Homeotic Gene Products Research Articles

Tribolium.

Tribolium.

Functional divergence within the APETALA3/PISTILLATA floral homeotic gene lineages.

Changes in homeotic gene expression patterns or in the functions of the encoded proteins are thought to play a prominent role in the evolution of new morphologies. The floral homeotic APETALA3 (AP3) and PISTILLATA (PI) genes encode MADS domain-containing transcription factors required to specify petal and stamen identities in Arabidopsis. We have previously shown that perianth expression of AP3 and PI homologs varies in different groups of angiosperms with diverse floral structures, suggesting that changes in expression may contribute to changing morphology. We have investigated the possibility that changes in the functions of the encoded gene products may also have played a role in the evolution of different floral morphologies. AP3 and PI are members of paralogous gene lineages and share extensive similarity along the length of the protein products. Genes within these lineages encode products with characteristic C-terminal motifs that we show are critical for functional specificity. In particular, the C terminus of AP3 is sufficient to confer AP3 functionality on the heterologous PI protein. Furthermore, we have shown that the evolution of the divergent AP3 C-terminal domain in the core eudicots is correlated with the acquisition of a role in specifying perianth structures. These results suggest that divergence in these sequence motifs has contributed to the evolution of distinct functions for these floral homeotic gene products.

China: Inabata — plastics colorants

This chapter discusses some aspects of the mammalian Hox gene family and mentions the results obtained with other animals, such as birds or amphibians. The first vertebrate homeobox-containing genes were isolated concomitantly in Xenopus and mouse after screening with probes derived from the Drosophila Antennupedia (Antp) and fushi tarazu (ftz) homeoboxes. As the homeobox was discovered during the cloning of insect homeotic genes, the first vertebrate genes isolated shown to encode homeodomain proteins were members of the Hox family—that is, genes containing a homeobox sequence related to that present in insect homeotics. Most of the information about the interactions between peptide growth factors, retinoids, and Hox genes during primary axis formation comes from the Xenopus embryo. In this system, peptide growth factors are able to induce regionally specified mesoderm from cultured blastula animal caps. Drosophila homeotic gene products are DNA sequence-specific transcription factors. So far, no available mouse mutant strain has been described to exhibit a pheno-type linked to alterations in a Hoxgene.

Biniou (FoxF), a central component in a regulatory network controlling visceral mesoderm development and midgut morphogenesis in Drosophila.

The subdivision of the lateral mesoderm into a visceral (splanchnic) and a somatic layer is a crucial event during early mesoderm development in both arthropod and vertebrate embryos. In Drosophila, this subdivision leads to the differential development of gut musculature versus body wall musculature. Here we report that biniou, the sole Drosophila representative of the FoxF subfamily of forkhead domain genes, has a key role in the development of the visceral mesoderm and the derived gut musculature. biniou expression is activated in the trunk visceral mesoderm primordia downstream of dpp, tinman, and bagpipe and is maintained in all types of developing gut muscles. We show that biniou activity is essential for maintaining the distinction between splanchnic and somatic mesoderm and for differentiation of the splanchnic mesoderm into midgut musculature. biniou is required not only for the activation of differentiation genes that are expressed ubiquitously in the trunk visceral mesoderm but also for the expression of dpp in parasegment 7, which governs proper midgut morphogenesis. Activation of dpp is mediated by specific Biniou binding sites in a dpp enhancer element, which suggests that Biniou serves as a tissue-specific cofactor of homeotic gene products in visceral mesoderm patterning. Based upon these and other data, we propose that the splanchnic mesoderm layers in Drosophila and vertebrate embryos are homologous structures whose development into gut musculature and other visceral organs is critically dependent on FoxF genes.

The Drosophila proboscis is specified by two Hox genes, proboscipedia and Sex combs reduced, via repression of leg and antennal appendage genes.

