The Planarian HOM/HOX Homeobox Genes (Plox) Expressed along the Anteroposterior Axis

In the freshwater planarian Dugesia japonica, five cDNAs for HOM/HOX homeobox genes were cloned and sequenced. Together with sequence data on HOM/HOX homeobox genes of platyhelminthes deposited in databases, comparison of the deduced amino acid sequences revealed that planarians have at least seven HOM/HOX homeobox genes, Plox1 to Plox7 ( anarian HOM/H homeobox genes). Whole-mount in situ hybridization and RT-PCR revealed that Plox4 and Plox5 were increasingly expressed along a spatial gradient in the posterior region of intact animals. During regeneration, Plox5 was expressed only in the posterior region of regenerating body pieces, suggesting that the gene is involved in the anteroposterior patterning in planarians. Plox5 was not found to be expressed in a blastema-specific manner, which contradicts a previous report (J. R. Bayascas, E. Castillo, A. M. Muños-Mármol, and E. Saló. Development 124, 141–148, 1997). X-ray irradiation experiments showed that Plox5 was expressed at least in some cells other than neoblasts, but that the induction of Plox5 expression during regeneration might require neoblasts.     

A rational nomenclature for vertebrate homeobox (HOX) genes.

Developmental fates along the anterior-posterior axes of animals are controlled by clustered homeotic genes which in vertebrates are called Hox genes. The gene clusters are similar and probably functionally homologous in animals as different as nematodes, flies, and mammals. A new set of names for Hox genes was recently agreed upon by many workers in the field. Remarkably, the order of the Hox genes along the chromosome reflects where they are expressed along the body axis. This simple principle is reflected in the new nomenclature system.     

The freshwater planarian Dugesia (G.) tigrina contains a great diversity of homeobox genes

To identify potential pattern control and cell determination and/or differentiation genes in the freshwater planarian Dugesial (G.) tigrina, we searched for homeobox genes of different types in the genome of this primitive metazoan. We applied two basic approaches: 1) Screening the cDNA library with degenerate oligonucleotides corresponding to the most conserved amino acid sequence from helix-3 of the homeodomain of each family; and 2) PCR amplification of genomic DNA or cDNA, using two sets of degenerated oligonucleotides corresponding to helices 1 and 3 of the homeodomain or two specific domains of the POU family. Using the first strategy we have identified and characterized two tissue-specific cell determination and/or differentiation NK-type homeobox genes. Using the second strategy we have identified several homeobox genes that belong to the HOM/Hox, paired (prd) or POU families.     

MIG-13 positions migrating cells along the anteroposterior body axis of C. elegans.

The C. elegans Q neuroblasts and their descendants migrate along the anteroposterior (A/P) body axis to positions that are not associated with any obvious landmarks. We find that a novel protein, MIG-13, is required to position these cells correctly. MIG-13 is a transmembrane protein whose expression is restricted to the anterior and central body regions by Hox gene activity. MIG-13 functions non-cell autonomously within these regions to promote migration toward the anterior: loss of mig-13 activity shifts the Q descendants toward the posterior, whereas increasing the level of MIG-13 shifts them anteriorly in a dose-dependent manner. Our findings suggest that MIG-13 is a component of a global A/P migration system, and that the level of MIG-13 determines where along the body axis these migrating cells stop.     

Hox gene expression reveals regionalization along the anteroposterior axis of the zebrafish notochord

 The vertebrate Hox genes have been shown to confer regional identity along the anteroposterior axis of the developing embryo, especially within the central nervous system (CNS) and the paraxial mesoderm. The notochord has been shown to play vital roles in patterning adjacent tissues along both the dorsoventral and mediolateral axes. However, the notochord’s role in imparting anteroposterior information to adjacent structures is less well understood, especially as the notochord shows no morphological distinctions along the anteroposterior axis and is not generally described as a segmental or compartmentalized structure. Here we report that four zebrafish hox genes: hoxb1, hoxb5, hoxc6 and hoxc8 are regionally expressed along the anteroposterior extent of the developing notochord. Notochord expression for each gene is transient, but maintains a definite, gene-specific anterior limit throughout its duration. The hox gene expression in the zebrafish notochord is spatially colinear with those genes lying most 3’ in the hox clusters having the most anterior limits. The expression patterns of these hox cluster genes in the zebrafish are the most direct molecular evidence for a system of anteroposterior regionalization of the notochord in any vertebrate studied to date. Received: 30 March 1998 / Accepted: 16 June 1998     

