Early development of the Drosophila mushroom body: the roles of eyeless and dachshund.

The mushroom body (MB) is a uniquely identifiable brain structure present in most arthropods. Functional studies have established its role in learning and memory. Here we describe the early embryonic origin of the four neuroblasts that give rise to the mushroom body and follow its morphogenesis through later embryonic stages. In the late embryo, axons of MB neurons lay down a characteristic pattern of pathways. eyeless (ey) and dachshund (dac) are expressed in the progenitor cells and neurons of the MB in the embryo and larva. In the larval brains of the hypomorphic ey(R) strain, we find that beside an overall reduction of MB neurons, one MB pathway, the medial lobe, is malformed or missing. Overexpression of eyeless in MBs under the control of an MB-specific promoter results in a converse type of axon pathway abnormality, i.e. malformation or loss of the dorsal lobe. In contrast, loss of dachshund results in deformation of the dorsal lobe, whereas no lobe abnormalities can be detected following dachshund overexpression. These results indicate that ey and dachshund may have a role in axon pathway selection during embryogenesis.

Phenotypic determination of epithelial appendages: genes, developmental pathways, and evolution.

Epithelial appendages are derivatives of epithelia that elaborate to form specialized structures and functions. The appendage can protrude out, such as in teeth and feathers, or invaginate in, such as in glands. The epithelia can be ectodermal, such as in hairs, or endodermal, such as in livers. Using feather as a prototype of epithelial appendage, we study the molecular signals involved in the successive stages of epithelial-mesenchymal interactions during morphogenesis. We propose that these form the basics of gene networks, which can be integrated to gene supernetwork and totinetwork. Because the unit of development is molecular pathway rather than single molecule, and the unit of morphogenesis is cell group rather than single cell, we make the analogy between genes/developmental pathways and words/sentences. The study of developmental pathways in epithelial appendage organogenesis will help us to understand the grammar of genes and the basic rules in constructing regulated new growth. This knowledge may contribute to the study of cancer biology (deregulated new growth) and organ regeneration.

Embryonic development of the Drosophila brain. I. Pattern of pioneer tracts.

The neuropile of the late embryonic Drosophila brain can be subdivided into a vertical component (cervical connective), a transverse component (supraesophageal commissure), and a horizontal component for which we propose the term protocerebral connective. The core of each neuropile component is formed by numerous axon fascicles, the trajectory of which follows an invariant pattern. In the present study we have used an antibody against the adhesion molecule Fasciclin II (FasII) that is expressed in a large number of early differentiating neurons of the Drosophila embryo to follow the development of the axon tracts of the brain. The FasII antigen appears on the surface of clusters of neuronal somata prior to axon outgrowth. These clusters, for which we propose the term fibre tract founder clusters, are laid out in a linear pattern that forms an almost uninterrupted longitudinal track reaching from the ventral nerve cord to the "tip" of the brain. After expressing FasII on their soma, neurons of the fibre tract founder clusters extend axons that grow along the surface of the founder clusters and form a simple system of pioneer tracts for each of the components of the brain neuropile. We have reconstructed the FasII-positive fibre tract founder clusters and their axons from optical sections and generated digital 3-D models that illustrate the spatial relationships of the pioneer tracts. Three fibre tract founder clusters, D/T, P1, and P3m, pioneer the cervical connective. P21 and P2m form a transverse track that pioneers the supraesophageal commissure. P4m and P41/P51/VP5m form two tracts that pioneer a medial and a lateral component of the protocerebral connective, respectively. Because FasII expression continues uninterruptedly into the larval period when the "rudiments" of many parts of the adult neuropile are readily identifiable, it was possible to assign several of the embryonic pioneer tracts to definitive neuropile components, including the median bundle, antennocerebral tract, mushroom body, and posterior optic tract.

Molecular histology in skin appendage morphogenesis.

