Nature versus nurture: asymmetric cell divisions in Drosophila bristle development.
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Animals↗
Biomedical subjects
J W Posakony
Publications and source records attributed to J W Posakony.
A small region surrounding the distal promoter of the hunchback gene directs maternal expression.
While maternally provided factors play essential roles in the earliest processes of embryogenesis, little is known about the control of female germline-specific gene expression. Primer extension analysis and in situ hybridization experiments demonstrate that in adult Drosophila females, transcripts of the gap gene hunchback (hb) are produced only by the distal (P1) promoter and that this expression is largely restricted to the ovarian nurse cells. A deletion analysis of the hb promoter using lacZ reporter constructs defines a 1.2-kb genomic DNA fragment surrounding the P1 promoter that is sufficient to reproduce the wild-type pattern of hb ovarian transcript accumulation. By contrast, the subfragments of this region we have tested fail to direct specific ovarian expression.
Animals↗
The sequence similarity of the Drosophila suppressor of hairless protein to the integrase domain has no functional significance in vivo.
Suppressor of Hairless [Su(H)] plays an essential role in neurogenesis in Drosophila by controlling successive alternative cell fate decisions in the developing adult epidermis. Analysis of the predicted amino acid sequence of the Su(H) protein revealed a weak similarity to the catalytic domain of a family of phage integrases and yeast recombinases. We present here the results of a site-directed mutagenesis of the integrase-related region of Su(H), which indicate that this sequence similarity has no functional significance in vivo. We suggest that the JK-RBP protein, encoded by the mouse homologue of Su(H), does not act as a recombinase, as originally proposed.
Animals↗
Antagonistic activities of Suppressor of Hairless and Hairless control alternative cell fates in the Drosophila adult epidermis.
Successive alternative cell fate choices in the imaginal disc epithelium lead to the differentiation of a relatively invariant pattern of multicellular adult sensory organs in Drosophila. We show here that the activity of Suppressor of Hairless is required for both the sensory organ precursor (SOP) versus epidermal cell fate decision, and for the trichogen (shaft) versus tormogen (socket) cell fate choice. Complete loss of Suppressor of Hairless function causes most proneural cluster cells to accumulate high levels of the achaete and Delta proteins and to adopt the SOP fate. Late or partial reduction in Suppressor of Hairless activity leads to the apparent transformation of the tormogen (socket) cell into a second trichogen (shaft) cell, producing a 'double shaft' phenotype. We find that overexpression of Suppressor of Hairless has the opposite phenotypic effects. SOP determination is prevented by an early excess of Suppressor of Hairless activity, while at a later stage, the trichogen (shaft) cell is transformed into a second tormogen (socket) cell, resulting in 'double socket' bristles. We conclude that, for two different cell fate decisions in adult sensory organ development, decreasing or increasing the level of Suppressor of Hairless function confers mutant phenotypes that closely resemble those associated with gain and loss of Hairless activity, respectively. These results, along with the intermediate SOP phenotype observed in Suppressor of Hairless; Hairless double mutant imaginal discs, suggest that the two genes act antagonistically to commit imaginal disc cells stably to alternative fates.
Animals↗
Dpbx, a new homeobox gene closely related to the human proto-oncogene pbx1 molecular structure and developmental expression.
Recently, a new class of homeodomain containing proteins, pbx1, pbx2, and pbx3 has been described. pbx proteins are most closely related to two yeast regulatory proteins, a1 and alpha 2. Here, we identify and characterize the pbx homolog in Drosophila, designated Dpbx. Dpbx is 95% identical to the pbx proteins within the homeodomain and, more remarkably, is 85% to 88% identical within a 201 amino acid region adjacent to the homeodomain. Cytologically, the Dpbx gene is located on the X chromosome at 14A. mRNA expression is both maternal and zygotic and occurs throughout the life cycle. Prior to full germband retraction, Dpbx is rather ubiquitously present and variations are minor. The most notable feature of Dpbx expression is that after germband retraction, high levels of Dpbx are observed in the anterior portion of the ventral nerve cord.
