In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes.

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick-translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling.

Homeo boxes in the study of development.

The body plan of Drosophila is determined to a large extent by homeotic genes, which specify the identity and spatial arrangement of the body segments. Homeotic genes share a characteristic DNA segment, the homeo box, which encodes a defined domain of the homeotic proteins. The homeo domain seems to mediate the binding to specific DNA sequences, whereby the homeotic proteins exert a gene regulatory function. By isolating the normal Antennapedia gene, fusing its protein-coding sequences to an inducible promoter, and reintroducing this fusion gene into the germline of flies, it has been possible to transform head structures into thoracic structures and to alter the body plan in a predicted way. Sequence homologies suggest that similar genetic mechanisms may control development in higher organisms.

Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila.

The Drosophila gene eyeless (ey) encodes a transcription factor with both a paired domain and a homeodomain. It is homologous to the mouse Small eye (Pax-6) gene and to the Aniridia gene in humans. These genes share extensive sequence identity, the position of three intron splice sites is conserved, and these genes are expressed similarly in the developing nervous system and in the eye during morphogenesis. Loss-of-function mutations in both the insect and in the mammalian genes have been shown to lead to a reduction or absence of eye structures, which suggests that ey functions in eye morphogenesis. By targeted expression of the ey complementary DNA in various imaginal disc primordia of Drosophila, ectopic eye structures were induced on the wings, the legs, and on the antennae. The ectopic eyes appeared morphologically normal and consisted of groups of fully differentiated ommatidia with a complete set of photoreceptor cells. These results support the proposition that ey is the master control gene for eye morphogenesis. Because homologous genes are present in vertebrates, ascidians, insects, cephalopods, and nemerteans, ey may function as a master control gene throughout the metazoa.

Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans.

A Drosophila gene that contains both a paired box and a homeobox and has extensive sequence homology to the mouse Pax-6 (Small eye) gene was isolated and mapped to chromosome IV in a region close to the eyeless locus. Two spontaneous mutations, ey2 and eyR, contain transposable element insertions into the cloned gene and affect gene expression, particularly in the eye primordia. This indicates that the cloned gene encodes ey. The finding that ey of Drosophila, Small eye of the mouse, and human Aniridia are encoded by homologous genes suggests that eye morphogenesis is under similar genetic control in both vertebrates and insects, in spite of the large differences in eye morphology and mode of development.

Occurrence of sialic acids in Drosophila melanogaster.

Sialylated oligosaccharides, which are cell type-specific and developmentally regulated, have been implicated in a variety of complex biological events. Their broad functional importance is reflected by their presence in a wide variety of phyla extending from Echinodermata through higher vertebrates. Here, sialic acids are detected throughout development in an insect, Drosophila. Homopolymers of alpha 2,8-linked sialic acid, polysialic acid, are developmentally regulated and only expressed during early Drosophila development.

Expression and function of the segmentation gene fushi tarazu during Drosophila neurogenesis.

Segmentation genes control cell identities during early pattern formation in Drosophila. One of these genes, fushi tarazu (ftz), is now shown also to control cell fate during neurogenesis. Early in development, ftz is expressed in a striped pattern at the blastoderm stage. Later, it is transiently expressed in a specific subset of neuronal precursor cells, neurons (such as aCC, pCC, RP1, and RP2), and glia in the developing central nervous system (CNS). The function of ftz in the CNS was determined by creating ftz mutant embryos that express ftz in the blastoderm stripes but not in the CNS. In the absence of ftz CNS expression, some neurons appear normal (for example, the aCC, pCC, and RP1), whereas the RP2 neuron extends its growth cone along an abnormal pathway, mimicking its sibling (RP1), suggesting a transformation in neuronal identity.

The homeobox in perspective.

The discovery of the homeobox marks the beginning of a new era in developmental biology in which a class of master control genes, which determine the body plan, have been identified. Their mechanism of action can now be studied at the molecular level and their occurrence seems to be much more universal than originally anticipated.

Developmental defects in brain segmentation caused by mutations of the homeobox genes orthodenticle and empty spiracles in Drosophila.

We have studied the roles of the homeobox genes orthodenticle (otd) and empty spiracles (ems) in embryonic brain development of Drosophila. The embryonic brain is composed of three segmental neuromeres. The otd gene is expressed predominantly in the anterior neuromere; expression of ems is restricted to the two posterior neuromeres. Mutation of otd eliminates the first (protocerebral) brain neuromere. Mutation of ems eliminates the second (deutocerebral) and third (tritocerebral) neuromeres. otd is also necessary for development of the dorsal protocerebrum of the adult brain. We conclude that these homeobox genes are required for the development of specific brain segments in Drosophila, and that the regionalized expression of their homologs in vertebrate brains suggests an evolutionarily conserved program for brain development.