Determination of cell fate along the anteroposterior axis of the Drosophila ventral midline

The Drosophila ventral midline has proven to be a useful model for understanding the function of central organizers during neurogenesis. The midline is similar to the vertebrate floor plate, in that it plays an essential role in cell fate determination in the lateral CNS and also, later, in axon pathfinding. Despite the importance of the midline, the specification of midline cell fates is still not well understood. Here, we show that most midline cells are determined not at the precursor cell stage, but as daughter cells. After the precursors divide, a combination of repression by Wingless and activation by Hedgehog induces expression of the proneural gene lethal of scute in the most anterior midline daughter cells of the neighbouring posterior segment. Hedgehog and Lethal of scute activate Engrailed in these anterior cells. Engrailed-positive midline cells develop into ventral unpaired median (VUM) neurons and the median neuroblast (MNB). Engrailed-negative midline cells develop into unpaired median interneurons (UMI), MP1 interneurons and midline glia.     

The BMP signaling gradient patterns dorsoventral tissues in a temporally progressive manner along the anteroposterior axis

Patterning of the vertebrate anteroposterior (AP) axis proceeds temporally from anterior to posterior. How dorsoventral (DV) axial patterning relates to AP temporal patterning is unknown. We examined the temporal activity of BMP signaling in patterning ventrolateral cell fates along the AP axis, using transgenes that rapidly turn "off" or "on" BMP signaling. We show that BMP signaling patterns rostral DV cell fates at the onset of gastrulation, whereas progressively more caudal DV cell fates are patterned at progressively later intervals during gastrulation. Increased BMP signal duration is not required to pattern more caudal DV cell fates; rather, distinct temporal intervals of signaling are required. This progressive action is regulated downstream of, or in parallel to, BMP signal transduction at the level of Smad1/5 phosphorylation. We propose that a temporal cue regulates a cell's competence to respond to BMP signaling, allowing the acquisition of a cell's DV and AP identity simultaneously.     

Structure and sequence of the human homeobox gene HOX7.

A cosmid containing the human sequence HOX7, homologous to the murine Hox-7 gene, was isolated from a genomic library, and the positions of the coding sequences were determined by hybridization. DNA sequence analysis demonstrated two exons that code for a homeodomain-containing protein of 297 amino acids. The open reading frame is interrupted by a single intron of approximately 1.6 kb, the splice donor and acceptor sites of which conform to known consensus sequences. The human HOX7 coding sequence has a very high degree of identity with the murine Hox-7 cDNA. Within the homeobox, the two sequences share 94% identity at the DNA level, all substitutions being silent. This high level of sequence similarity is not confined to the homeodomain; overall the human and murine HOX7 gene products show 80% identity at the amino acid level. Both the 5' and 3' untranslated regions also show significant similarity to the murine gene, with 79 and 70% sequence identity, respectively. The sequence upstream of the coding sequence of exon 1 contains a GC-rich putative promoter region. There is no TATA box, but a CCAAT and numerous GC boxes are present. The region encompassing the promoter region, exon 1, and the 5' region of exon 2 have a higher than expected frequency of CpG dinucleotides; numerous sites for rare-cutter restriction enzymes are present, a characteristic of HTF islands.     

Structural analysis of the human HOX4A homeobox gene.

The HOX4A gene, one of a cluster of homeobox-containing genes on human chromosome 2, has been isolated by screening a genomic cosmid library with the HOX4B cDNA probe. The amino acid sequence was predicted according to the conceptual translation of 13 homology groups of human HOX genes (1). The HOX4A gene consists of at least two exons separated by a long intron of 1860 bp. The HOX4A protein predicted from the nucleotide sequence of the HOX4A gene is comprised of 416 amino acid residues. Comparison of the predicted HOX4A protein with the HOX2G protein revealed three regions of sequence similarity: an N-terminal octapeptide, a hexapeptide (pre-box) upstream of the homeodomain, and the homeodomain at the C-terminus.     

Involvement of the Chox-4 chicken homeobox genes in determination of anteroposterior axial polarity during limb development.

