High-resolution, three-dimensional mapping of gene expression using GeneExpressMap (GEM).

The analysis of temporal and spatial patterns of gene expression is critically important for many kinds of developmental studies, including the construction of gene regulatory networks. Recently, multiplex, fluorescent, whole mount in situ hybridization (multiplex F-WMISH), applied in combination with confocal microscopy, has emerged as the method of choice for high-resolution, three-dimensional (3D) mapping of gene expression patterns in developing tissues. We have developed an image analysis tool, GeneExpressMap (GEM), that facilitates the rapid, 3D analysis of multiplex F-WMISH data at single-cell resolution. GEM assigns F-WMISH signal to individual cells based upon the proximity of cytoplasmic hybridization signal to cell nuclei. Here, we describe the features of GEM and, as a test of its utility, we use GEM to analyze patterns of regulatory gene expression in the non-skeletogenic mesoderm of the early sea urchin embryo. GEM greatly extends the power of multiplex F-WMISH for analyzing patterns of gene expression and is a valuable tool for gene network analysis and many other kinds of developmental studies.

Object relations in borderline adolescents.

Although pathological object relations is a core aspect of borderline psychopathology, few studies have examined borderline object relations empirically, and none has focused on borderline adolescents. The present study examined four dimensions of object relations, as measured by the Thematic Apperception Test, in a sample of adolescent borderlines, psychiatric comparison subjects, and normals. These dimensions are complexity of object representations, affect-tone of relationship paradigms, capacity for emotional investment in relationships and moral standards, and understanding of social causality. Borderlines differed significantly from both comparison groups in several distinct ways, supporting some aspects of psychoanalytic theories of borderline object relations, while challenging others. Borderline adolescents have a malevolent object world, a relative incapacity to invest in others in a non-need-gratifying way, and a tendency to attribute motivation to others in simple, illogical, and idiosyncratic ways. Their object representations, however, can be quite complex, suggesting something other than a preoedipal arrest.

Object relations in childhood and adolescence: the development of working representations.

This article describes two studies based on an integration of theory and research in object relations and developmental social cognition. The following four dimensions were assessed in 2nd and 5th graders and in 9th and 12th graders using Thematic Apperception Test (TAT) responses and Ss' descriptions of meaningful interpersonal episodes: complexity of representations of people, affect-tone of relationship paradigms, capacity for emotional investment in relationships and moral standards, and understanding of social causality. In both studies, developmental differences emerged on all measures except affect-tone, as expected. Results document object-relational development beyond the preoedipal years and suggest the diagnostic importance of distinguishing multiple dimensions of object relations rather than considering object relations as a single or unitary developmental line.

Physical and sexual abuse in adolescent girls with borderline personality disorder.

Recent investigations suggest that a history of childhood sexual abuse may be associated with borderline personality disorder. This study compares a group of female adolescents diagnosed as borderline with a control group for history of physical and sexual abuse. History of both forms of abuse, particularly sexual, distinguishes the two groups. Results point to the impact of repeated trauma on character structure, cognitive functioning, and Axis II symptomatology.

A clonal analysis of secondary mesenchyme cell fates in the sea urchin embryo.

The secondary mesenchyme cells (SMCs) give rise to most of the mesoderm of the sea urchin embryo. Although the early embryonic lineage of these cells has been described, the mechanisms that cause SMCs to become restricted to a particular mesodermal cell fate are unknown. To begin to address this question, we performed a clonal analysis of the fates of SMC precursors in the vegetal plate by labeling single cells with the fluorescent dye DiI (C18). Our data show that some presumptive SMCs remain pluripotent at the late blastula stage, since some cells labeled at this stage gave rise to more than one mesodermal cell type. Surprisingly, however, most labeled cells gave rise to homogeneous clones composed of a single cell type. This observation indicates that either many SMC precursors are restricted in their fate before the start of gastrulation or that all the progeny of a single vegetal plate cell are influenced by the same instructional signals during gastrulation, despite the cell divisions and extensive cell movements that occur during this time. The percentage of clones composed of a single cell type increased during the blastula stage, supporting the view that the process of SMC fate specification begins before the onset of gastrulation.

Mesodermal cell interactions in the sea urchin embryo: properties of skeletogenic secondary mesenchyme cells.

