests that inflammatory processes that involve prostaglandins, nitric oxide, cytokines, as well as systemic and paracrine endocrine mediators may enhance uterine contractility, promote ripening of the cervx, and thus constitute an integrative hypothesis for the initiation of labor. Methods: Techniques to study the uterus and cervix of pregnant and virgin C3H/HeN mice included light and fluorescence microscopy. Tissues were processed by histochemistry and immunofluorescence. Analytic approaches to enumerae macrophages and assess activation included quantitative stereologic morphometry and laser scanning cytometry. Results: The transition between relative quiescene of the uterus and enhanced contractility involed migration of macrophages from the uterine endometrium and activation of macrophages in the cervix. Before birth, macrophages migrae into the cervix and are activated in the myometrium. Conclusion: Immune cell trafficking and activation are part of the initial mechanism that promotes ripening of the cervix, enhances uterine contractility, and initiates parturition. Markers for the conclusion of pregnancy may have diagnostic or therapeutic value to assess the normal progress of labor or identify women at risk of preterm labor.

Topographic organization of the retinocollicular projection in the neonatal rat.

The topographic order of the retinocollicular projection in the rat was examined from birth until maturity. Small, localized deposits of rhodamine-filled latex microspheres were placed into the superior colliculus at different locations. To minimize labeling fibers of passage deposit sites were typically, although not exclusively, placed into the caudal-lateral pole of the colliculus. Examination of the area and density of labeled cells in the retinae of these animals led to the following conclusions: (1) At each age examined, the location of the majority of labeled cells was observed to be in appropriate topographic register with the deposit site in the superior colliculus. (2) Confirming the work of previous investigators, errors in topographic projection were observed. These were present in both the contralateral and ipsilateral retinae and decreased with increasing postnatal age. The mature pattern was present by P10. (3) Quantitatively, the number of retinal ganglion cells terminating nontopographically within the colliculus constituted a relatively minor proportion of the total number of labeled cells in both retinae. It is concluded that the majority of the retinal ganglion cells make topographically appropriate terminations within the superior colliculus during development.

Number and Distribution of Retinal Ganglion Cells in Anubis Baboons (Papio anubis)

The number of retinal ganglion cells in Papio anubis was determined from light microscopic observations of wholemounted and vertically sectioned retinal tissue and electron microscopic examination of optic nerve cross sections. The total number of ganglion cells ranged from 1.41 to 1.81 million (mean 1.58 million, n = 6, SD = 169,927) per retina. The distribution of ganglion cells in cresyl violet stained wholemounts was also examined. Isodensity contours were almost circular perifoveally, but became horizontally elongated outside of the central retina, providing strong evidence for a visual streak. Ganglion cell somata within the streak were found to be significantly smaller than those outside of the streak in comparing regions of equal density. Finally, the distribution of blood vessels within the retina formed a watershed pattern with its crux centered on the ridge of this horizontally oriented high-density zone. Combined, these features indicate that anubis baboons possess a visual streak specialization as reported for lagomorphs, felines, and several primate species. Further, the visual streak appears more pronounced in anubis baboons than in any other primate species studied to date, with the possible exception of Homo sapiens, a similarly ground-dwelling/foraging and secondarily terrestrial species.

Increased innervation and ripening of the prepartum murine cervix.

Objective: Ripening of the cervix before birth is coincident with reduced collagen content and leukocyte immigration, characteristics that are analogous to a neurogenic inflammatory-like process. We sought to assess the morphologic relationship between innervation and remodeling of the peripatum cervix. Methods: Cervix was obtained from C3H/HeN mice on days 15 and 18 of pregnancy, 1 day postpartum, and from non-pregnant controls. Tissues were immersion-fixed, paraffin-embedded, and some sections stained with Picrosirius red to assess collagen content and complexity of organization. By image analysis of optical density, collagen content and structure were significantly decreased by the day before birth. Other sections were processed to visualize nerve fibers by immunohistochemistry with antibodies against neuron-specific epitopes, PGP9.5, peripherin, as well as brain nitric oxide synthase (bNOS), calcitonin gene-related peptide (CGRP), and other neuropeptides. Fiber density was assessed stereologically and normalized to cell density in non-pregnant cervix to correct for tissue hypertrophy due to reproductive status. Results: In groups of non-pregnant, day 15 pregnant, and postpartum mice, cervix contained nerve fibers tghat were immunoreactive for the pan-neural markers PGP9.5 and peripherin. Punctate and beaded varicosities were sparsely distributed in stroma, subepithelium, and in proximity to vascular structures. By day 18 of pregnancy, 1 day before birth, fiber density was increased fourfold or more compared to other groups. bNOS fiberrs and, to a lesser extent, CGRP accounted for most of the increased innervation of the murine cervix by the day before birth, a period when macrophage numbers are enhanced. Conclusions: The findings suggest that increased bNOS and CGRP innervation contribute to early inflammatory-like processes that ripen the cervix before birth.

