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Hox genes (also known as homeotic genes) are a group of related genes that control the body plan of an embryo along the anterior-posterior (head-tail) axis. After the embryonic segments have formed, the Hox proteins determine the type of segment structures (e.g. legs, antennae, and wings in fruit flies or the different types of vertebrae in humans) that will form on a given segment. Hox proteins thus confer segmental identity, but do not form the actual segments themselves.
Hox genes are defined as having the following properties:
The products of Hox genes are Hox proteins. Hox proteins are transcription factors, which are proteins that are capable of binding to specific nucleotide sequences on the DNA called enhancers where they either activate or repress genes. The same Hox protein can act as a repressor at one gene and an activator at another. The ability of Hox proteins to bind DNA is conferred by a part of the protein referred to as the homeodomain. The homeodomain is a 60-amino-acid-long DNA-binding domain (encoded by its corresponding 180-base-pair DNA sequence, the homeobox). This amino acid sequence folds into a "helix-turn-helix" (i.e. homeodomain fold) motif that is stabilized by a third helix. The consensus polypeptide chain is (typical intron position noted with dashes):
The homeodomain protein motif is highly conserved across vast evolutionary distances. In addition, homeodomains of individual Hox proteins usually exhibit greater similarity to homeodomains in other species than to proteins encoded by adjacent genes within their own Hox cluster. These two observations led to the suggestions that Hox gene clusters evolved from a single Hox gene via tandem duplication and subsequent divergence and that a prototypic Hox gene cluster containing at least seven different Hox genes was present in the common ancestor of all bilaterian animals.
The functional conservation of Hox proteins can be demonstrated by the fact that a fly can function perfectly well with a chicken Hox protein in place of its own. So, despite having a last common ancestor that lived over 670 million years ago, the chicken and fly version of the same Hox gene can actually take each other's places when swapped.
Drosophila melanogaster is an important model for understanding body plan generation and evolution. The general principles of Hox gene function and logic elucidated in flies will apply to all Bilaterian organisms, including humans. Drosophila, like all insects, has 8 Hox genes. These are clustered into two complexes, both of which are located on chromosome 3. The Antennapedia complex (not to be confused with the Antp gene) consists of 5 genes: labial (lab), proboscipedia (pb), Deformed (Dfd) Sex combs reduced (Scr) and Antennapedia (Antp). The Bithorax complex, named after the Ultrabithorax gene, consists of the remaining 3 genes: Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (abd-B).
The lab gene is the most anteriorly expressed gene. It is expressed in the head, primarily in the intercalary segment (an appendage-less segment between the antenna and mandible), and also in the midgut. Loss of function of lab results in the failure of the Drosophila embryo to internalize the mouth and head structures that initially develop on the outside of its body (a process called head involution). Failure of head involution disrupts or deletes the salivary glands, pharynx. The lab gene was initially so named because it disrupted the labial appendage; however, the lab gene is not expressed in the labial segment, and the labial appendage phenotype is likely a result of the broad disorganization resulting from the failure of head involution.
The pb gene is responsible for the formation of the labial and maxillary palps. There is evidence that pb interacts with Scr.
The Dfd gene is responsible for the formation of the maxillary and mandibular segments in the larval head. The mutant phenotypes of Deformed are similar to those of labial. Loss of function of Deformed in the embryo results in a failure of head involution (see labial gene), with a loss of larval head structures. Mutations in the adult have either deletions of parts of the head or transformations of head to thoracic identity.
The Scr gene is responsible for cephalic and thoracic development in Drosophila embryo and adult.
The 2nd thoracic segment, or T2, develops a pair of legs and a pair of wings. The Antp gene specifies this identity by promoting leg formation and allowing (but not directly activating) wing formation. A dominant Antp mutation, caused by a chromosomal inversion, causes Antp to be expressed in the antennal imaginal disc, so that, instead of forming an antenna, the disc makes a leg, resulting in a leg coming out of the fly's head.
The third thoracic segment, or T3, bears a pair of legs and a pair of halteres (highly reduced wings that function in balancing during flight). Ubx patterns T3 largely by repressing genes involved in wing formation. The wing blade is composed of two layers of cells that adhere tightly to one another, and are supplied with nutrient by several wing veins. One of the many genes that Ubx represses is blistered, which activates proteins involved in cell-cell adhesion, and spalt, which patterns the placement of wing veins. In Ubx loss-of-function mutants, Ubx no longer represses wing genes, and the halteres develop as a second pair of wings, resulting in the famous four-winged flies. When Ubx is misexpressed in the 2nd thoracic segment, such as occurs in flies with the "Cbx" enhancer mutation, it represses wing genes, and the wings develop as halteres, resulting in a four-haltered fly.