An interaction between the two principal populations of mesodermal cells in the sea urchin embryo, primary and secondary mesenchyme cells (PMCs and SMCs, respectively), regulates SMC fates and the process of skeletogenesis. In the undisturbed embryo, skeletal elements are produced exclusively by PMCs. Certain SMCs also have the ability to express a skeletogenic phenotype; however, signals transmitted by the PMCs direct these cells into alternative developmental pathways. In this study, a combination of fluorescent cell-labeling methods, embryo microsurgery and cell-specific molecular markers have been used to study the lineage, numbers, normal fate(s) and developmental potential of the skeletogenic SMCs. Previous fate-mapping studies have shown that SMCs are derived from the veg2 layer of blastomeres of the 64-cell-stage embryo and from the small micromeres. By specifically labeling the small micromeres with 5-bromodeoxyuridine, we demonstrate that descendants of these cells do not participate in skeletogenesis in PMC-depleted larvae, even though they are the closest lineal relatives of PMCs. Skeletogenic SMCs are therefore derived exclusively from the veg2 blastomeres. Because the SMCs are a heterogeneous population of cells, we have sought to gain information concerning the normal fate(s) of skeletogenic SMCs by determining whether specific cell types are reduced or absent in PMC(-) larvae. Of the four known SMC derivatives: pigment cells, blastocoelar (basal) cells, muscle cells and coelomic pouch cells, only pigment cells show a major reduction (> 50%) in number following SMC skeletogenesis. We therefore propose that the PMC-derived signal regulates a developmental switch, directing SMCs to adopt a pigment cell phenotype instead of a default (skeletogenic) fate. Ablation of SMCs at the late gastrula stage does not result in the recruitment of any additional skeletogenic cells, demonstrating that, by this stage, the number of SMCs with skeletogenic potential is restricted to 60-70 cells. Previous studies showed that during their switch to a skeletogenic fate, SMCs alter their migratory behavior and cell surface properties. In this study, we demonstrate that during conversion, SMCs become insensitive to the PMC-derived signal, while at the same time they acquire PMC-specific signaling properties.

A critical period for conversion of ectodermal cells to a neural crest fate.

Previously, we found that interactions between neural and nonneural ectoderm can generate neural crest cells, with both the ectodermal and the neuroepithelial cells contributing to induced population (M. A. J. Selleck and M. Bronner-Fraser, 1995, Development 121, 525-538). To further characterize the ability of ectodermal cells to form neural crest, we have challenged their normal fate by transplanting them into the neural tube. To ensure that the ectoderm was from nonneural regions, we utilized extraembryonic ectoderm (the proamnion) and transplanted it into the presumptive midbrain of 1. 5-day-old chick embryos. We observed that the grafted ectoderm has the capacity to adopt a neural crest fate, responding within a few hours of surgery by turning on neural crest markers HNK-1 and Slug. However, the competence of the ectoderm to respond to neural crest-inducing signals is time limited, declining rapidly in donors older than the 10-somite stage. Similarly, the inductive capacity of the host midbrain declines in a time-dependent fashion. Our results show that extraembryonic ectoderm has the capacity to form neural crest cells given proper inducing signals, expressing both morphological and molecular markers characteristic of neural crest cells.

A fate map of the vegetal plate of the sea urchin (Lytechinus variegatus) mesenchyme blastula.

Previous lineage tracing experiments have shown that the vegetal blastomers of cleavage stage embryos give rise to all the mesoderm and endoderm of the sea urchin larva. In these studies, vegetal blastomers were labeled no later than the sixth cleavage division (60-64 cell stage). In an earlier study we showed that single cells in the vegetal plate of the blastula stage Lytechinus variegatus embryo could be labeled in situ with the fluorescent, lipophilic dye, DiI(C18), and that cells labeled in the central region of the vegetal plate of the mesenchyme blastula primarily gave rise to homogeneous clones consisting of a single secondary mesenchyme cell (SMC) type (Ruffins and Ettensohn (1993) Dev. Biol. 160, 285-288). Our clonal labeling showed that a detailed fate map could be generated using the DiI(C18) labeling technique. Such a fate map could provide information about the spatial relationships between the precursors of specific mesodermal and endodermal cell types and information concerning the movements of these cells during gastrulation and later embryogenesis. We have used this method to construct the first detailed fate map of the vegetal plate of the sea urchin embryo. Ours is a latitudinal map; mapping from the plate center, where the mesodermal precursors reside, through the region which contains the endodermal precursors and across the ectodermal boundary. We found that the precursors of certain SMC types are segregated in the mesenchyme blastula stage vegetal plate and that prospective germ layers reside within specific boundaries. To determine whether the vegetal plate is radially symmetrical with respect to mesodermal cell fates, single blastomeres of four cell stage embryos were injected with lysyl-rhodamine dextran (LRD). The resulting ectodermal labeling patterns were classified and correlated with the SMC types labeled. This analysis indicates that the dorsal and ventral blastomers do not contribute equally to SMC derivatives in L. variegatus.