We have isolated and identified four chicken homeobox genes in the upstream region of the Chox-4 complex. The Chox-4g and -4f genes, at the 5' extremity of the complex, were expressed locally in the vicinity of the zone of polarizing activity (ZPA) at early stages of limb development, substantiating the involvement of the genes in anteroposterior axis determination. To confirm their function, we implanted a bead containing retinoic acid, or the ZPA itself, in the anterior margin of the limb bud, leading to formation of mirror-image duplicated digits, and observed the resultant change in gene expression. Expression of the Chox-4g and -4f genes was induced in the new digit-forming region. Those results suggest that positional information assigned by a ZPA morphogen is imprinted on cellular memory by expression of the Chox-4 genes to maintain positional signaling along the anteroposterior axis in the limb field.     

Cloning and sequencing of the human homeobox gene HOX4A.

The HOX4A gene, one of the homeobox-containing genes on human chromosome 2, has been isolated by screening a genomic cosmid library with a HOX4B cDNA probe. The HOX4A gene consists of at least two exons separated by a long intron of 1860 bp. According to conceptual translation, the HOX4A protein is predicted to be composed of 416 amino acid residues. Interestingly, the HOX4A protein has a sequence, Pro-Ala-Ser-Gln-Ser-Pro-Glu-Arg-Ser, eight amino acids downstream from the homeodomain, which is similar to that containing a phosphorylation site in pp60c-src, Pro-Ala-Ser-Gln-Thr-Pro-Asn-Lys-Thr. However, the HOX2G protein, which exhibits a paralogous relationship with the HOX4A protein, does not possess the sequence which is similar to that in pp60c-src. A comparison of the predicted HOX4A protein with the HOX2G protein revealed four regions of amino acid sequence similarities: an N-terminal tetrapeptide, a pentapeptide (pre-box) upstream of the homeodomain, the homeodomain and a C-terminal octapeptide.     

Genes that control dorsoventral polarity affect gene expression along the anteroposterior axis of the Drosophila embryo

At least 13 genes control the establishment of dorsoventral polarity in the Drosophila embryo and more than 30 genes control the anteroposterior pattern of body segments. Each group of genes is thought to control pattern formation along one body axis, independently of the other group. We have used the expression of the fushi tarazu (ftz) segmentation gene as a positional marker to investigate the relationship between the dorsoventral and anteroposterior axes. The ftz gene is normally expressed in seven transverse stripes. Changes in the striped pattern in embryos mutant for other genes (or progeny of females homozygous for maternal-effect mutations) can reveal alterations of cell fate resulting from such mutations. We show that in the absence of any of ten maternal-effect dorsoventral polarity gene functions, the characteristic stripes of ftz protein are altered. Normally there is a difference between ftz stripe spacing on the dorsal and ventral sides of the embryo; in dorsalized mutant embryos the ftz stripes appear to be altered so that dorsal-type spacing occurs on all sides of the embryo. These results indicate that cells respond to dorsoventral positional information in establishing early patterns of gene expression along the anteroposterior axis and that there may be more significant interactions between the different axes of positional information than previously determined.     

Differential regulation by retinoic acid of the homeobox genes of the four HOX loci in human embryonal carcinoma cells.

We studied the expression of 38 human homeobox genes belonging to the four HOX complex loci in embryonal carcinoma (EC) cells induced to differentiate by culturing them in a medium containing retinoic acid (RA). Genes located at the 3' end of each one of the four HOX loci are activated by RA in a sequential order colinear with their 3' to 5' arrangement in the cluster: 3' HOX genes respond early to the drug while upstream genes respond progressively later. Among the genes located at the 5' end of HOX loci RNase protection analysis reveals that one HOX3 gene and four HOX4 genes are weakly expressed in EC stem cells and downregulated upon treatment with 10(-5) M RA. While activation of early responding genes does not require continuous protein synthesis, the observed timing and polarity of gene activation is disrupted in the absence of protein synthesis.     

Involvement of the Xenopus homeobox gene Xhox3 in pattern formation along the anterior-posterior axis

The Xenopus homeobox gene Xhox3 shows a graded expression in the axial mesoderm, with the highest concentration in the posterior end of frog gastrula and neurula embryos. To investigate the function of the Xhox3 gene, synthetic Xhox3 mRNA was injected into different regions of developing embryos. In particular, Xhox3 was supplied in excess to anterior cells, which normally have the lowest levels of Xhox3 RNA. The results show that injection of Xhox3, but not control, mRNA into prospective anterior regions of developing embryos produces a series of graded axial defects. The injected embryos gastrulate normally but fail to form anterior (head) structures. Our findings suggest that Xhox3 is involved in establishing anterior-posterior cell identities during pattern formation of the axial mesoderm in early embryonic development.