Amino Acid Sequence↗
Suppressor of Hairless, the Drosophila homolog of the mouse recombination signal-binding protein gene, controls sensory organ cell fates.
Suppressor of Hairless (Su(H)) is required at two stages of adult sensory organ development in Drosophila. Complete loss of Su(H) function results in a "neurogenic" phenotype in imaginal discs, in which too many cells adopt the sensory organ precursor cell fate. Su(H) is also involved in controlling the fates of sensillum accessory cells and is specifically expressed in two of these cells. Su(H) is the Drosophila homolog of the mouse J kappa RBP gene, whose product binds specifically to the recombination signal sequence of immunoglobulin J kappa segments. The Su(H) and J kappa RBP proteins are 82% identical over most of their length, and share with bacteriophage integrates and yeast recombinases a motif that includes residues directly involved in catalyzing recombination.
Amino Acid Sequence↗
Spatial regulation of proneural gene activity: auto- and cross-activation of achaete is antagonized by extramacrochaetae.
The spatially restricted activities of achaete (ac) and scute (sc) are thought to define proneural clusters of potential sensory organ precursor cells in the imaginal discs of Drosophila. These genes encode transcriptional regulators of the basic helix-loop-helix (bHLH) class. We have found that direct, positive transcriptional autoregulation by the ac protein and cross-regulation by sc are essential for high-level expression of the ac promoter in the proneural cluster pattern and that autoactivation is important for the bristle-promoting function of the ac gene. These auto- and cross-regulatory activities are antagonized in a dose-dependent manner by the inhibitory HLH protein encoded by the extramacrochaetae (emc) gene. We have found that emc is expressed in the wing imaginal disc in a pattern strongly complementary to that of the proneural clusters. Our results indicate that emc plays an essential early role in defining territories of bristle-forming potential.
Animals↗
The Drosophila gene Hairless encodes a novel basic protein that controls alternative cell fates in adult sensory organ development.
The mechanosensory bristles of adult Drosophila are composed of four cells that, in most cases, are progeny of a single sensory organ precursor (SOP) cell. Two sister cells in this lineage, the trichogen and tormogen, produce the external shaft and socket of the bristle, respectively. Loss-of-function mutations of Hairless (H) confer two distinct mutant phenotypes on adult bristles. The bristle loss phenotype results from the failure to specify and/or execute the SOP cell fate; the double socket phenotype results from the transformation of the trichogen (shaft) cell into a second tormogen (socket) cell. We have found that the H gene encodes a novel basic protein with a predicted molecular mass of 109 kD. Basal levels of expression of a transgene (P[Hs-H]) in which the H protein-coding region is under the control of the Hsp70 promoter are sufficient to provide full rescue of H mutant phenotypes. Heat shock treatment of P[Hs-H] transgenic animals as late larvae and early pupae produces a tormogen-to-trichogen (double shaft) cell fate transformation, as well as bristle multiplication and loss phenotypes very similar to those caused by loss-of-function mutations in the neurogenic gene Notch. Our results indicate that the SOP cell fate requires H to antagonize the activity of the neurogenic group of genes and that the expression of distinct cell fates by the trichogen/tormogen sister cell pair depends on an asymmetry in their levels of H+ activity or in their thresholds for response to H.
Amino Acid Sequence↗
Hairless is required for the development of adult sensory organ precursor cells in Drosophila.
