Identification of a regulatory allele of teashirt (tsh) in Drosophila melanogaster that affects wing hinge development. An adult-specific tsh enhancer in Drosophila.

A cis-acting regulatory element defined herein is required to drive teashirt (tsh) expression in the regions of the developing adult that give rise to proximal wing and haltere tissues. Loss of this expression results in the fusion of the proximal structures of the wing and halteres to the thoracic cuticle. This represents the first description of a viable adult-specific regulatory allele of tsh with a visible phenotype, and it enlarges our understanding of the expression of tsh and its function during the development of the adult.

Altered retinoic acid sensitivity and temporal expression of Hox genes in polycomb-M33-deficient mice.

The Polycomb group genes are required for the correct expression of the homeotic complex genes and segment specification during Drosophila embryogenesis and larval development. In mouse, inactivation studies of several Polycomb group genes indicate that they are also involved in Hox gene regulation. We have used our previously generated M33 mutants to study the function of M33, the mouse homologue of the Polycomb gene of Drosophila. In this paper, we show that in the absence of M33, the window of Hoxd4 retinoic acid (RA) responsiveness is opened earlier and that Hoxd11 gene expression is activated earlier in development This indicates that M33 antagonizes the RA pathway and has a function in the establishment of the early temporal sequence of activation of Hox genes. Despite the early activation, A-P boundaries are correct in later stages, indicating a separate control mechanism for early aspects of Hox regulation. This raises a number of interesting issues with respect to the roles of both Pc-G proteins and Hox regulatory mechanisms. We propose that a function of the M33 protein is to control the accessibility of retinoic acid response elements in the vicinity of Hox genes regulatory regions by direct or indirect mechanisms or both. This could provide a means for preventing ectopic transactivation early in development and be part of the molecular basis for temporal colinearity of Hox gene expression.

Altered lymphoid development in mice deficient for the mAF4 proto-oncogene.

Some chromosomal translocations in acute leukemias involve the fusion of the trithorax-related protein Mll (also called HRX, All1, Htrx,) with a variety of heterologous proteins. In acute lymphoblastic leukemia associated with the t(4;11)(q21;q23) translocation, the 4q21 gene that fuses with Mll is AF4. To gain insight into the potential role of AF4 in leukemogenesis and development, this gene was inactivated by homologous recombination in mice. As expected from the tissue distribution of the AF4 transcript, development of both B and T cells is affected in AF4 mutant mice. A severe reduction of the thymic double positive CD4/CD8 (CD4(+)/CD8(+)) population was observed; in addition most double- and single-positive cells expressed lower levels of CD4 and CD8 coreceptors. Most importantly, the reconstitution of the double-positive compartment by expansion of the double-negative cell compartment was severely impaired in these mutant mice. In the bone marrow pre-B and mature B-cell numbers are reduced. These results demonstrate that the function of the mAF4 gene is critical for normal lymphocyte development. This raises the possibility that the disruption of the normal AF4 gene or its association with Mll function by translocation may orient the oncogenic process toward the lymphoid lineage. This represents the first functional study using a knock-out strategy on one of the Mll partner genes in translocation-associated leukemias. (Blood. 2000;96:705-710)

Vertebrate orthologues of the Drosophila region-specific patterning gene teashirt.

In Drosophila the teashirt gene, coding for a zinc finger protein, is active in specific body parts for patterning. For example, Teashirt is required in the trunk (thorax and abdomen) tagmata of the embryo, parts of the intestine and the proximal parts of appendages. Here we report the isolation of vertebrate cDNAs related to teashirt. As in Drosophila, human and murine proteins possess three widely spaced zinc finger motifs. Additionally, we describe the expression patterns of the two murine genes. Both genes show regionalized patterns of expression, in the trunk, in the developing limbs and the gut.

Genetic interactions and dosage effects of Polycomb group genes in mice.

In Drosophila and mouse, Polycomb group genes are involved in the maintenance of homeotic gene expression patterns throughout development. Here we report the skeletal phenotypes of compound mutants for two Polycomb group genes bmi1 and M33. We show that mice deficient for both bmi1 and M33 present stronger homeotic transformations of the axial skeleton as compared to each single Polycomb group mutant, indicating strong dosage interactions between those two genes. These skeletal transformations are accompanied with an enhanced shift of the anterior limit of expression of several Hox genes in the somitic mesoderm. Our results demonstrate that in mice the Polycomb group genes act in synergy to control the nested expression pattern of some Hox genes in somitic mesodermal tissues during development.

Altered cellular proliferation and mesoderm patterning in Polycomb-M33-deficient mice.