The proboscis is one of the most highly modified appendages in Drosophila melanogaster. However, the phenotypes of proboscipedia (pb) mutants, which transform the proboscis into leg or antenna, indicate a basic homology among these limbs. Recent genetic studies have revealed a developmental system for patterning appendages and identified several genes required for limb development. Among these are: extradenticle (exd), homothorax (hth), dachshund (dac), Distal-less (Dll) and spalt (sal). These limb genes have not been well studied in wild-type mouthparts and their role if any in this appendage is not well understood. Here we demonstrate that the homeotic gene products Proboscipedia (Pb) and Sex combs reduced (Scr) regulate the limb genes in the labial disc to give rise to a unique type of appendage, the proboscis. Pb inhibits exd, dac and sal expression and downregulates DLL: This observation explains the ability of Pb to inhibit the effects of ectopically expressed trunk Hox genes in the proboscis, to suppress leg identity in the trunk and to transform antenna to maxillary palp. Scr suppresses sal expression and also downregulates Dll in the labial discs; discs mutant for both pb and Scr give rise to complete antennae, further demonstrating appendage homology. In the labial disc, Pb positively regulates transcription of Scr, whereas in the embryo, Scr positively regulates pb. Additionally, our results suggests a revised fate map of the labial disc. We conclude that the proboscis constitutes a genetically distinct type of appendage whose morphogenesis does not require several important components of leg and/or antennal patterning systems, but retains distal segmental homology with these appendages.

Dysregulation of EGF family of growth factors and COX-2 in the uterus during the preattachment and attachment reactions of the blastocyst with the luminal epithelium correlates with implantation failure in LIF-deficient mice.

Various mediators, including cytokines, growth factors, homeotic gene products, and prostaglandins (PGs), participate in the implantation process in an autocrine, paracrine, or juxtacrine manner. However, interactions among these factors that result in successful implantation are not clearly understood. Leukemia inhibitory factor (LIF), a pleiotropic cytokine, was shown to be expressed in uterine glands on day 4 morning before implantation and is critical to this process in mice. However, the mechanism by which LIF executes its effects in implantation remains unknown. Moreover, interactions of LIF with other implantation-specific molecules have not yet been defined. Using normal and delayed implantation models, we herein show that LIF is not only expressed in progesterone (P4)-primed uterine glands before implantation in response to nidatory estrogen, it is also induced in stromal cells surrounding the active blastocyst at the time of the attachment reaction. This suggests that LIF has biphasic effects: first in the preparation of the receptive uterus and subsequently in the attachment reaction. The mechanism by which LIF participates in these events was addressed using LIF-deficient mice. We observed that while uterine cell-specific proliferation, steroid hormone responsiveness, and expression patterns of several genes are normal, specific members of the EGF family of growth factors, such as amphiregulin (Ar), heparin-binding EGF-like growth factor (HB-EGF), and epiregulin, are not expressed in LIF(-/-) uteri before and during the anticipated time of implantation, although EGF receptor family members (erbBs) are expressed correctly. Furthermore, cyclooxygenase-2 (COX-2), an inducible rate-limiting enzyme for PG synthesis and essential for implantation, is aberrantly expressed in the uterus surrounding the blastocyst in LIF(-/-) mice. These results suggest that dysregulation of specific EGF-like growth factors and COX-2 in the uterus contributes, at least partially, to implantation failure in LIF(-/-) mice. Since estrogen is essential for uterine receptivity, LIF induction, and blastocyst activation, it is possible that the nidatory estrogen effects in the P4-primed uterus for implantation are mediated via LIF signaling. However, we observed that LIF can only partially resume implantation in P4-primed, delayed implanting mice in the absence of estrogen, suggesting LIF induction is one of many functions that are executed by estrogen for implantation.

Molecular mechanism of polyhomeotic activation by Engrailed.