In Drosophila, abdominal-A (abd-A) is expressed along most of the abdomen, from abdominal segment 1 (A1) to A8. Expression of abdominal-A is necessary to specify the identity of most of the abdominal segments. A major function of abd-A in insects is to repress limb formation. In abd-A loss-of-function mutants, abdominal segments A2 through A8 are transformed into an identity more like A1. When abd-A is ectopically expressed throughout the embryo, all segments anterior of A4 are transformed to an A4-like abdominal identity. The abd-A gene also affects the pattern of cuticle generation in the ectoderm, and pattern of muscle generation in the mesoderm.
Abd-B is transcribed in two different forms, a regulatory protein, and a morphogenic protein. Regulatory Abd-B suppress embryonic ventral epidermal structures in the 8th and 9th segment of the Drosophila abdomen. Both the regulatory protein, and the morphogenic protein are involved in the development of the tail segment.
Proteins with a high degree of sequence similarity are also generally assumed to exhibit a high degree of functional similarity, i.e. Hox proteins with identical homeodomains are assumed to have identical DNA-binding properties (unless additional sequences are known to influence DNA-binding). To identify the set of proteins between two different species that are most likely to be most similar in function, classification schemes are used. For Hox proteins, three different classification schemes exist: phylogenetic inference based, synteny based, and sequence similarity based. The three classification schemes provide conflicting information for Hox proteins expressed in the middle of the body axis (Hox6-8 and Antp, Ubx and Abd-A). A combined approach used phylogenetic inference based information of the different species and plotted the protein sequence types onto the phylogenetic tree of the species. The approach identified the proteins that best represent ancestral forms (Hox7 and Antp) and the proteins that represent new, derived versions (or were lost in an ancestor and are now missing in numerous species).
Hox genes act at many levels within developmental gene hierarchies: at the "executive" level they regulate genes that in turn regulate large networks of other genes (like the gene pathway that forms an appendage). They also directly regulate what are called realisator genes or effector genes that act at the bottom of such hierarchies to ultimately form the tissues, structures, and organs of each segment. Segmentation involves such processes as morphogenesis (differentiation of precursor cells into their terminal specialized cells), the tight association of groups of cells with similar fates, the sculpting of structures and segment boundaries via programmed cell death, and the movement of cells from where they are first born to where they will ultimately function, so it is not surprising that the target genes of Hox genes promote cell division, cell adhesion, apoptosis, and cell migration.
|Organism||Target gene||Normal function of target gene||Regulated by|
|Drosophila||distal-less||activates gene pathway for limb formation||ULTRABITHORAX|
|distal-less||activates gene pathway for limb formation||ABDOMINAL-A|
|decapentaplegic||triggers cell shape changes in the gut that are |
required for normal visceral morphology
|reaper||Apoptosis: localized cell death creates the segmental |
boundary between the maxilla and mandible of the head
|decapentaplegic||prevents the above cell changes in more posterior |
|Mouse||EphA7||Cell adhesion: causes tight association of cells in |
distal limb that will form digit, carpal and tarsal bones
|Cdkn1a||Cell cycle: differentiation of myelomonocyte cells into |
monocytes (white blood cells), with cell cycle arrest
The DNA sequence that is bound by the homeodomain protein contains the nucleotide sequence TAAT, with the 5' terminal T being the most important for binding. This sequence is conserved in nearly all sites recognized by homeodomains, and probably distinguishes such locations as DNA binding sites. The base pairs following this initial sequence are used to distinguish between homeodomain proteins, all of which have similar recognition sites. For instance, the nucleotide following the TAAT sequence is recognized by the amino acid at position 9 of the homeodomain protein. In the maternal protein Bicoid, this position is occupied by lysine, which recognizes and binds to the nucleotide guanine. In Antennapedia, this position is occupied by glutamine, which recognizes and binds to adenine. If the lysine in Bicoid is replaced by glutamine, the resulting protein will recognize Antennapedia-binding enhancer sites.
However, all homeodomain-containing transcription factors bind essentially the same DNA sequence. The sequence bound by the homeodomain of a Hox protein is only 6 nucleotides long, and such a short sequence would be found at random many times throughout the genome, far more than the number of actual functional sites. Especially for Hox proteins, which produce such dramatic changes in morphology when misexpressed, this raises the question of how each transcription factor can produce such specific and different outcomes if they all bind the same sequence. One mechanism that introduces greater DNA sequence specificity to Hox proteins is to bind protein cofactors. Two such Hox cofactors are Extradenticle (Exd) and Homothorax (Hth). Exd and Hth bind to Hox proteins and appear to induce conformational changes in the Hox protein that increase its specificity.
