Beverly E. Faulkner-Jones

Separate Progenitors for Radial and Tangential Cell Dispersion during Development of the Cerebral Neocortex

Cell lineage analyses suggest that cortical neuroblasts are capable of undertaking both radial and tangential modes of cell movement. However, it is unclear whether distinct progenitors are committed to generating neuroblasts that disperse exclusively in either radial or tangential directions. Using highly unbalanced mouse stem cell chimeras, we have identified certain progenitors that are committed to one mode of cell dispersion only. Radially dispersed neurons expressed glutamate, the neurochemical signature of excitatory pyramidal cells. In contrast, tangential progenitors gave rise to widely scattered neurons that are predominantly GABAergic. These results suggest lineage-based mechanisms for early specification of certain progenitors to distinct dispersion pathways and neuronal phenotypes.

Clonal expansion and cell dispersion in the developing mouse retina.

The present study has used two different approaches for labelling progenitor cells at the optic vesicle stage in order to examine patterns of clonal expansion and cellular dispersion within the developing retina. X‐inactivation transgenic mice and chimeric mice expressing the lacZ reporter transgene were examined during development and in adulthood to study the radial and tangential dispersion of proliferating neuroepithelial cells and postmitotic retinal cells of known identities. Chimeric retinas were used to measure tangential dispersion distances, while transgenic retinas were used to assess the frequency of tangential dispersion for individual populations of retinal neurons. Tangential dispersion is shown to be a universal feature of particular retinal cell types, being contrasted with the strictly radial dispersion of other cells. Tangential dispersion is a relatively short‐distance phenomenon, with distinct dispersion distances characteristic for cone, horizontal, amacrine and ganglion cells. Embryonic and postnatal retinas show that tangential dispersion occurs at different times for these distinct cell types, associated with their times of differentiation rather than their neurogenetic periods. These developmental results rule out the possibility that tangential dispersion is due to a passive displacement produced by the proliferation of later‐born cells, or to the lateral dispersion of a dividing sibling; rather, they are consistent with the hypothesis that tangential dispersion plays a role in the establishment of the orderly spatial distribution of retinal mosaics.

Fine Needle Aspiration Biopsy with Adjunct Immunohistochemistry in Intraocular Tumor Management

OBJECTIVE To assess the effectiveness of fine needle aspiration biopsy (FNAB), with and without immunohistochemistry (IHC), in the management of solid intraocular tumors. STUDY DESIGN Thirty-three consecutive adults undergoing FNAB of suspected intraocular tumors were studied. Clinical, cytologic and histologic diagnoses were correlated. The positive predictive value, sensitivity and specificity of FNAB for detecting malignancy, the effect of lHC on the final cytologic diagnosis and the number of patients in whom clinical management was altered as a result of cytologic evaluation were determined. RESULTS The positive predictive value was 96% with and 93% without adjunct IHC. The sensitivity and specificity of FNAB for detecting malignancy were 96% and 83%, respectively, with IHC. Without IHC, the sensitivity was unaltered, but the specificity was 67%. IHC confirmed the morphologic diagnosis in 75% of cases, made a diagnosis in 12.5% and changed a malignant diagnosis from carcinoma to melanoma in 6% of cases. The planned management was changed by the FNAB findings in 24% of patients. In 3 patients (9%), IHC was essential for diagnosis and management. No patients exhibited local tumor dissemination or recurrence associated with the biopsy. CONCLUSION FNAB is a safe, sensitive and specific method of establishing a tissue diagnosis in a subset of patients with solid intraocular tumors. The routine use of immunohistochemical stain ing increases the diagnostic utility of the technique and may change clinical management.