The Drosophila Engrailed homeoprotein has been shown to activate directly a Polycomb-group gene, polyhomeotic, during embryogenesis. The molecular mechanism involved in this activation has been studied. Two different types of Engrailed-binding fragments have been detected within the polyhomeotic locus. The P1 and D1 fragments contain several 'TTAATTGCAT' motifs, whereas the D2 fragment contains a long 'TAAT' stretch to which multiple copies of Engrailed bind cooperatively. Another homeodomain-containing protein, Extradenticle, establishes protein-protein interactions with Engrailed on the D2 fragment. We have shown by CAT assays that both types of Engrailed-binding sites (P1 or D1 and D2), as well as Extradenticle, are necessary to obtain activation by Engrailed. In vivo, we have also shown that normal polyhomeotic expression depends on extradenticle expression. Moreover, in the absence of Extradenticle, overexpression of Engrailed protein represses polyhomeotic expression.

Interaction between spineless-aristapedia gene and genes from Antennapedia and bithorax complexes of Drosophila melanogaster.

Mutations in the spineless-aristapedia (ssa) gene of Drosophila melanogaster are pleiotropic, and their classical manifestations include a reduction in size of all bristles (spineless phenotype), transformation of distal parts of antennae into tarsal segments of the mesothoracic leg (aristapedia phenotype), and, in extreme alleles, fusion of tarsal segments on all six legs and the transformed aristaes. We isolated a new allele, which is a severe loss-of-function mutation and, in addition to the above-mentioned features, is characterized by amplification of sex combs on the first leg. This phenotype can be caused by a change in the expression of the Sex combs reduced (Scr) gene of the ANTP-C. Identification of this phenotype, together with observed variations in the extent of the fusion of tarsal segments in the legs of different segments, raised the possibility that ssa interacts with homeotic genes controlling the identity of segments. This possibility was tested in genetical experiments using flies with loss-of-function mutations in several homeotic genes and flies transformed by heat shock-driven homeotic genes. Analysis of adult phenotypes of different ssa alleles in the background of under-, over-, or ectopic expression of some genes of BX-C and ANT-C suggests that the ssa product is required to prevent the effect of the homeotic gene products in the distal segments of the appendages.

Morphological and biochemical characterization of spikelet development in naked oats (Avena sativa)

summaryThe dominant gene N‐1 in cultivated oats gives rise to a phenotype characterized by a thin papery lemma from which the grain readily threshes free, a multiflorous habit, and rachillas that are elongated in comparison to those of husked oats. This study was undertaken in order to identify the stages at which morphological and biochemical development of naked and husked oats begin to diverge, and to look for correlated differences in gene products (mRNAs and proteins). Spikelet morphology in naked and husked oats was shown to diverge early in panicle development: the first visible difference was premature cessation of growth of the third and subsequent florets in husked oats. Lignin deposition in the lemma occurred later and followed a similar time‐course in both genotypes, though in naked oats it was restricted to the vascular bundles. Mature lemmas of the naked genotype closely resembled glumes in appearance and in lignification pattern. Genotypic differences in extractable polypeptide complements were apparent only late in development, coinciding with the onset of lignification, and were not associated with comparable differences in translatable mRNA profiles. The normal (n‐1) allele at the naked locus is likely to encode a transcription factor with properties similar to those of previously‐characterized homeotic gene products in plants.

Nuclear localization of the Arabidopsis APETALA3 and PISTILLATA homeotic gene products depends on their simultaneous expression.

The Arabidopsis APETALA3 (AP3) and PISTILLATA (PI) proteins are thought to act as transcription factors and are required for specifying floral organ identities. To define the nuclear localization signals within these proteins, we generated translational fusions of the coding regions of AP3 and PI to the bacterial uidA gene that encodes beta-glucuronidase (GUS). Transient transformation assays of either the AP3-GUS or PI-GUS fusion protein alone resulted in cytoplasmic localization of GUS activity. However, coexpression of AP3-GUS with PI, or PI-GUS with AP3, resulted in nuclear localization of GUS activity. Stable transformation with these fusion proteins in Arabidopsis showed similar results. The nuclear colocalization signals in AP3 and PI were mapped to the amino-terminal regions of each protein. These observations suggest that the interaction of the AP3 and PI gene products results in the formation of a protein complex that generates or exposes a colocalization signal required to translocate the resulting complex into the nucleus. The colocalization phenomenon that we have described represents a novel mechanism to coordinate the functions of transcription factors within the nucleus.