Reduction of the wild-type activity of the gene Hairless (H) results in two major phenotypic effects on the mechanosensory bristles of adult Drosophila. Bristles are either 'lost' (i.e. the shaft and socket fail to appear) or they exhibit a 'double socket' phenotype, in which the shaft is apparently transformed into a second socket. Analysis of the phenotypes conferred by a series of H mutant genotypes demonstrates (1) that different sensilla exhibit different patterns of response to decreasing levels of H+ function, and (2) that the 'bristle loss' phenotype results from greater loss of H+ function than the 'double socket' phenotype. The systematic study of H allelic combinations enabled us to identify genotypes that reliably produce specific mutant defects in particular positions on the bodies of adult flies. This permitted us to investigate the cellular development of sensilla in these same positions in larvae and pupae and thereby establish the developmental basis for the mutant phenotypes. We have found that H is required for at least two steps of adult sensillum development. In positions where 'double socket' microchaetes appear on the notum of H mutant flies, sensillum precursor cells are present in the developing pupa and divide normally, but their progeny adopt an aberrant spatial arrangement and fail to differentiate correctly. In regions of the notum exhibiting 'bristle loss' in adult H mutants, we were unable at the appropriate stages of development to detect sensillum-specific cell types, the precursor cell divisions that generate them, or the primary precursor cells themselves. Thus, the H 'bristle loss' phenotype appears to reflect a very early defect in sensillum development, namely the failure to specify and/or execute the sensory organ precursor cell fate. This finding indicates that H is one of a small number of identified genes for which the loss-of-function phenotype is the failure of sensillum precursor cell development.
Alleles↗
The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes.
In Drosophila, a group of regulatory proteins of the helix-loop-helix (HLH) class play an essential role in conferring upon cells in the developing adult epidermis the competence to give rise to sensory organs. Proteins encoded by the daughterless (da) gene and three genes of the achaete-scute complex (AS-C) act positively in the determination of the sensory organ precursor cell fate, while the extramacrochaetae (emc) and hairy (h) gene products act as negative regulators. In the region upstream of the achaete gene of the AS-C, we have identified three 'E box' consensus sequences that are bound specifically in vitro by hetero-oligomeric complexes consisting of the da protein and an AS-C protein. We have used this DNA-binding activity to investigate the biochemical basis of the negative regulatory function of emc. Under the conditions of our experiments, the emc protein, but not the h protein, is able to antagonize specifically the in vitro DNA-binding activity of da/AS-C and putative da/da protein complexes. We interpret these results as follows: the heterodimerization capacity of the emc protein (conferred by its HLH domain) allows it to act in vivo as a competitive inhibitor of the formation of functional DNA-binding protein complexes by the da and AS-C proteins, thereby reducing the effective level of their transcriptional regulatory activity within the cell.
Animals↗
extramacrochaetae, a negative regulator of sensory organ development in Drosophila, defines a new class of helix-loop-helix proteins.
The function of the extramacrochaetae (emc) gene is required to establish the normal spatial pattern of adult sensory organs in Drosophila. emc acts to suppress sensory organ development in certain regions of the body surface, apparently by antagonizing the function of the achaete and scute genes of the achaetescute complex (AS.C). We have found that emc encodes a novel member of the helix-loop-helix (HLH) family of proteins. The emc protein shares the dimerization domain of other HLH proteins but lacks their DNA binding motif. We propose a model in which the emc protein negatively regulates sensory organ determination by forming heterodimers with the HLH proteins encoded by the AS-C and/or daughterless, thereby altering or interfering with their activity.
Alleles↗
A dual function of the Notch gene in Drosophila sensillum development.
We have investigated the function of the neurogenic gene Notch (N) during development of the adult sensilla of Drosophila. Heat pulses were applied to flies carrying the temperature-sensitive Notch allele Nts1 at different larval and pupal stages. We can show that the reduction of Notch+ function during a short interval prior to the onset of sensillum precursor division, resulting from a heat pulse between 0 and 14 hr after puparium formation (apf), leads to an increase in microchaete precursors at the expense of epidermal cells. The structure and cellular composition of the sensilla produced by these supernumerary precursors are normal. Later heat pulses which include the interval immediately after sensillum precursor division (14-20 hr apf) lead, among the progeny of the sensillum precursors, to a hyperplasia of sensory neurons, at the expense of accessory cells. The resulting "sensilla" consist of neurons only and lack the external cuticular structures (i.e., shaft, socket). These results demonstrate that similar mechanisms both of which involve the function of the Notch gene may be operating to sort out (premitotic) sensillum precursors from epidermal precursors and (postmitotic) sensory neurons from accessory cells. They further show that in postmitotic sensillum cells the differentiative fate is not yet irreversibly fixed, but presumably requires cell-cell interaction to become established.