Induction and axonal localization of epithelial/epidermal fatty acid‐binding protein in retinal ganglion cells are associated with axon development and regeneration

Epithelial/epidermal fatty acid‐binding protein (E‐FABP) is induced in peripheral neurons during nerve regeneration and is found at high levels in central neurons during neuronal migration and development. Furthermore, E‐FABP expression is required for normal neurite outgrowth in PC12 cells treated with nerve growth factor (NGF). The present study examined whether E‐FABP plays a role in retinal ganglion cell (RGC) differentiation and axon growth. Rat retinal tissues from embryonic (E) and postnatal (P) development through adulthood were examined using immunocytochemical labeling with E‐FABP and growth‐associated protein 43 (GAP‐43) antibodies. E‐FABP colocalized with GAP‐43 at E14 through P10. At E14, E‐FABP immunoreactivity was confined to the somas of GAP‐43‐positive cells in the ganglion cell layer, but it was localized to their axons by E15. The axons in the optic nerve were GAP‐43‐positive and E‐FABP‐negative on E15, but the two proteins were colocalized by E18. Retinal cultures at E15 confirmed that E‐FABP and GAP‐43 colocalize in RGCs. Postnatally, labeling was present between P1 and P10 but decreased at older ages and was minimally present or absent in adult animals. Western immunoblotting revealed that at E18, P1, and P10 E‐FABP levels were at least fourfold greater than those in the adult. By P15, protein levels were only twofold greater, with adult levels reached by P31. Furthermore, E‐FABP could be reinduced during axon regeneration. Dissociated P15 retinal cells cultured in the presence of brain‐derived neurotrophic factor, ciliary neurotrophic factor, and basic fibroblast growth factor exhibited sixfold more GAP‐43 and E‐FABP double‐positive RGCs (cell body and axons) than controls. Moreover, all GAP‐43‐immunoreactive RGCs were also positive for E‐FABP. Taken together, these results indicate the following: 1) E‐FABP is expressed in RGCs as they reached the ganglion cell layer and 2) E‐FABP plays a functional role in the elaboration of RGC axons in both development and regeneration. J. Neurosci. Res. 66:396–405, 2001.

Progesterone withdrawal promotes remodeling processes in the nonpregnant mouse cervix.

Abstract Prepartum cervical ripening is associated with remodeling of collagen structure and with inflammation. Progesterone withdrawal is critical for parturition, but the effects of progesterone decline on cervical morphology are unknown. The present study tested the hypothesis that progesterone withdrawal promotes processes associated with remodeling of the cervix. Adult, virgin, female C57BL/6 mice received silastic capsules with oil vehicle or estradiol plus progesterone to parallel concentrations in circulation during pregnancy. After 17 days of estradiol and progesterone treatment, the progesterone implant was removed from one group. Mice in each group were killed 15, 18, or 19 days after placement of capsules. Sections of cervix were stained for collagen, and the densities of macrophages, neutrophils, and area with nerve fibers were assessed. Treatment with gonadal steroids promoted hypertrophy of the cervix, as well as reduced collagen and increased area with nerve fibers compared with vehicle-treated controls. Removal of the progesterone capsule did not affect hypertrophy or innervation, but it did reduce collagen. By contrast, significantly more macrophages and neutrophils were present in the cervix on Days 18 and 19 (i.e., by 24 and 48 h after withdrawal of the progesterone capsule); the immune cell census was equivalent to that in vehicle controls. Findings indicate that gonadal steroids, comparable to those during pregnancy, promote hypertrophy and suppress immigration of immune cells in the cervix. Therefore, in a nonpregnant murine model for parturition, progesterone withdrawal is suggested to recruit immune cells and processes that remodel the cervix.

Topographic organization in the retinocollicular pathway of the fetal cat demonstrated by retrograde labeling of ganglion cells