Classical histological studies have demonstrated the cellular organization of skin appendages and helped us appreciate the intricate structures and function of skin appendages. At this juncture, questions can be directed to determine how these cellular organizations are achieved. How do cells rearrange themselves to form the complex cyto-architecture of skin appendages? What are the molecular bases of the morphogenesis and histogenesis of skin appendages? Recently, many new molecules expressed in a spatial and temporal specific manner during the formation of skin appendages were identified by molecular biological approaches. In this review, novel molecular techniques that are useful in skin appendage research are discussed. The distribution of exemplary molecules from different categories including growth factors, intracellular signaling molecules, homeobox genes, adhesion molecules, and extracellular matrix molecules are summarized in a diagram using feather and hair as models. We hope that these results will serve as the ground work for completing the molecular mapping of skin appendages which will refine and re-define our understanding of the developmental process beyond relying on morphological criteria. We also hope that the listed protocols will help those who are interested in this venture. This new molecular histology of skin appendages is the foundation for forming new hypotheses on how molecules are mechanistically involved in skin appendage development and for designing experiments to test them. This may also lead to the modulation of healing and regeneration processes in future treatment modalities.

FGF induces new feather buds from developing avian skin.

Induction of skin appendages involves a cascade of molecular events. The fibroblast growth factor (FGF) family of peptide growth factors is involved in cell proliferation and morphogenesis. We explored the role of the FGFs during skin appendage induction using developing chicken feather buds as a model. FGF-1, FGF-2, or FGF-4 was added directly to the culture medium or was released from pre-soaked Affigel blue beads. Near the midline, FGFs led to fusion of developing feather buds, representing FGFs' ability to expand feather bud domains in developing skin. In lateral regions of the explant where feather placodes have not formed, FGF treatment produces a zone of condensation and a region with an increased number of feather buds. In ventral epidermis that is normally apteric (without feathers), FGFs can also induce new feather buds. Like normal feather buds, the newly induced buds express Shh. The expression of Grb, Ras, Raf, and Erk, intracellular signaling molecules known to be downstream to tyrosine kinase receptors such as the FGF receptor, was enriched in feather bud domains. Genistein, an inhibitor of tyrosine kinase, suppressed feather bud formation and the effect of FGF. These results indicate that there are varied responses to FGFs depending on epithelial competence. All the phenotypic responses, however, show that FGFs facilitate the formation of skin appendage domains.

cAMP, an activator of protein kinase A, suppresses the expression of sonic hedgehog.

In Drosophila, it has been shown that protein kinase A and hedgehog have antagonistic actions during the formation of imaginal disks. In vertebrate skin, sonic hedgehog is expressed specifically in the feather bud epithelia. using an in vitro explant culture model we showed that dibutyryl cAMP, a protein kinase A (PKA) activator, suppresses the expression of Sonic hedgehog, (Shh) and continuous feather growth. The results suggest that Shh and PKA also have antagonistic action during vertebrate skin morphogenesis.

Protein kinase A and protein kinase C modulators have reciprocal effects on mesenchymal condensation during skin appendage morphogenesis.

The molecular signaling of secondary induction is a fundamental process in organogenesis during embryonic development. To study the signal transduction pathways involved, we used developing chicken skin as a model and focused on the roles of intracellular signaling during feather morphogenesis. Protein kinase C (PKC) immunoreactivity increases in the whole layer of forming dermis around H and H stage 30. This is followed by a gradual and highly localized decrease of PKC expression immediately beneath each forming feather germ. In contrast, cAMP response element binding protein (CREB) is ubiquitously expressed in both epithelium and mesenchyme. From stage 29 on, phosphorylated CREB (P-CREB), reflecting the activity of protein kinase A (PKA), begins to be seen in placode but not in interplacode epithelia. P-CREB is also expressed in bud mesenchyme transiently between stages 33 and 36, but not in the interbud mesenchyme. The presence and activity of PKC, PKA, and P-CREB in developing chicken skin are further characterized by immunoblot, kinase activity, and gel shift assays. To explore their physiological significance, embryonic chicken dorsal skin explants were treated with different modulators in medium or in beads for localized effects. The results showed that PKA activators and PKC inhibitors can expand a feather bud domain by enhancing dermal condensation, while PKC activators and PKA inhibitors can expand interbud domains. Neural cell adhesion molecule (N-CAM) is involved in dermal condensation. We observed that activation of PKA causes diffused expression of N-CAM in mesenchyme while activation of PKC causes the disappearance of N-CAM in precondensed mesenchymal regions. A model of how the well-concerted PKA and PKC signaling may be involved in the formation and size regulation of dermal condensation is presented.