Alleles↗
Sensillum development in the absence of cell division: the sensillum phenotype of the Drosophila mutant string.
We have investigated sensillum development in Drosophila embryos homozygous for mutations in the locus string (stg). In these embryos, cell division is blocked following blastoderm formation. This permits a study of the differentiative fate of undivided precursor cells, in particular those giving rise to the larval sensory organs (sensilla). Of the different cell fates normally represented in the sensilla (i.e., sensory neuron, thecogen cell, trichogen cell, tormogen cell, glia cell), only the phenotype of sensory neurons is expressed morphologically in stg embryos, suggesting that the neuronal fate predominates over the fates of the nonneuronal accessory cells. Consistent with this finding, the P element-lacZ insertion A1-2nd-29, which is a marker for trichogen and tormogen cells in the wild-type embryo, is not expressed in the body wall of the stg embryo. Some sensillum precursor cells appear to express a mixed fate in stg mutants: They express antigens (recognized by the monoclonal antibodies 22C10 and 21A6) which in the wild-type appear in separate cells (sensory neurons and thecogen cell, respectively). The differentiation of undivided cells in stg embryos is not restricted to the peripheral nervous system; in all types of tissues analyzed in this study (e.g., epidermis, intestine, muscle, CNS), precursor cells express characteristics normally exhibited by their progeny.
Animals↗
Development of adult sensilla on the wing and notum of Drosophila melanogaster.
We have investigated the temporal pattern of appearance, cell lineage, and cytodifferentiation of selected sensory organs (sensilla) of adult Drosophila. This analysis was facilitated by the discovery that the monoclonal antibody 22C10 labels not only the neuron of the developing sensillum organ, but the accessory cells as well. The precursors of the macrochaetes and the recurved (chemosensory) bristles of the wing margin divide around and shortly after puparium formation, while those of the microchaetes and the stout and slender (mechanosensory) bristles of the wing margin divide between 9 h and 18 h after puparium formation (apf). The onset of sensillum differentiation follows the terminal precursor division within a few hours. Four of the cells in an individual microchaete organ are clonally related: A single first-order precursor cell divides to produce two second-order precursors; one of these divides into the neuron and thecogen cell, the other into the trichogen cell and tormogen cell. Along the anterior wing margin, two rounds of division generate the cells of the mechanosensory sensilla; here, no strict clonal relationship seems to exist between the cells of an individual sensillum. At the time of sensillum precursor division, many other, non-sensillum-producing cells within the notum and wing proliferate as well. This mitotic activity follows a spatially non-random pattern.
Animals↗
The Drosophila gene knirps-related is a member of the steroid-receptor gene superfamily.
Molecular cloning has demonstrated that the receptors for steroid, retinoid and thyroid hormones are part of a large superfamily of nuclear regulatory proteins. In vertebrates these molecules regulate diverse biological processes such as pattern formation, cellular differentiation and homeostasis. The universal necessity for embryonic and adult cells to respond to their external environment might mean that members of this family pre-date the divergence of vertebrates and invertebrates. We have screened a Drosophila genomic library for steroid receptor homologues using a human retinoic acid receptor complementary DNA as a hybridization probe. Several clones were recovered, one of which mapped to chromosomal position 77E1-2, the cytological location of the gap segmentation gene knirps. Sequence analysis of a cDNA clone representing the human retinoic acid receptor homologue showed similarity of the predicted protein to the vertebrate steroid receptors, as well as to the predicted knirps gene product. In situ hybridization of a cDNA probe to wild-type embryos revealed a uniform distribution of transcripts that were apparently maternally derived. Zygotic transcript accumulation begins in a broad anteroventral domain before the cellular blastoderm stage. At the cellular blastoderm stage two additional circumferential bands of transcript appear.