In Drosophila, the trithorax-group and the Polycomb-group genes are necessary to maintain the expression of the homeobox genes in the appropriate segments. Loss-of-function mutations in those groups of genes lead to misexpression of the homeotic genes resulting in segmental homeotic transformations. Recently, mouse homologues of the Polycomb-group genes were identified including M33, the murine counterpart of Polycomb. In this report, M33 was targeted in mice by homologous recombination in embryonic stem (ES) cells to assess its function during development. Homozygous M33 (-/-) mice show greatly retarded growth, homeotic transformations of the axial skeleton, sternal and limb malformations and a failure to expand in vitro of several cell types including lymphocytes and fibroblasts. In addition, M33 null mutant mice show an aggravation of the skeletal malformations when treated to RA at embryonic day 7.5, leading to the hypothesis that, during development, the M33 gene might play a role in defining access to retinoic acid response elements localised in the regulatory regions of several Hox genes.

Transcriptional regulation of the Drosophila homeotic gene teashirt by the homeodomain protein Fushi tarazu.

The Drosophila melanogaster gene teashirt (tsh) is essential for segment identity of the embryonic thorax and abdomen. A deletion 3' to the tsh transcription unit causes the loss of tsh early expression in the even-numbered parasegments, and the corresponding larval cuticular patterns are disrupted. tsh function in the odd-numbered parasegments in these mutants is normal by both criteria. The in vivo activities of genomic fragments from the deleted region were tested in transgenic embryos. A 2.0 kb enhancer from the 3' region acts mainly in the even-numbered parasegments and is dependent on fushi tarazu (ftz) activity, which encodes a homeodomain protein required for the development of even-numbered parasegments. Ftz protein binds in vitro to four distinct sequences in a 220 bp sub-fragment; these and neighboring sequences are conserved in the equivalent enhancer isolated from Drosophila virilis. Tsh protein produced under the control of the 220 bp enhancer partially rescues a null tsh mutation, with its strongest effect in the even-numbered parasegments. Mutation of the Ftz binding sites partially abrogates the capacity for rescue. These results suggest a composite mechanism for regulation of tsh, with different activators such as ftz contributing to the overall pattern of expression of this key regulator.

Homeotic response elements are tightly linked to tissue-specific elements in a transcriptional enhancer of the teashirt gene.

Along the anterior-posterior axis of animal embryos, the choice of cell fates, and the organization of morphogenesis, is regulated by transcription factors encoded by clustered homeotic or 'Hox' genes. Hox genes function in both epidermis and internal tissues by regulating the transcription of target genes in a position- and tissue-specific manner. Hox proteins can have distinct targets in different tissues; the mechanisms underlying tissue and homeotic protein specificity are unknown. Light may be shed by studying the organization of target gene enhancers. In flies, one of the target genes is teashirt (tsh), which encodes a zinc finger protein. tsh itself is a homeotic gene that controls trunk versus head development. We identified a tsh gene enhancer that is differentially activated by Hox proteins in epidermis and mesoderm. Sites where Antennapedia (Antp) and Ultrabithorax (Ubx) proteins bind in vitro were mapped within evolutionarily conserved sequences. Although Antp and Ubx bind to identical sites in vitro, Antp activates the tsh enhancer only in epidermis while Ubx activates the tsh enhancer in both epidermis and in somatic mesoderm. We show that the DNA elements driving tissue-specific transcriptional activation by Antp and Ubx are separable. Next to the homeotic protein-binding sites are extensive conserved sequences likely to control tissue activation by different homeodomain proteins. We propose that local interactions between homeotic proteins and other factors effect activation of targets in proper cell types.

The gene teashirt is required for the development of Drosophila embryonic trunk segments and encodes a protein with widely spaced zinc finger motifs.

We have discovered a reporter gene insertion that is expressed in the trunk region of Drosophila embryos. Genetic and molecular details of a new regulatory gene neighboring the reporter gene insertion, which we call teashirt (tsh), are described. In situ hybridization of a tsh probe to embryos shows that this gene is expressed in a way similar to the reporter gene. Mutations of tsh show that the gene is required for normal development of the ventral trunk region of embryos, which correlates with the spatial expression of the gene in the anteroposterior axis but not in the dorsoventral axis. Sequencing of a tsh cDNA shows that the putative protein possesses three distantly spaced CX2CX12HX5H zinc finger motifs.

Parallel computation of multiple biological sequence comparisons.

A parallel implementation of an efficient method for comparison of multiple DNA sequences is presented. The method is described in terms of a conceptual tree data structure for the sequences to be compared. The parallel algorithm shows efficient utilization of processors on an Encore Multimax computer in a sample comparison of 11 sequences totaling over 4000 bases. Timing data show the strong influence of computer system details on this parallel program. Also presented is a graphics program for displaying multiple sequence comparison output data. The display is capable of representing large volumes of multiple sequence comparison data in a single plot. The program has several additional features that allow closer examination of subsets of sequences. A display of matches from the sample comparison reflects the known structure of these sequences.