A role for mel-18, a Polycomb group-related vertebrate gene, during theanteroposterior specification of the axial skeleton.

Segment identity in both invertebrates and vertebrates is conferred by spatially restricted distribution of homeotic gene products. In Drosophila, the expression of Homeobox genes during embryogenesis is initially induced by segmentation gene products and then maintained by Polycomb group and Trithorax group gene products. Polycomb group gene homologs are conserved in vertebrates. Murine mel-18 and closely related bmi-1 are homologous to posterior sex combs and suppressor two of zeste. Mel-18 protein mediates a transcriptional repression via direct binding to specific DNA sequences. To gain further insight into the function of Mel-18, we have inactivated the mel-18 locus by homologous recombination. Mice lacking mel-18 survive to birth and die around 4 weeks after birth after exhibiting strong growth retardation. Similar to the Drosophila posterior sex combs mutant, posterior transformations of the axial skeleton were reproducibly observed in mel-18 mutants. The homeotic transformations were correlated with ectopic expression of Homeobox cluster genes along the anteroposterior axis in the developing paraxial mesoderm. Surprisingly, mel-18-deficient phenotypes are reminiscent of bmi-1 mutants. These results indicate that the vertebrate Polycomb group genes mel-18 and bmi-1, like Drosophila Polycomb group gene products, might play a crucial role in maintaining the silent state of Homeobox gene expression during paraxial mesoderm development.

Muscle development and attachment to the epidermis is accompanied by expression of beta 3 and beta 1 tubulin isotypes, respectively.

In Drosophila beta tubulins are encoded by a small gene family whose members are differentially expressed in a highly cell and tissue specific manner. Here we focus on the expression of the beta 3 tubulin isotype during mesoderm differentiation and beta 1 tubulin expression in the apodemes during embryonic development. The beta 3 tubulin isotype is first detectable at the extended germband stage shortly before the separation of somatic and visceral derivatives. Comparing the distribution of the beta 3 mRNA and the beta 3 isotype shows that the transcription of the beta 3 tubulin gene is cell type specifically repressed during differentiation of individual mesodermal derivatives, from which the dorsal vessel remains transcriptionally active until shortly before hatching. In contrast the beta 3 tubulin protein is detectable in all mesodermal derivatives. The beta 3 tubulin is an excellent marker to study mesoderm differentiation on a regulatory and cellular level using both genetics and molecular biology. In the visceral mesoderm, the expression of the beta 3 tubulin gene is regulated by homeotic gene products, while other transactivators regulate expression in the dorsal vessel and the body wall musculature. In the somatic mesoderm, the beta 3 tubulin allows to visualize myotube formation and insertion into the epidermis. This contact to the epidermal attachment sites (apodemes) induces beta 1 tubulin expression, as can be seen in double staining experiments. We determined a 14bp cis-regulatory enhancer element guiding expression of the beta 1 tubulin gene in these attachment sites. Using the beta 1 and beta 3 tubulin isotypes as markers we started to isolate mutants which are disturbed in muscle formation.

PZF, a cDNA Isolated from Lotus japonicus and Soybean Root Nodule Libraries, Encodes a New Plant Member of the RING-Finger Family of Zinc-Binding Proteins