Early migrating neural crest cells can form ventral neural tube derivatives when challenged by transplantation.

Once neural crest cells undergo an epithelial-mesenchymal transition to leave the neural tube, it has been classically assumed that they are fated to differentiate within the neural crest lineage. To test this idea, we challenged the developmental potential of recently emigrated neural crest cells by transplanting them into the ventral portion of the neural tube at the open neural plate stage. Newly migrating neural crest cells were isolated in tissue culture, labeled with the lipophilic dye DiI, and microinjected into the ventral portion of the neural plate. After 2 days, some neural crest cells became incorporated into the neuroepithelium in positions characteristic of floor plate cells and motor neurons. Some of the labeled cells within the ventral neural tube expressed FP-1, characteristic of floor plate cells. A few labeled cells were found in positions characteristic of motor neurons and expressed islet-1. In contrast, neural crest cells transplanted onto neural crest pathways expressed the HNK-1 epitope, but no ventral neural tube markers. Injection of neural crest cells into the mesenchyme adjacent to the notochord or culturing them in the presence of Sonic hedgehog failed to elicit FP-1 expression. These results suggest that migrating neural crest cells are flexible in their fate and retain the ability to form neural tube derivatives even after emigrating from the neural tube.

Three-dimensional digital mouse atlas using high-resolution MRI.

We present an archetypal digital atlas of the mouse embryo based on microscopic magnetic resonance imaging. The atlas is composed of three modules: (1) images of fixed embryos 6 to 15.5 days postconception (dpc) [Theiler Stages (TS) 8 to 24]; (2) an annotated atlas of the anterior portion of a 13.5 dpc (TS 22) mouse with anatomical structures delineated and linked to explanatory files; and (3) three-dimensional renderings of the entire 13.5 dpc embryo and specific organ systems. The explanatory files include brief descriptions of the structure at each volume element in the image and links to 3D reconstructions, allowing visualization of the shape of the isolated structures. These files can also contain or be linked to other types of information and data including detailed anatomical and physiological information about structures with pointers to online references, relationships between structures, temporal characteristics (cell lineage patterns, size, and shape changes), and gene expression patterns (both spatial and temporal). As an example, we have "painted" in the expression pattern of Dlx5/Dlx6 genes. This digital atlas provides a means to put specific data within the context of normal specimen anatomy, to analyze the information in 3D, and to examine relationships between different types of information.

Dorsal hindbrain ablation results in rerouting of neural crest migration and changes in gene expression, but normal hyoid development.

Our previous studies have shown that hindbrain neural tube cells can regulate to form neural crest cells for a limited time after neural fold removal (Scherson, T., Serbedzija, G., Fraser, S. E. and Bronner-Fraser, M. (1993). Development 188, 1049-1061; Sechrist, J., Nieto, M. A., Zamanian, R. T. and Bronner-Fraser, M. (1995). Development 121, 4103-4115). In the present study, we ablated the dorsal hindbrain at later stages to examine possible alterations in migratory behavior and/or gene expression in neural crest populations rostral and caudal to the operated region. The results were compared with those obtained by misdirecting neural crest cells via rhombomere rotation. Following surgical ablation of dorsal r5 and r6 prior to the 10 somite stage, r4 neural crest cells migrate along normal pathways toward the second branchial arch. Similarly, r7 neural crest cells migrate primarily to the fourth branchial arch. When analogous ablations are performed at the 10-12 somite stage, however, a marked increase in the numbers of DiI/Hoxa-3-positive cells from r7 are observed within the third branchial arch. In addition, some DiI-labeled r4 cells migrate into the depleted hindbrain region and the third branchial arch. During their migration, a subset of these r4 cells up-regulate Hoxa-3, a transcript they do not normally express. Krox20 transcript levels were augmented after ablation in a population of neural crest cells migrating from r4, caudal r3 and rostral r3. Long-term survivors of bilateral ablations possess normal neural crest-derived cartilage of the hyoid complex, suggesting that misrouted r4 and r7 cells contribute to cranial derivatives appropriate for their new location. In contrast, misdirecting of the neural crest by rostrocaudal rotation of r4 through r6 results in a reduction of Hoxa-3 expression in the third branchial arch and corresponding deficits in third arch-derived structures of the hyoid apparatus. These results demonstrate that neural crest/tube progenitors in the hindbrain can compensate by altering migratory trajectories and patterns of gene expression when the adjacent neural crest is removed, but fail to compensate appropriately when the existing neural crest is misrouted by neural tube rotation.