Supercomputers and biological sequence comparison algorithms.

Comparison of biological (DNA or protein) sequences provides insight into molecular structure, function, and homology and is increasingly important as the available databases become larger and more numerous. One method of increasing the speed of the calculations is to perform them in parallel. We present the results of initial investigations using two dynamic programming algorithms on the Intel iPSC hypercube and the Connection Machine as well as an inexpensive, heuristically-based algorithm on the Encore Multimax.

Phorbol ester serves as a coepibolin in the spreading of primary guinea pig epidermal cells.

In earlier studies, it was shown that the human plasma-spreading glycoprotein, epibolin (the 65 kD species of serum-spreading factor or vitronectin), requires a second plasma component, termed coepibolin, in order to support maximal dissociated epidermal cell spreading in tissue culture. Whereas epibolin alone in defined medium supports some cell spreading, the purified plasma coepibolin preparations do not effect spreading in the absence of epibolin. Although not yet entirely purified, coepibolin associates with some plasma fractions but not with others; it is certainly not a property of all proteins, e.g., while bovine serum albumin (BSA) has coepibolin activity, ovalbumin does not. The data presented here show that the phorbol ester, 12-tetra-decanoyl-1-phorbol-13-acetate (TPA) can act as a potent coepibolin and support maximal spreading over a concentration range of 10-100 ng/ml. In the absence of epibolin TPA does not stimulate the spreading of epidermal cells when given alone or in the presence of BSA or ovalbumin. Coepibolin activity appears to associate with tumor-promoting activity in that the phorbol derivative, phorbol-12,13-didecanoate, shows coepibolin activity, while its inactive non-tumor-promoting isomer, phorbol-4 alpha-phorbol-12,13-didecanoate, does not. These data suggest that the proteinaceous plasma-derived cofactor acts in a fashion similar to TPA and that this as yet unexplained mechanism of TPA action is important to the full expression of epibolin and to the early phase of epidermal cell spreading.

Cation dependence of guinea pig epidermal cell spreading.

To understand the earliest phases of epidermal cell spreading we have sought a defined in vitro system. We studied the divalent cation dependence of guinea pig epidermal cell spreading in media containing varying concentrations of cations. No spreading occurred in calcium-magnesium-free Dulbecco's modified Eagle's medium (CMF-DME) in the presence of cation-free fetal bovine serum; however, significant spreading occurred if the medium was supplemented with Mg++ plus Ca++ or Mg++ alone. Supplementing with Ca++ alone led to much less spreading. These cations in CMF-DME did not support spreading in the absence of serum or the presence of serum albumin. Assaying cell spreading in a simple salt solution consisting of NaCl, KCl, Tris buffer, pH 7.4 plus dialyzed serum and a series of divalent cation supplements (Ca++, Mg++, Mn++, Co++, Zn++, Ni++), showed that only Mg++ and Mn++, and to a lesser extent, Ca++, supported cell spreading. In contrast to Mg++, however, Mn++ could support spreading in the absence of whole serum if serum albumin were present. Although Mn++ plus serum albumin supported more rapid spreading at lower cation concentrations than Mg++ plus serum, equal concentrations of Ca++ completely blocked the Mn++ effect. In contrast to the increasing cell spreading, which occurred in Mg++-containing medium with time, cell death occurred in Mn++-containing medium by 24 h. Consonant with studies from other laboratories, human foreskin fibroblasts spread in Mn++-containing salt solution in the absence of protein supplements. These experiments indicate for epidermal cell spreading that Mg++ is the important cation in tissue culture media, that under proper cation conditions epidermal cells do not need a specific spreading protein (i.e. a protein that has been demonstrated to support cell spreading), that Mn++ and Mg++-induced spreading seem to represent different mechanisms, that fibroblastic and epidermal cells have different cation requirements for in vitro spreading, and that the crucial role cations play in cell spreading remains to be elucidated.

Fifty-two medical student suicides.

The authors surveyed all U.S. medical schools to ascertain the frequency with which medical students attempt suicide, complete suicide, and seek psychiatric treatment. In the classes of 1974-1981 the annual suicide rate for male students was 15.6 per 100,000, which is comparable to their agemates in the national population. The rate for female students equaled that of the male students but was three to four times that of their agemates. Seventy-six percent of the suicides were committed by sophomore and junior students, and 50% were committed in November, December, or January. The authors discuss four steps schools can take in suicidal prevention.