Low-stringency screening of a soybean root nodule cDNA library (106 recombinants screened) with a probe encoding the bovine rasGAP catalytic domain (Marshall et al., 1989) permitted the isolation of a rare cDNA that contains a 500-bp region of 68% similarity to the rasGAP cDNA. This partia1 soybean cDNA was used to isolate a full-length cDNA of 2.1 kb from the root nodule cDNA library of the model legume Lotus japonicus (Handberg and Stougaard, 1992). The full-length cDNA contains only one long ORF coding for a translation product of 60 kD. Searching the GenBank and EMBL data bases with FASTA (Pearson and Lipman, 1989) and BLAST (Altschul et al., 1990) revealed only little overall similarity to known proteins. The most prominent characteristic is the presence of a putative C3HC4 Zn-finger domain, the RING-finger domain (Lovering et al., 1993), suggesting that the plant proteins deduced from the cDNA sequences have a regulatory function. Consequently, the soybean and L. japonicus cDNAs were designated Gmpzf and Ljpzf, respectively. The deduced proteins were named Pzf (Table I). Most RING-finger family members appear to be regulatory transcription factors or DNA handling tools. The group comprises severa1 mammalian oncoproteins, homeotic gene products in Drosopkila, and DNA repair proteins in yeast and mammals. Furthermore, many members are encoded by immediate-early genes of a variety of viruses (Lovering et al., 1993). Biochemical evidence for Zn2+ and DNA binding due to the C3HC4 domain was established for some members, such as Me118 (Tagawa et al., 1990), RINGl (Lovering et al., 1993), and COPl (von Arnim and Deng, 1993). The only plant RING-finger family member identified so far is AtCOPl from Arabidopsis tkaliana (Deng et al., 1992). This putative transcription factor also possesses homology to p subunits of heterotrimeric G proteins. Thus, COPl may link phytochrome-mediated signal reception directly to regulation of transcriptional activities. The Ljpzf protein sequence contains an upstream HKRK motif (aa 141-145) that might serve as a NLS. The protein also contains other regions of clustered basic residues that might be involved in nucleic acid binding. The protein is

Engrailed-mediated repression of Ultrabithorax is necessary for the parasegment 6 identity in Drosophila

The homeotic genes of Drosophila are expressed in overlapping domains along the anterior-to-posterior axis and specify the distinct morphological patterns of each parasegment. Within single parasegments, the levels of homeotic gene expression are often modulated, in part because of cross-regulation by other homeotic gene products. However, the functional significance of different levels of homeotic gene expression is unclear. Here modulations in Ultrabithorax (Ubx) expression within parasegment 6 are examined. Specifically, Ubx is shown to be down-regulated in the posterior compartment of this parasegment by engrailed (en). The significance of Ubx repression by en was demonstrated by characterizing the expression of the Ubx target gene, Distal-less (Dll). In the posterior compartment of parasegment 6, Dll is normally expressed in a small cluster of cells. If Ubx is expressed uniformly via a heat-shock promoter, Dll is inappropriately repressed in these posterior compartment cells. In the anterior compartment of parasegment 6, Dll is normally repressed by high levels of Ubx. However, if en is expressed uniformly via a heat-shock promoter, Ubx is repressed and Dll is derepressed. Because Dll is required for the development of larval sensory structures, these results demonstrate that en-mediated repression of Ubx in the posterior compartment is necessary for the morphology of parasegment 6. Thus, different levels of homeotic gene expression can be important for their segmental patterning functions.

Cell-type-specific mechanisms of transcriptional repression by the homeotic gene products UBX and ABD-A in Drosophila embryos.

The homeotic genes of Drosophila melanogaster, which are required for specification of segmental identities, encode proteins capable of regulating gene expression. We have chosen to study the organization and function of a regulatory target in an attempt to learn how homeotic gene products provide appropriate transcriptional controls. We identified 30 common binding sites for the proteins encoded by the Ultrabithorax (Ubx) and abdominal-A (abd-A) genes within a negatively regulated target, the P2 promoter of the Antennapedia (Antp) gene. By systematically mutagenizing binding sites and observing the resulting P2 expression pattern in embryos, we have found evidence for cell-type-specific interactions that are mediated by these sequences. In certain neuronal cells, UBX and ABD-A proteins appear to repress by competing for common binding sites with another homeodomain protein, which we propose to be ANTP acting to induce P2 transcription in an autoregulatory manner. In sets of cells that contribute to the tracheal system, UBX and ABD-A repress by counteracting the function of a factor acting at independent sites. The latter mechanism of repression requires only that multiple homeodomain binding sequences be present and is not dependent on any particular binding site.

