Sonic hedgehog control of size and shape in midbrain pattern formation.

Little is known about how patterns of cell types are organized to form brain structures of appropriate size and shape. To study this process, we employed in vivo electroporation during midbrain development to create ectopic sources of Sonic Hedgehog, a signaling molecule previously shown to specify different neuronal cell types in a concentration-dependent manner in vitro. We provide direct evidence that a Sonic Hedgehog source can control pattern at a distance in brain development and demonstrate that the size, shape, and orientation of the cell populations produced depend on the geometry of the morphogen source. Thus, a single regulatory molecule can coordinate tissue size and shape with cell-type identity in brain development.

Patterning the mammalian cerebral cortex.

When and how is the area map of the cerebral cortex set up during development? Recent studies indicate that regional pattern emerges early in cortical neurogenesis, and that this pattern does not require cues from extrinsic innervation. Studies of mutant mice indicate a role for embryonic signaling centers and for specific transcription factors in regionalizing the cortex. Thus, it is increasingly probable that the cortex is partitioned using the same types of mechanisms--and in some cases, the same gene families--that are used in patterning other parts of the embryo. This emerging model is likely to be the basis for many future studies. However, new evidence also confirms the special nature of the cerebral cortex, in that cues from developing connections appear to modify and refine the final area map.

Dorsoventral patterning of the telencephalon is disrupted in the mouse mutant extra-toes(J).

Little is known about the mechanisms that control the development of regional identity in the mammalian telencephalon. The Gli family of transcription factor genes is involved in the regulation of pattern at many sites in the embryo and is expressed in the embryonic mouse telencephalon. We have analyzed telencephalic patterning in the extra-toes (J) (Xt(J)) mouse mutant, which carries a deletion in the Gli family member Gli3. We report that dorsoventral patterning of the telencephalon is dramatically disrupted in the Xt(J) mutant. Specific dorsal telencephalic cell types and gene expression patterns are lost in homozygous Xt(J) mutants, and features of ventral telencephalic identity develop ectopically in the dorsal telencephalon. This partial ventralization of the dorsal telencephalon does not appear to be induced by an expansion of Sonic hedgehog expression in the telencephalon, but may be due to a loss of Bmp and Wnt gene expression in a putative dorsal telencephalic signaling center, the cortical hem. Our findings suggest that in dorsal telencephalon Gli3 is needed to repress ventral telencephalic identity.

The hem of the embryonic cerebral cortex is defined by the expression of multiple Wnt genes and is compromised in Gli3-deficient mice.

In the developing vertebrate CNS, members of the Wnt gene family are characteristically expressed at signaling centers that pattern adjacent parts of the neural tube. To identify candidate signaling centers in the telencephalon, we isolated Wnt gene fragments from cDNA derived from embryonic mouse telencephalon. In situ hybridization experiments demonstrate that one of the isolated Wnt genes, Wnt7a, is broadly expressed in the embryonic telencephalon. By contrast, three others, Wnt3a, 5a and a novel mouse Wnt gene, Wnt2b, are expressed only at the medial edge of the telencephalon, defining the hem of the cerebral cortex. The Wnt-rich cortical hem is a transient, neuron-containing, neuroepithelial structure that forms a boundary between the hippocampus and the telencephalic choroid plexus epithelium (CPe) throughout their embryonic development. Indicating a close developmental relationship between the cortical hem and the CPe, Wnt gene expression is upregulated in the cortical hem both before and just as the CPe begins to form, and persists until birth. In addition, although the cortical hem does not show features of differentiated CPe, such as expression of transthyretin mRNA, the CPe and cortical hem are linked by shared expression of members of the Bmp and Msx gene families. In the extra-toesJ (XtJ) mouse mutant, telencephalic CPe fails to develop. We show that Wnt gene expression is deficient at the cortical hem in XtJ/XtJ mice, but that the expression of other telencephalic developmental control genes, including Wnt7a, is maintained. The XtJ mutant carries a deletion in Gli3, a vertebrate homolog of the Drosophila gene cubitus interruptus (ci), which encodes a transcriptional regulator of the Drosophila Wnt gene, wingless. Our observations indicate that Gli3 participates in Wnt gene regulation in the vertebrate telencephalon, and suggest that the loss of telencephalic choroid plexus in XtJ mice is due to defects in the cortical hem that include Wnt gene misregulation.

Isoform-specific immunological detection of newt retinoic acid receptor delta 1 in normal and regenerating limbs.