New perspectives on eye evolution.

The highly complex eyes of vertebrates, insects and molluscs have long been considered to be of independent evolutionary origin. Recently, however, Pax-6, a highly conserved transcription factor, has been identified as a key regulator of eye development in both mammals and flies. Homologues of Pax-6 have also been identified in species from other phyla, including molluscs. The wide variety of eyes in the animal kingdom may, therefore, have evolved from a single ancestral photosensitive origin.

Prefrontal-cingulate interactions in action monitoring.

We found that medial frontal cortex activity associated with action monitoring (detecting errors and behavioral conflict) depended on activity in the lateral prefrontal cortex. We recorded the error-related negativity (ERN), an event-related brain potential proposed to reflect anterior cingulate action monitoring, from individuals with lateral prefrontal damage or age-matched or young control participants. In controls, error trials generated greater ERN activity than correct trials. In individuals with lateral prefrontal damage, however, correct-trial ERN activity was equal to error-trial ERN activity. Lateral prefrontal damage also affected corrective behavior. Thus the lateral prefrontal cortex seemed to interact with the anterior cingulate cortex in monitoring behavior and in guiding compensatory systems.

The anterior cingulate cortex lends a hand in response selection.

The anterior cingulate cortex is involved in decisions between conflicting response tendencies. This executive function seems to involve separate pathways for manual and verbal responses.

Pax 6: mastering eye morphogenesis and eye evolution.

Pax 6 genes from various animal phyla are capable of inducing ectopic eye development, indicating that Pax 6 is a master control gene for eye morphogenesis. It is proposed that the various eye-types found in metazoa are derived from a common prototype, monophyletically, by a mechanism called intercalary evolution.

The structure of the homeodomain and its functional implications.

The three-dimensional structure of the homeodomain, as determined by nuclear magnetic resonance spectroscopy, reveals the presence of a helix-turn-helix motif, similar to the one found in prokaryotic gene regulatory proteins. Isolated homeodomains bind with high affinity to specific DNA sequences. Thus, the structure-function relationship is highly conserved in evolution.

Sequence requirement for expression of the Drosophila melanogaster heat shock protein hsp22 gene during heat shock and normal development.

A 14-base-pair sequence element present in almost all Drosophila melanogaster heat shock genes has been implicated in the heat inducibility of transcription. The D. melanogaster gene encoding the smallest heat shock protein, hsp22, contains within its 5' flanking sequences three such repeats, two close to the transcription start site and a distally located third one 101 base pairs further upstream. Deletion analyses reveal that the 5' flanking sequences required for full expression of the hsp22 gene extend beyond the distal repeat. Deletion of the furthest upstream repeat results in a five to sixfold reduction of gene expression. The small heat shock genes are transiently expressed in the late third instar larval and early pupal stages without external stimulation. A deletion of 5' flanking sequences to position -194, which includes two nucleotides of the distal heat shock element, has no effect on the developmental expression, whereas removal of an additional 18 nucleotides, including 12 nucleotides of the distal heat shock element, severely reduces developmental expression.

Functions of the medial frontal cortex in the processing of conflict and errors.

A principal function of the medial frontal cortex, in particular the anterior cingulate cortex (ACC), is to monitor action. The error-related negativity (ERN, or N(E)), an event-related brain potential, reflects medial frontal action-monitoring processes. Specifically, the error-detection theory of the ERN states that the ERN reflects ACC processing that is directly related to detecting the error. This theory predicts that ERN and ACC activity should increase directly with the dissimilarity of the error from the correct response, with similarity defined with respect to the common movement features of the responses. In contrast, the conflict-detection theory claims that ACC and ERN activity represent the detection of response conflict. This theory predicts that the activity should increase directly with the similarity of the error and the correct response. To test these theories, we investigated the effects of response similarity and conflict on the ERN, using a task that involved hand and foot movements. ERN activity was largest under conditions of high response conflict, where the error was similar to the correct response. This finding favors the conflict-detection theory over the error-detection theory, although the ERN was not associated with posterror slowing, as predicted by proponents of both theories. Discrepancies between our results and those of past studies may stem from the use in previous studies of four-finger response tasks which are subject to unique physiological and biomechanical constraints. We conclude that the ERN reflects medial frontal activity involved in the detection or affective processing of response conflict.

Homeodomain proteins in development and therapy.