Developmental Genes and Ocular Malformation Syndromes

How does a fertilized ovum develop into a well-formed human being? What coordinates the fate of genetically identical embryonic stem cells, turning some into limbs and others into eyes or parts thereof? What synchronizes the myriad decision-requiring events in fetal development? What reparative processes exist to compensate for mishaps? What is behind the miracle of the perfect eye and body? Several families of so-called homeotic genes are responsible for the orchestration and regulation of normal development of biologic systems. In the last decade new landmarks have been set in our understanding of embryology with the discovery, among others, of the homeobox independently by McGinnis and associates! and Scott and Weiner. 5 The homeobox is a DNA segment, 183 base pairs in length, which codes for a protein that constitutes the DNA binding region or homeodomain of some homeotic gene products. The proteins coded for by homeobox-containing genes regulate DNA transcription by binding to downstream (toward the 3' end of the gene) genes and also regulate their own production. Homeobox sequences in different homeotic genes within and between genera and species are so similar or conserved that they are likely to have evolved from common ancestral genes by a process of tandem duplication, or side-by-side duplication.v? It is also hypothesized that after duplication, mutations in some respective genes result in diversification of function and specific body segment expressivity. Homeobox-containing genes are arranged in clusters on one or more chromosomes (one in Drosophila and four in mouse and man). These genes regulate the development of body segments cephalad to caudad, depending on their location in the gene cluster. The developing embryo at the multiple somite stage consists of a tandem array of duplicated body segments. These primordial segments form different body parts under the control of homeotic genes. There are clear-cut boundaries and synchronized expression of

Homeotic genes have specific functional roles in the establishment of the Drosophila embryonic peripheral nervous system.

The Drosophila embryonic peripheral nervous system (PNS) contains segment-specific spatial patterns of sensory organs which derive from the ectoderm. Many studies have established that the homeotic genes of Drosophila control segment specific characteristics of the epidermis, and more recently these genes have also been shown to control gut morphogenesis through their expression in the visceral mesoderm (Tremml, G. and Bienz, M. (1989), EMBO J. 8, 2677-2685). We report here the roles of homeotic genes in establishing the spatial patterns of sensory organs in the embryonic PNS. The PNS was examined in embryos homozygous for mutations in the homeotic genes Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B) with antibodies that label specific subsets of sensory organs. Our results suggest that the homeotic genes have specific roles in establishing the correct spatial patterns of sensory organs in their normal domains of expression. In addition, we also report the effects of ectopic expression of the homeotic genes labial (lab), Deformed (Dfd), Scr, Antp or Ubx on the normal development of sensory organs in the embryonic PNS. Interestingly, while previous studies have concluded that ectopic expression of the homeotic genes Dfd, Scr and Antp has no effect on the segmental identity of the abdominal segments, our results demonstrate that this is not true. We show that ectopic expression of these genes does result in the disruption of the developing PNS in the abdomen. Our results are suggestive of a role for the homeotic gene products in regulating genes which are necessary for generating sensory progenitor cells in the developing PNS.

Function of the apetala-1 gene during Arabidopsis floral development.

We have characterized the floral phenotypes produced by the recessive homeotic apetala 1-1 (ap1-1) mutation in Arabidopsis. Plants homozygous for this mutation display a homeotic conversion of sepsis into brachts and the concomitant formation of floral buds in the axil of each transformed sepal. In addition, these flowers lack petals. We show that the loss of petal phenotype is due to the failure of petal primordia to be initiated. We have also constructed double mutant combinations with ap1 and other mutations affecting floral development. Based on these results, we suggest that the AP1 and the apetala 2 (AP2) genes may encode similar functions that are required to define the pattern of where floral organs arise, as well as for determinate development of the floral meristem. We propose that the AP1 and AP2 gene products act in concert with the product of the agamous (AG) locus to establish a determinate floral meristem, whereas other homeotic gene products are required for cells to differentiate correctly according to their position. These results extend the proposed role of the homeotic genes in floral development and suggest new models for the establishment of floral pattern.

Homeotic gene products as growth factors.

Homeotic gene products as growth factors.