Retinoic acid (RA) exerts a variety of effects on the regenerating urodele limb including positional respecification of the blastema. The major RA receptor expressed in the newt limb and blastema is the delta 1 isoform and, in order to detect delta 1 in this context, we have made five affinity-purified antibodies against fusion proteins and peptides from non-overlapping regions of the molecule. These antibodies have been evaluated by reaction with transfected COS-7 cells, newt limb cells in culture and newt limb tissue sections. The most informative antibodies were RP6, directed against N-terminal region A sequence, and RP8, directed against C-terminal sequence. In western blots of blastemal extracts, delta 1 protein was detected as two major bands of immunoreactivity at positions consistent with the employment of two candidate methionine initiators identified by cDNA sequencing. Staining of adult limb sections with RP6 and RP8 showed reactivity in half of the nuclei in epidermal and mesenchymal tissues, a heterogeneity that was observed with adjacent nuclei in muscle fibres. In the regenerating limb, nuclei in the blastemal mesenchyme and wound epidermis were strongly reactive, although no axial variation in expression was detected.

Delta retinoic acid receptor isoform delta 1 is distinguished by its exceptional N-terminal sequence and abundance in the limb regeneration blastema.

In amphibian limb regeneration memory for position in the proximal-distal axis can be respecified by retinoic acid. The favoured candidates to mediate this effect are the retinoic acid receptors (RARs) and of the RARs identified in the regeneration blastema, the delta receptor is the most abundant. The presence in blastemal mesenchyme of at least two delta receptor isoforms, delta 1 and delta 2, alternatively spliced at the A-B junction, was demonstrated in expression studies and by PCR cloning. The delta 1 receptor is abundant in regenerative structures such as the limb and tail, whereas the delta 2 and alpha receptors show a more uniform pattern of expression across adult newt tissues. Full-length cloning of the delta 1 receptor established the presence of an unusually long open reading frame and N-terminal sequence that appears unique among vertebrate retinoic acid receptors. Transient transfection of expression constructs into COS cells followed by Western blotting confirmed the existence of at least three potential initiation sites for delta 1 translation. The possibility that delta 1 RAR expression may specify positional memory directly was tested in RNase protection experiments. delta 1 receptor message is increased on amputation, but does not exhibit a pronounced differential distribution along the proximal-distal axis in normal and regenerating limbs, nor does it show a persistent alteration in expression levels following a dose of retinoic acid sufficient to respecify position. The possibility that the morphogenetic effects of RA may be mediated through receptor interactions is raised by the finding that single mesenchymal blastemal cells in culture can express multiple RAR subtypes (delta 1 and alpha) and isoforms (delta 1 and delta 2).

Retinoids and their targets in vertebrate development.

The elaboration of the effect of retinoic acid on limb morphogenesis has prompted renewed investigation into the teratology of retinoic acid treatment, with the hope that such analysis might give insight into mechanisms of vertebrate patterning. Retinoids, their nuclear receptors and their cytoplasmic binding proteins are now known to be deployed throughout development, but the extent to which they are natural agents of morphogenesis remains obscure. The study of retinoic acid receptors may offer molecular insight into gene regulation underlying vertebrate pattern formation.

Compartmental organization of the thalamostriatal connection in the cat.

The compartmental organization of the thalamostriatal connection in the cat was studied by labelling thalamic fibers in anterograde axonal transport experiments and comparing their striatal distributions with the arrangement of striosomes and matrix tissue identified by histochemical staining methods. When analyzed according to their principal compartmental targets in dorsal striatum, the thalamic deposits indicated the existence of medial and lateral divisions within the thalamostriatal projection. Nuclei of the medial division, which includes parts of the thalamic midline, projected primarily to striosomes. The lateral division, which embraces the anterior and posterior intralaminar groups, the rostral ventral tier nuclei, and parts of the posterior lateral nuclear complex, predominantly innervated matrix tissue. In the dorsal division of the nucleus accumbens, the medial system preferentially terminated in zones that stain heavily in butyrylcholinesterase and substance P preparations, but fibers from both the medial and the lateral systems largely avoided the histochemically marked compartments such as the border islands of the nucleus accumbens that are seen elsewhere in the ventral striatum. Medial division: Thalamic deposits involving the paraventricular and rhomboid nuclei of the thalamic midline elicited labelling of striosomes and, invariably, ventral extrastriosomal matrix, the nucleus accumbens, and the amygdala. This projection was topographically organized: rostral thalamic deposits elicited labelling in the medial caudate nucleus and the medial nucleus accumbens. More caudal injections produced more lateral labelling. Lateral division: The lateral division is composed of at least three projection systems distinguished by their patterns of matrix innervation. Deposits involving the anterior intralaminar nuclei and the striatally projecting cells located lateral to the stria medullaris (anterior intralaminar complex) produced an even, diffuse labelling of the matrix tissue and weak labelling of the striosomes. Injections placed in the ventroanterior, ventrolateral, and ventromedial nuclei (rostral ventral complex) elicited fibrous labelling of matrix tissue that often showed nonstriosomal inhomogeneities. Deposits involving the centromedian and parafascicular nuclei (posterior intralaminar complex) produced a highly variable pattern of matrix labelling that included both homogeneous and decidedly patchy innervations of the extrastriosomal matrix. Each of these lateral thalamostriatal systems showed a similar spatial organization, whereby dorsoventral and mediolateral thalamic axes were roughly preserved in the projection to striatum.