Amino Acid Sequence↗
Identification of DNA sequences required for the regulation of Drosophila alcohol dehydrogenase gene expression.
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Alcohol Dehydrogenase↗
Interspersed sequence organization and developmental representation of cloned poly(A) RNAs from sea urchin eggs.
A random primed complementary DNA (cDNA) clone library constructed from total maternal poly(A) RNA of sea urchin eggs was screened with two cloned genomic repetitive sequence probes. Sets of cDNA clones reacting with each of these repetitive sequences were recovered. Most of the cloned transcripts included both single copy and repeat sequence elements. Except for the shared repeat sequence element, both the repetitive and single copy regions of the members of each set of clones failed to crossreact. Single copy probes linked to the repeats on the cloned maternal RNAs are represented in an asymmetric manner. It follows that many different genomic members of a given dispersed repeat sequence family are represented in the maternal RNA. RNA gel blots carried out with several repeat probes display about 10 to 20 prominent maternal poly(A) RNAs containing transcripts of each repetitive sequence family. The interspersed maternal transcripts are 3000 to 15,000 bases in length. Maternal transcripts reacting with single copy probes derived from the cloned cDNAs persist during embryonic development, and in some cases appear to be augmented by similar, newly synthesized embryo transcripts. Two examples were found in which additional transcripts of different length appear at specific developmental stages. The transcribed single copy regions are highly polymorphic in the genomes of different individual sea urchins, and comparisons of closely related sea urchin species showed that both the prevalence and length of specific maternal transcripts change rapidly during evolution. Nucleotide sequences of two homologous repeat elements occurring on different cloned transcripts displayed translation stop codons in every possible reading frame. These repeat sequences display structural features suggesting that there has been evolutionary transposition into transcription units active during oogenesis. The repeat elements and their flanking single copy regions reside either in very long 3' or 5'-terminal sequences, or in unprocessed intervening sequences in the maternal poly(A) RNA. These findings lead us to the proposal that the majority of the cytoplasmic poly(A) RNA in echinoderm eggs and early embryos is similar in form to RNAs that occur in the nucleus rather than to the messenger RNA of later cells.
Animals↗
Mitochondrial DNA sequences in the nuclear genome of Strongylocentrotus purpuratus.
Two sea urchin embryo complementary DNA clones representing mitochondrial 16 S ribosomal RNA and cytochrome oxidase subunit I messenger RNA have been characterized. The cloned cDNAs are colinear with sea urchin mitochondrial DNA, and their identification is based on cross-hybridization with known restriction fragments of human mitochondrial DNA, and on nucleotide sequence determinations. The mitochondrial cDNA clones also displayed an unexpected reaction with specific genomic DNA sequences in gel blot hybridizations. Genomic phage lambda recombinants containing sequences hybridizing with the mitochondrial clones were isolated and the arrangement of these sequences was determined. The genomic region studied contains a sequence homologous with the 3' end of the mitochondrial 16 S rRNA gene, flanked on one side by what is possibly a complete copy of the cytochrome oxidase subunit I gene, and on the other by a duplication of a fragment of this gene. The nucleotide sequence divergence between the mitochondrial and nuclear homologues of the cytochrome oxidase subunit I gene varies for different regions of the gene, from about 13% to 25%, while there is about 8% sequence divergence between nuclear and mitochondrial versions of the 3' 16S rRNA sequence. The structure of the genomic mitochondrial sequence homologues indicates that during sea urchin evolution there occurred a germ-line transposition of a fragment of the mitochondrial genome into the nuclear DNA, followed by rearrangements and single nucleotide substitutions.
Animals↗