Homeobox genes Msx-1 and Msx-2 are associated with induction and growth of skin appendages.

The mechanism involved in the morphogenesis of skin appendages is a fundamental issue underlying the development and healing of skin. To identify molecules involved in the induction and growth of skin appendages, we studied the expression of two homeobox genes, Msx-1 and Msx-2, during embryonic chicken skin development. We found that i) both Msx-1 and Msx-2 are early markers of epithelial placodes for skin appendages; ii) both Msx-1 and Msx-2 are expressed in the growing feather bud epithelia but not in the interbud epithelia; iii) although mostly overlapping, there are differences between the expression of the two Msx genes, Msx-1 being expressed more toward the anterior whereas Msx-2 is expressed more toward the distal feather bud; iv) there is no body-position-specific expression pattern as was observed for members of the Hox A-D clusters; v) in the feather follicle, Msx-1 and 2 are expressed in the collar and barb ridge epithelia, both regions of continuous cell proliferation; vi) when feather-bud growth was inhibited by forskolin, an activator of adenylyl cyclase, the expression of both genes was reduced. These results showed that Msx genes are specifically expressed in epithelial domains destined to become skin appendages. Its function in skin-appendage morphogenesis may be twofold, first in making epithelial cells competent to become skin appendages and, second, in making epithelial cells maintain their potential for continuous growth.

Neurofilament protein heterotetramers as assembly intermediates.

Evidence is presented for the existence of a soluble heterotetramer containing the low and middle molecular weight neurofilament (NF) proteins, NF-L and NF-M, and one containing the low and high molecular weight proteins, NF-L and NF-H, and for their role in filament assembly. When a mixture of either pair of proteins was renatured in 2 M urea, 20 mM Tris, pH 7.2, a new band representing a complex was observed in native gel electrophoresis. No new band was observed with a mixture of NF-M and NF-H. Two-dimensional gel electrophoresis showed that treatment of the complexes with SDS caused them to dissociate into their constituent polypeptide chains. Native neurofilaments dissociated in 2 M urea into a mixture of LM and LH complexes. Titration of NF-L with NF-M indicated that complex formation was complete at an approximately equimolar ratio of the two proteins. The LM complex had a sedimentation coefficient, s20,w, of 4.4 S, consistent with a tetrameric structure. Dialysis of a solution of the LM complex against 50 mM 4-morpholineethanesulfonic acid, 0.17 M NaCl, pH 6.25, led to the formation of 10-nm filaments in good yield. These results suggest that NF protein heterooligomers are intermediates in NF assembly and disassembly.

Genomic structure, chromosomal location, and evolution of the mouse Hox 8 gene.

We isolated genomic clones containing the mouse Hox 8 gene, a member of the msh gene family. We show that Hox 8 comprises two exons of approximately 600 and 691 bp separated by a 3.5-kb intron, and that it cosegregates with previously mapped markers in the distal region of mouse chromosome 13. In midgestation embryos, the Hox 8 gene produces transcripts of 1.4 and 2.2 kb. Both transcripts are present in facial tissues of the newborn mouse, though the ratio of the 2.2-kb transcript to the 1.4-kb transcript is reduced relative to the ratio observed for midgestation embryos. An alignment of the homeobox sequences of previously characterized members of the msh family revealed three subclasses: Hox 7-like genes, Hox 8-like genes, and msh-like genes. Both the Hox 7-like genes and Hox 8-like genes are present throughout the vertebrates. Representatives of the third subclass, the msh-like genes, are found in a protostome (Drosophila) and a deuterostome (Ciona) and are thus likely to be phylogenetically widespread. To investigate the distribution of Hox 8-like genes outside the chordates, we used the polymerase chain reaction and degenerate Hox 8 primers to screen genomic DNA of the purple sea urchin (Strongylocentrotus purpuratus, Phylum Echinodermata). We isolated a gene with greater sequence similarity to mouse Hox 8 than to members of the Hox 7 or msh subfamilies, demonstrating that the Hox 8 subfamily has been in existence at least since the echinoderms diverged from the lineage that gave rise to the chordates.(ABSTRACT TRUNCATED AT 250 WORDS)