Computational Pathology to Discriminate Benign from Malignant Intraductal Proliferations of the Breast

The categorization of intraductal proliferative lesions of the breast based on routine light microscopic examination of histopathologic sections is in many cases challenging, even for experienced pathologists. The development of computational tools to aid pathologists in the characterization of these lesions would have great diagnostic and clinical value. As a first step to address this issue, we evaluated the ability of computational image analysis to accurately classify DCIS and UDH and to stratify nuclear grade within DCIS. Using 116 breast biopsies diagnosed as DCIS or UDH from the Massachusetts General Hospital (MGH), we developed a computational method to extract 392 features corresponding to the mean and standard deviation in nuclear size and shape, intensity, and texture across 8 color channels. We used L1-regularized logistic regression to build classification models to discriminate DCIS from UDH. The top-performing model contained 22 active features and achieved an AUC of 0.95 in cross-validation on the MGH data-set. We applied this model to an external validation set of 51 breast biopsies diagnosed as DCIS or UDH from the Beth Israel Deaconess Medical Center, and the model achieved an AUC of 0.86. The top-performing model contained active features from all color-spaces and from the three classes of features (morphology, intensity, and texture), suggesting the value of each for prediction. We built models to stratify grade within DCIS and obtained strong performance for stratifying low nuclear grade vs. high nuclear grade DCIS (AUC = 0.98 in cross-validation) with only moderate performance for discriminating low nuclear grade vs. intermediate nuclear grade and intermediate nuclear grade vs. high nuclear grade DCIS (AUC = 0.83 and 0.69, respectively). These data show that computational pathology models can robustly discriminate benign from malignant intraductal proliferative lesions of the breast and may aid pathologists in the diagnosis and classification of these lesions.

Pattern formation in the cerebellum of murine embryonic stem cell chimeras

The cerebellar cortex is subdivided into an elaborate, stereotyped array of transverse zones and parasagittal stripes. It has been speculated that (i) all Purkinje cells derive from 10 to 20 precursors allocated early in embryogenesis and (ii) that pattern formation is based on cell lineage restriction in the founder pool. These hypotheses have been tested by clonal analysis of embryonic stem cell chimeras. Neither speculation is supported: the analysis suggests that Purkinje cells derive from a founder population of > 102 precursors, and that neither cerebellar transverse developmental boundaries nor parasagittal stripes have a clonal origin. We conclude that early lineage restriction plays no role in cerebellar pattern formation.

Interactive Histology of Large-Scale Biomedical Image Stacks

Histology is the study of the structure of biological tissue using microscopy techniques. As digital imaging technology advances, high resolution microscopy of large tissue volumes is becoming feasible; however, new interactive tools are needed to explore and analyze the enormous datasets. In this paper we present a visualization framework that specifically targets interactive examination of arbitrarily large image stacks. Our framework is built upon two core techniques: display-aware processing and GPU-accelerated texture compression. With display-aware processing, only the currently visible image tiles are fetched and aligned on-the-fly, reducing memory bandwidth and minimizing the need for time-consuming global pre-processing. Our novel texture compression scheme for GPUs is tailored for quick browsing of image stacks. We evaluate the usability of our viewer for two histology applications: digital pathology and visualization of neural structure at nanoscale-resolution in serial electron micrographs.

Cloning and expression of mouse Cadherin-7, a type-II cadherin isolated from the developing eye.

We report the molecular cloning of Cadherin-7 from the embryonic mouse eye. The deduced amino acid sequence shows it to be a type-II cadherin similar to Xenopus F-cadherin and chick Cadherin-7. The mouse Cadherin-7 gene maps to chromosome 1, outside the conserved linkage group of cadherin genes on chromosome 8. Cadherin-7 is expressed throughout the entire period of neural development and mRNA levels are developmentally regulated in both the embryonic and the postnatal central nervous system (CNS). In adult mice, Cadherin-7 expression is restricted to the CNS, with highest levels in the retina. In the developing eye, Cadherin-7 mRNA is found only in the neural retina. It is expressed by all retinal neuroblasts from E11 onward, but becomes progressively restricted to neurons in the inner neuroblast and developing ganglion cell layers (GCL). In the adult retina it is confined to subpopulations of cells in the GCL and to amacrine cells in the inner part of the inner nuclear layer. This expression pattern suggests a role for Cadherin-7 in mouse retinal development, particularly in the formation and maintenance of the GCL.

Lymphoid aggregates in bone marrow: study of eventual outcome.