The topographic organization of the developing retinocollicular pathway was assessed by making focal deposits of a retrograde tracer (usually rhodamine latex beads) into the superficial layers of the superior colliculus of fetal cats at known gestational ages. Subsequently, the distributions of labeled cells in the contralateral and ipsilateral retinas were examined. At all stages of development, a high density of labeled cells was found in a delimited area (core region) of both retinas. The locations of the retinal regions containing the high density of labeled cells varied with the locus of the tracer deposit in the superior colliculus in a manner consistent with the topographic organization of the mature cats retinocollicular pathway. Additionally, some labeled ganglion cells, considered to be ectopic, were found to be scattered throughout the contralateral and ipsilateral fetal retinas. Such ectopic cells were few in number throughout prenatal development. For every 100 cells projecting to the appropriate region of the colliculus, we estimate that less than one ganglion cell makes a gross projection error. The incidence of ectopic cells did not differ between the contralateral and ipsilateral retina, even though the overall density of crossed labeled cells was always greater than that of uncrossed labeled cells. In the youngest fetal animals, tracer deposits into the caudal portion of the superior colliculus resulted in a core region of labeled cells in the contralateral nasal retina as well as in the nasal ipsilateral retina. Such uncrossed nasal cells, not seen in more mature animals, appear to innervate the appropriate topographic location of the superior colliculus, but on the wrong side of the brain. Most likely, these uncrossed nasal ganglion cells contribute to the widespread distribution of the ipsilateral retinocollicular pathway observed in fetal cats after intraocular injections of anterograde tracers (Williams and Chalupa, 1982). Collectively, our findings demonstrate that the developing retinocollicular pathway of the fetal cat is characterized by a remarkable degree of topographic precision.

Early dendritic outgrowth of primate retinal ganglion cells.

The pattern of dendritic stratification of retinal ganglion cells in the fetal monkey (Macaca mulatta) was examined using horseradish peroxidase and retinal explants. Ganglion cells in the rhesus monkey are born between embryonic day (E) 30-70 (LaVail et al., 1983). At E60, E67, and E68, approximately 50% of all ganglion cells within the central 3.0 mm of the retina had dendritic arbors that were unistratified within the inner plexiform layer (IPL), while the remaining 50% had bistratified arbors. Unistratified cells had relatively flat arbors that ramified within a restricted portion of the IPL. In contrast, bistratified cells had one portion of the arbor that branched in the inner half of the IPL and a second portion that branched in the outer half of the IPL. Relatively few bistratified cells were encountered in the central 1.0 mm of the retina but were more numerous with increasing eccentricity. At E81, E90, and E110, the dendritic arbors of ganglion cells increased in both area and complexity, but occupied a relatively small percentage of the total depth of the IPL. The bistratified cells encountered at these fetal ages were typically located in the far retinal periphery. Between E125-E140, the dendritic arbors of individual ganglion cells increased in area and depth to occupy a greater proportion of the total IPL than at earlier fetal ages. These observations suggest that ganglion cells in the macaque undergo at least three stages of dendritic stratification: (1) an initial period of dendritic growth during which the cells have either unistratified or bistratified dendritic arbors; (2) a loss of the majority of bistratified cells through cell death or remodeling of the arbor; and (3) growth or expansion of the arbor to occupy a greater percentage of the total depth of the IPL. The first two stages are similar to recent observations in the fetal cat (Maslim & Stone, 1988) with the exception that dendritic development in the primate lacks an initial diffuse ingrowth to the IPL. Additionally, primate ganglion cells undergo a third stage of dendritic growth in late fetal development during which the arbor occupies a greater proportion of the depth of the IPL.

Morphogenesis of retinal ganglion cells during formation of the fovea in the Rhesus macaque

The morphology of retinal ganglion cells within the central retina during formation of the fovea was examined in retinal explants with horseradish-peroxidase histochemistry. A foveal depression was first apparent in retinal wholemounts at embryonic day 112 (E112; gestational term is approximately 165 days). At earlier fetal ages, the site of the future fovea was identified by several criteria that included peak density of ganglion cells, lack of blood vessels in the inner retinal layers, arcuate fiber bundles, and the absence of rod outer segments in the photoreceptor layer. Prior to E112, the terminal dendritic arbor of retinal ganglion cells within the central retina extended into the inner plexiform layer and were located directly beneath their somas of origin or at most were slightly displaced from it. For example, at E90 the mean horizontal displacement of the geometric center of the dendritic arbor from the somas of cells within 600 microns of the estimated center of the future fovea was 4.1 microns (S.D. 2.7, range 1.0-10.0, n = 97). Following formation of the foveal depression the dendritic arbors of cells were significantly displaced from their somas. For example, at E138 the mean displacement was 41.2 microns (S.D. 12.2, range 12.0-56.0, n = 97). The displacement of the dendritic arbor which occurred during this period was not accounted for by areal growth of the dendritic arbor, the somas, or the retina, but was produced by the lengthening of the primary dendritic trunk. Moreover, no significant displacement was observed within the remaining 1.5-6.5 mm of the central retina. These observations provide evidence supporting early speculations that the formation of the foveal pit occurs, in part, by the radial migration of ganglion cells from the center of the fovea during its formation. Our analyses suggest that this migration occurs by the lengthening of the primary dendrite presumably by the addition of membrane. This migration is in a direction opposite to the inward movement of photoreceptors that occurs during late fetal and early postnatal periods (Packer et al., 1990, Journal of Comparative Neurology 298, 472-493).