Homeobox genes encode transcriptional regulators found in all organisms ranging from yeast to humans. In Drosophila, a specific class of homeobox genes, the homeotic genes, specifies the identity of certain spatial units of development. Their genomic organization, in Drosophila, as well as in vertebrates, is uniquely connected with their expression which follows a 5'-posterior-3'-anterior rule along the longitudinal body axis. The 180-bp homeobox is part of the coding sequence of these genes, and the sequence of 60 amino acids it encodes is referred to as the homeodomain. Structural analyses have shown that homeodomains consist of a helix-turn-helix motif that binds the DNA by inserting the recognition helix into the major groove of the DNA and its amino-terminal arm into the adjacent minor groove. Developmental as well as gene regulatory functions of homeobox genes are discussed, with special emphasis on one group, the Antennapedia (Antp) class homeobox genes and a representative 60-amino acid Antennapedia peptide (pAntp). In cultured neuronal cells, pAntp translocates through the membrane specifically and efficiently and accumulates in the nucleus. The internalization process is followed by a strong induction of neuronal morphological differentiation, which raises the possibility that motoneuron growth is controlled by homeodomain proteins. It has been demonstrated that chimeric peptide molecules encompassing pAntp are also captured by cultured neurons and conveyed to their nuclei. This may be of enormous interest for the internalization of drugs.

Nuclear magnetic resonance solution structure of the fushi tarazu homeodomain from Drosophila and comparison with the Antennapedia homeodomain.

The three-dimensional structure of a recombinant 70-residue polypeptide containing the complete fushi tarazu (ftz) homeodomain from Drosophila melanogaster has been determined by nuclear magnetic resonance (NMR) spectroscopy in solution. On the basis of 915 upper distance constraints derived from nuclear Overhauser effects and 178 dihedral angle constraints, a group of 20 conformers representing the solution structure of the ftz homeodomain was computed with the program DIANA and energy-minimized with the program OPAL. The average of the pairwise root-mean-square deviations of the individual NMR conformers relative to the mean coordinates is 0.50 A for the backbone atoms N, C alpha and C' of residues 8 to 53. The molecular architecture includes three helices comprising the residues 10 to 21, 28 to 38, and 42 to 52, a loop of residues 22 to 27 between the helices I and II, and a turn of residues 39 to 41 linking the helices II and III. Comparisons with the structure of the mutant Antennapedia homeodomain with Cys39 replaced by Ser, Antp (C39S), shows that the two proteins contain the same molecular fold for residues 8 to 53, whereas the more flexible fourth helix comprising residues 53 to 59 in the Antp (C39S) homeodomain has no counterpart in the ftz homeodomain. Considering that important intermolecular interactions in the DNA complexes with the Antp, engrailed and Mat alpha 2 homeodomains involve the fourth helix, it was rather unexpected that the stability of the complex of ftz with the BS2 operator site was found to be comparable to or even somewhat higher than that of the Antp complex with BS2. Another difference is that the Antp homeodomain is more stable with respect to thermal denaturation, with denaturation temperatures at pH 4.8 of 27 degrees C and 48 degrees C, respectively, for ftz and Antp.

Determination of the three-dimensional structure of the Antennapedia homeodomain from Drosophila in solution by 1H nuclear magnetic resonance spectroscopy.

The determination of the three-dimensional structure of the Antennapedia homeodomain from Drosophila in solution is described. The techniques used are 1H nuclear magnetic resonance spectroscopy for the data collection, and calculation of the protein structure with the program DISMAN followed by restrained energy minimization with a modified version of the program AMBER. A group of 19 conformers characterizes a well-defined structure for residues 7 to 59, with an average root-mean-square distance from the backbone atoms of 0.6 A relative to the mean of the 19 structures. The structure contains a helix from residues 10 to 21, a helix-turn-helix motif from residues 28 to 52, which is similar to those reported for several prokaryotic repressor proteins, and a somewhat flexible fourth helix from residues 53 to 59, which essentially forms an extension of the presumed recognition helix, residues 42 to 52. The helices enclose a structurally well-defined molecular core of hydrophobic amino acid side-chains.

Characterization and expression analysis of an ancestor-type Pax gene in the hydrozoan jellyfish Podocoryne carnea.

We characterized a Pax gene from the hydrozoan Podocoryne carnea. It is most similar to cnidarian Pax-B genes and encodes a paired domain, a homeodomain and an octapeptide. Expression analysis demonstrates the presence of Pax-B transcripts in eggs, the ectoderm of the planula larva and in a few scattered cells in the apical polyp ectoderm. In developing and mature medusae, Pax-B is localized in particular endodermal cells, oriented toward the outside. Pax-B is not expressed in muscle cells. However, if isolated striated muscle tissue is activated for transdifferentiation, the gene is expressed within 1 h, before new cell types, such as smooth muscle and nerve cells, have formed. The expression data indicate that Pax-B is involved in nerve cell differentiation.