The practical importance of finding a morphologically benign lymphoid aggregate in the bone marrow of patients without known lymphoproliferative disease was assessed in 786 consecutive patients who had had 951 iliac crest bone marrow biopsies performed. Of these, 430 patients known to have lymphoproliferative disease at the time of biopsy were excluded. Of 356 patients, 86 (aggregate group) had at least one lymphoid aggregate in their biopsy specimen biopsy specimen (82 morphologically benign, three suspicious, and one malignant). Another 86 patients without aggregates (control group) were matched by age and sex. Both groups were followed up until death, or for a mean of 21.9 and 22.9 months, respectively, to assess their outcome. Eighteen (22%) of the 82 patients with morphologically benign lymphoid aggregates were later proved to have lymphoproliferative disease compared with none of the 86 control patients. Another 12 patients in the aggregate group and seven in the control group were suspected of having a lymphoproliferative disease on clinical grounds, so that altogether 30 (37%) and seven (8%), respectively, developed confirmed or suspected lymphoproliferative disease. In both cases the differences were highly significant (p less than 0.001). It is suggested that lymphoid aggregates in clinical biopsy material may not be a physiological finding and should alert pathologists or haematologists to the possibility of lymphoproliferative disease.

Multiple cadherin mRNA expression and developmental regulation of a novel cadherin in the developing mouse eye.

Cadherins form a large family of transmembrane glycoproteins whose members include the classical cadherins, the desmosomal cadherins, and the protocadherins. The classical cadherins mediate homophilic cell-cell adhesion and are key regulators of many morphogenetic processes. More than a dozen classical cadherins are expressed in both the developing and the mature central nervous system. Although individual cadherins have been identified in the retina of various species, we wished to determine the range of cadherins expressed at distinct developmental stages in the mouse retina. Using a PCR-based cloning strategy, we detected 10 different classical cadherin mRNAs of both type I and type II subtypes. The most abundant cDNA was that encoding the type II cadherin, Cadherin-11. The other type II cadherins detected were VE- and T2-cadherin and Cadherin-6 and -12. Four type I cadherins, N-, R-, P-, and E-cadherin, were also present. One cadherin cDNA encoded a novel cadherin, called EY-cadherin for cloned from eye. EY-cadherin is most closely related to human Cadherin-14 (93% identical). EY-cadherin mRNA was detected in the adult mouse eye, brain, and testis with a 20-fold increase in expression levels in the embryonic head from E11 to E19 and a 50-fold increase in expression levels in the postnatal eye from PN1 to PN16. Multiple cadherin gene expression is consistent with the hypothesis that different cadherins regulate morphogenetic processes, such as neuronal migration and lamination, and determine the specific interneuronal connections found in the mature retina.

Transcription and translation of two glutamate decarboxylase genes in the ileum of rat, mouse and guinea pig.

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter, synthesised from glutamate by glutamate decarboxylase (GAD), in the central nervous system. Two forms of GAD, designated GAD 65 and GAD 67, are encoded by distinct genes and have been demonstrated in the mammalian brain. GABA has been postulated to be synthesised in neurons of the enteric nervous system (ENS), but evidence for its role as an enteric neurotransmitter is equivocal. We therefore aimed to determine whether GAD 65 and GAD 67 messenger RNAs (mRNAs) and proteins were expressed in the ileum of mice, rats and guinea pigs. Using an RNase protection assay, both GAD 65 and GAD 67 mRNAs were detected in the rodent small intestine. Antisera specific for GAD 65 or GAD 67, used in immunoblot analyses, revealed GAD 65-like and GAD 67-like immunoreactivity in rat and guinea pig ileum. Anti-GAD 65 antisera detected a major band of 65 kDa. Anti-GAD 67 antisera detected a major band of 55 kDa, which probably represented a breakdown product, and a minor band of 67 kDa. Analysis of immunoblot extracts of rat and guinea pig ileum revealed more GAD 67-like than GAD 65-like immunoreactivity. GAD enzymatic activity was high in the rat and guinea-pig brain, and low in the whole and dissected ileum. These results demonstrate that both GAD 65 and GAD 67 genes are transcribed and translated in the ileum of three rodent species and lend indirect support to the postulate that GABA is synthesised by neurons of the ENS and intestinal endocrine cells.