Interactive Fly, Drosophila

Fish Abd-B homologs

The structural organization and expression of Abd-B related zebrafish HoxA cluster genes (Hoxa-9, Hoxa-10, Hoxa-11 and Hoxa-13) have been examined, as well as the structure and organization of Evx-2 (Drosophila homolog: Even-skipped), a gene closely linked to the HoxD complex. The genomic organization of Hoxa genes in fish resembles that of tetrapods albeit intergenic distances are shorter. During development of the fish trunk, Hoxa genes are coordinately expressed, whereas in pectoral fins, they display transcript domains similar to those observed in developing tetrapod limbs. Likewise, the Evx-2 gene seems to respond to both Hox- and Evx-types of regulation. During fin development, this latter gene is expressed as are the neighbouring Hox genes (that is complying with colinearity), in contrast Evx-2's expression in the central nervous system, which does not comply with colinearity and extends up to anterior parts of the brain. These results are discussed in the context of the functional evolution of Hoxa versus Hoxd genes and their different roles in building up paired appendages (Sordino, 1996).

Fibroblast growth factor (Fgf) and retinoic acid (RA) signals control the formation and anteroposterior patterning of posterior hindbrain. They are also involved in development processes in other regions of the embryo. Therefore, responsiveness to Fgf and RA signals must be controlled in a context-dependent manner. Inhibiting the caudal-related genes cdx1a and cdx4 in zebrafish embryos caused ectopic expression of genes that are normally expressed in the posterior hindbrain and anterior spinal cord, and ectopic formation of the hindbrain motor and commissure neurons in the posteriormost neural tissue. Combinational marker analyses suggest mirror-image duplication in the Cdx1a/4-defective embryos, and cell transplantation analysis further revealed that Cdx1a and Cdx4 repress a posterior hindbrain-specific gene expression cell-autonomously in the posterior neural tissue. Expression of fgfs and retinaldehyde dehydrogenase 2 suggested that in the Cdx1a/4-defective embryos, the Fgf and RA signaling activities overlap in the posterior body and display opposing gradients, compared with those in the hindbrain region. Fgf and RA signals were required for ectopic expression. Expression of the posterior hox genes hoxb7a, hoxa9a or hoxb9a, which function downstream of Cdx1a/4, or activator fusion genes of hoxa9a or hoxb9a (VP16-hoxa9a, VP16-hoxb9a) suppressed this loss-of-function phenotype. These data suggest that Cdx suppresses the posterior hindbrain fate through regulation of the posterior hox genes; the posterior Hox proteins function as transcriptional activators and indirectly repress the ectopic expression of the posterior hindbrain genes in the posterior neural tissue. These results indicate that the Cdx-Hox code modifies tissue competence to respond to Fgfs and RA in neural tissue (Shumizu, 2006).

Understanding the evolutionary transformation of fish fins into tetrapod limbs is a fundamental problem in biology. The search for antecedents of tetrapod digits in fish has remained controversial because the distal skeletons of limbs and fins differ structurally, developmentally, and histologically. Moreover, comparisons of fins with limbs have been limited by a relative paucity of data on the cellular and molecular processes underlying the development of the fin skeleton. This study provides a functional analysis, using CRISPR/Cas9 and fate mapping, of 5' hox genes and enhancers in zebrafish that are indispensable for the development of the wrists and digits of tetrapods. Cells marked by the activity of an autopodial hoxa13 enhancer exclusively form elements of the fin fold, including the osteoblasts of the dermal rays. In hox13 knockout fish, a marked reduction and loss of fin rays was found to be associated with an increased number of endochondral distal radials. These discoveries reveal a cellular and genetic connection between the fin rays of fish and the digits of tetrapods and suggest that digits originated via the transition of distal cellular fates (Nakamura, 2016).

Amphibian Abdominal-B homologs

The expression of Hox complex genes in correct spatial and temporal order is critical to patterning of the body axis and limbs during embryonic development. In order to understand the role such genes play in appendage regeneration, the expression of two 5' Hox complex genes, Hoxb13 and Hoxc10, were compared during development and regeneration of the body axis and the limbs of axolotls. In contrast to higher vertebrates, Hoxb13 is expressed not only in the tip of the developing tail, but also in the distal mesenchyme of developing hind limbs, and at low levels in developing forelimbs. Hoxc10 is expressed as two transcripts during both development and regeneration. The short transcript (Hoxc10S) is expressed in the tip of the developing tail, in developing hind limbs, and at low levels in developing forelimbs. The long transcript (Hoxc10L) is expressed in a similar pattern, with the exception that no expression in developing forelimbs could be detected. Hoxb13 and both transcripts of Hoxc10 are expressed at high levels in the regenerating spinal cord during tail regeneration, and in both regenerating hind limbs and forelimbs. The up-regulation of expression of these genes during forelimb regeneration, relative to the very low levels of expression during forelimb development, suggests that they play a critical and perhaps unique role in regeneration. This is particularly true for Hoxc10L, which is not expressed during forelimb development, but is expressed during forelimb regeneration; thus making it the first truly 'regeneration-specific' gene transcript identified to date (Carlson, 2001).