A simple ordering of neocortical areas established by the compartmental organization of their striatal projections.

The compartmental organization of corticostriatal projections from the fronto-orbito-insular cortex was studied in the cat. Cortical areas in this field were found to have a highly organized projection to the striatum, selectively innervating striosomes dorsally and predominantly avoiding them ventrally within their striatal fields of termination. These observations have two important implications for striatal processing. First, some cortical areas preferentially terminate in different compartments in different parts of the striatum. Therefore, the sources of input to striosomes and matrix are not categorical but switch according to the striatal region considered. Second, three properties of the bicompartmental termination pattern--one-dimensionality, common polarization, and multiple positions at which the pattern switched from "fills" to "avoids"--allowed us to order the corticostriatal projections with respect to one another. This ordering of the striatal projections of cortical areas implies an ordering of the cortical areas themselves, one that is independent of transcortical connections. For the corticostriatal projections described in this report, the ordering is [parietal, dorsomedial prefrontal, ventrolateral prefrontal, insular, rostral temporal] cortex. Our analysis suggests that a major function of striatal compartmentalization is to segregate and then bring together inputs from cortical areas at different positions in this ordering. The ordering may also serve as a simple format for specifying corticostriatal connections in development.

Hepatic fibrosis with the use of methotrexate for juvenile rheumatoid arthritis.

Methotrexate (MTX) is currently under study for use in juvenile rheumatoid arthritis. One complication of MTX is hepatotoxicity. Although liver function tests may be abnormal with its use, in this setting they do not correlate well with the development of hepatic fibrosis. Periodic liver biopsy is required to monitor for the hepatotoxic changes secondary to MTX. We describe and discuss the case of a 17-year-old woman who developed evidence of hepatic fibrosis after 3 years of MTX therapy.

Predicting outcome at the knee in juvenile rheumatoid arthritis.

This study was undertaken to correlate long-term severe radiographic changes with clinical profiles in an attempt to identify a group of patients at risk. Knee radiographs of 100 juvenile rheumatoid arthritis (JRA) patients with 1-20 years of follow-up studies were reviewed for changes leading to severe disability. Eighteen children had evidence of destructive changes an average of 4.3 years after onset of JRA. 23 patients had reactive changes superimposed on destructive changes seen 9.7 years after onset of disease. 92% of the children with reactive and destructive knee changes had radiographic evidence of significant JRA at another joint. Polyarticular disease with significant JRA involvement of another joint seems to indicate a group of children at higher risk for destructive and reactive changes at the knee.

Identification of a novel retinoic acid receptor in regenerative tissues of the newt.

In urodele amphibians, the progenitor cells that regenerate amputated limbs (known as the blastema) normally replace only the missing structures. After systemic delivery of retinoic acid (RA), more proximal structures are also formed, indicating that RA can control position specification in the proximal-distal axis of the regenerating limb. According to dose and experimental context, retinoids can also re-specify the anteroposterior axis of the limb, induce deletions of skeletal elements, or block re-growth completely. To study the molecular basis of these morphogenetic effects, we screened complementary DNA libraries of newt regenerative tissues (limbs and tails) for hormone nuclear receptors activated by RA. Two functional retinoic acid receptors (RARs) were identified, one of which is the newt homologue of the human alpha-receptor (RAR alpha). The second receptor, called RAR delta, is novel. Sequence analysis suggests that the composite newt RAR previously reported is chimaeric, consisting of 5'RAR-beta-like and 3' RAR delta clones. We conclude that multiple RARs are expressed during limb regeneration in amphibians and suggest that receptor heterogeneity may underlie the different effects of retinoids on limb morphogenesis.

Connective tissue activation. XXXIII. Biologically active cleavage products of CTAP-III from human platelets.

Evidence for three new isoforms of CTAP-III from human platelets is presented; two NH2-terminal cleavage products were identified, CTAP-III (des 1-13) and CTAP-III (des 1-15). CTAP-III (des 1-13) has a pI of 8.6 and is a relatively stable proteolytic cleavage product that retains the capacity to stimulate [14C]GAG synthesis in human synovial cell cultures. CTAP-III (des 1-15) appears to be an elastase or chymotrypsin cleavage product and identical to NAP-2, an entity thought to have neutrophil activating properties.

Factor VIII related antigen and childhood rheumatic diseases.

Factor VIII related antigen (FVIIIRAg) levels were measured in the plasma of 63 children with rheumatic diseases and 20 controls. High levels were found in patients with systemic juvenile arthritis, systemic lupus erythematosus, dermatomyositis and systemic forms of vasculitis. The amount of circulating FVIIIRAg seemed to be independent of values for erythrocyte sedimentation rate, C-reactive protein and fibrinogen, implying that it was not just another acute phase reactant. Rather, a high level of circulating FVIIIRAg most likely reflects the presence of vascular endothelial injury, and this test may be useful in monitoring disease activity in children with rheumatic diseases in which vasculitis is present.