Transactivation domains of Abdominal-B homologs

Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions that ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analyses. The transcriptional activation domains have been identified for three human Hox proteins, HOXB1, HOXB3, and HOXD9, that interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery, depending on the structure of their target regulatory elements (Vigano, 1998).

A conventional deletion analysis on the 270-aa N terminus of HOXD9 shows that the first 75 residues contain a potential transcriptional activator when tested in the context of a Gal4 chimeric protein. In contrast, this region is dispensable when the activity of the protein is tested on the HCR (Hox control region, a 90-bp, ATTA-rich autoregulatory element identified in the HOXD9 locus), a context in which most of the activating function appears to be located within residues 76 to 264. The regions identified by the two alternative assays share no obvious characteristics with canonical eukaryotic activator domains and are only loosely conserved among different vertebrate species. The activation domain of another posteriorly expressed Hox protein, HOXD8, can be localized to a similar sub-N-terminal region. HOXD8 and HOXD9 bind the multiple ATTA-containing sites within the HCR as monomers in a noncooperative fashion, while Gal4-DBD chimeras bind the Gal4-responsive element (UAS) as a homodimer, a context that could force the HOXD9 N-terminal region to assume a different structural conformation and unmask a potential activating function in the N-terminal 75 residues. For the HOXB1-PBX heterodimer, the analysis carried out on the natural autoregulatory element identifies a transcriptional activation domain in a Ser-Pro-rich, 52-residue sub-N-terminal region. This region also contains most of the HOXB1 transcriptional activity when tested as a Gal4-DBD chimera, a possible indication that the 52-residue region assumes a similar conformation or activates transcription by a similar mechanism, either in the context of a homodimer or in that of a HOX-PBX heterodimer. The activity of HOXB3 was tested in three different contexts, i.e., upon binding DNA as a monomer to an ATTA-containing element: as a HOX-PBX heterodimer to a bipartite HOX-PBX core element, and as a Gal4-DBD chimeric homodimer to the Gal4-responsive element. Although in the context of a monomer the transcriptional activity is spread over the entire protein sequence, only the C terminus contains a potent activator domain in the context of a Gal4 homodimer or of a PBX heterodimer. The 71-residue C terminus is relatively highly conserved in the mammalian group 3 Hox proteins (Vigano, 1998).

Functional interactions between Abdominal-B homologs

Hox genes show related sequences and overlapping expression domains that often reflect functional redundancy as well as a common evolutionary origin. To accurately define their functions, it has become necessary to compare phenotypes of mice with single and multiple Hox gene mutations. A focus was placed on two Abd-B-type genes, Hoxa-10 and Hoxa-11, which are coexpressed in developing vertebrae, limbs, and reproductive tracts. To assess possible functional redundancy between these two genes, Hoxa-10/Hoxa-11 transheterozygotes were produced by genetic intercrosses and analyzed. This compound mutation results in synergistic defects in transheterozygous limbs and reproductive tracts, but not in vertebrae. In the forelimb, distal radial/ulnar thickening and pisiform/triangular carpal fusion are observed in 35% and 21% of transheterozygotes, respectively, but are effectively absent in Hoxa-10 and Hoxa-11 +/- forelimbs. In the hindlimb, distal tibial/fibular thickening and loss of tibial/fibular fusion were observed in greater than 80% of transheterozygotes but in no Hoxa-10 or Hoxa-11 +/- hindlimbs, and all transheterozygotes display reduced medial patellar sesamoids, compared to modest incidences in Hoxa-10 and Hoxa-11 +/- mutants. Furthermore, while the reproductive tracts of Hoxa-10 and Hoxa-11 single heterozygous mutants of both sexes are primarily unaffected, male transheterozygotes display cryptorchidism and abnormal tortuosity of the ductus deferens, and female transheterozygotes exhibit abnormal uterotubal junctions and narrowing of the uterus. In addition the targeted mutations of Hoxa-10 and Hoxa-11 each affect the expression of the other gene in the developing prevertebra and reproductive tracts. These results provide a measure of the functional redundancy of Hoxa-10 and Hoxa-11 and a deeper understanding of the phenotypes resulting in the single mutants and help elucidate the regulatory interactions between these two genes (Branford, 2000).

Linkage of Hoxd13 and Evx-2

The mouse Evx-2 gene is located in the Hox immediate vicinity of the Hoxd-13 gene, the most posteriorly expressed gene of the HOXD complex. While the Evx-1 gene is also physically linked to the HOXA complex, it is more distantly located from the corresponding Hoxa-13 gene. The expression of Evx-2 during development has been analyzed and it has been compared to that of Evx-1 and Hoxd-13. Even though Evx-2 is expressed in the developing CNS in a pattern resembling that of other Evx-related genes, the overall expression profile is similar to that of the neighboring limbs and genitalia. It is proposed that the acquisition of expression features typical of Hox genes, together with the disappearance of some expression traits common to Evx genes, is due to the close physical linkage of Evx-2 to the HOXD complex, which results in Evx-2 expression being partly controlled by mechanisms acting in the HOX complex. This transposition of the Evx-2 gene next to the Hoxd-13 gene may have occurred soon after the large scale duplications of the HOX complexes. A scheme is proposed to account for the functional evolution of eve-related genes in the context of their linkage to the HOM/Hox complexes (Dolle, 1994).

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