Just as Hox genes regulate realisator genes, they are in turn regulated themselves by gap genes and pair-rule genes, which are in their turn regulated by maternally-supplied mRNA. This results in a transcription factor cascade: maternal factors activate gap or pair-rule genes; gap and pair-rule genes activate Hox genes; then, finally, Hox genes activate realisator genes that cause the segments in the developing embryo to differentiate. Regulation is achieved via protein concentration gradients, called morphogenic fields. For example, high concentrations of one maternal protein and low concentrations of others will turn on a specific set of gap or pair-rule genes. In flies, stripe 2 in the embryo is activated by the maternal proteins Bicoid and Hunchback, but repressed by the gap proteins Giant and Kruppel. Thus, stripe 2 will only form wherever there is Bicoid and Hunchback, but not where there is Giant and Kruppel.
Non-coding RNA (ncRNA) has been shown to be abundant in Hox clusters. In humans, 231 ncRNA may be present. One of these, HOTAIR, silences in trans (it is transcribed from the HOXC cluster and inhibits late HOXD genes) by binding to Polycomb-group proteins (PRC2).
In higher animals including humans, retinoic acid regulates differential expression of Hox genes along the anteroposterior axis. Genes in the 3' ends of Hox clusters are induced by retinoic acid resulting in expression domains that extend more anteriorly in the body compared to 5' Hox genes that are not induced by retinoic acid resulting in expression domains that remain more posterior.
Quantitative PCR has shown several trends regarding colinearity: the system is in equilibrium and the total number of transcripts depends on the number of genes present according to a linear relationship.
In some organisms, especially vertebrates, the various Hox genes are situated very close to one another on the chromosome in groups or clusters. Interestingly, the order of the genes on the chromosome is the same as the expression of the genes in the developing embryo, with the first gene being expressed in the anterior end of the developing organism. The reason for this colinearity is not yet completely understood. The diagram above shows the relationship between the genes and protein expression in flies.
The Hox genes are named for the homeotic phenotypes that result when their function is disrupted, wherein one segment develops with the identity of another (e.g. legs where antenna should be). Hox genes in different phyla have been given different names, which has led to confusion about nomenclature. The complement of Hox genes in Drosophila is made up of two clusters, the Antennapedia complex and the Bithorax complex, which together were historically referred to as the HOM-C (for Homeotic Complex). Although historically HOM-C genes have referred to Drosophila homologues, while Hox genes referred to vertebrate homologues, this distinction is no longer made, and both HOM-C and Hox genes are called Hox genes.
Humans have Hox genes in four clusters:
|HOXA||chromosome 7||HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13|
|HOXB||chromosome 17||HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXB13|
|HOXC||chromosome 12||HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12, HOXC13|
|HOXD||chromosome 2||HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12, HOXD13|
The Hox genes are so named because mutations in them cause homeotic transformations. Homeotic transformations were first identified and studied by William Bateson in 1894, who coined the term "homeosis". After the rediscovery of Mendel's genetic principles, Bateson and others realized that some examples of homeosis in ﬂoral organs and animal skeletons could be attributed to variation in genes.
Deﬁnitive evidence for a genetic basis of some homeotic transformations was obtained by isolating homeotic mutants. The ﬁrst homeotic mutant was found by Calvin Bridges in Thomas Hunt Morgan's laboratory in 1915. This mutant shows a partial duplication of the thorax and was therefore named Bithorax (bx). It transforms the third thoracic segment (T3) toward the second (T2). Bithorax arose spontaneously in the laboratory and has been maintained continuously as a laboratory stock ever since.
The genetic studies by Morgan and others provided the foundation for the systematic analyses of Edward B. Lewis and Thomas Kaufman, which provided preliminary definitions of the many homeotic genes of the Bithorax and Antennapedia complexes, and also showed that the mutant phenotypes for most of these genes could be traced back to patterning defects in the embryonic body plan.
Ed Lewis, Christiane Nüsslein-Volhard and Eric F. Wieschaus identified and classified 15 genes of key importance in determining the body plan and the formation of body segments of the fruit fly Drosophila melanogaster.[when?] For their work, Lewis, Nüsslein-Volhard, and Wieschaus were awarded the Nobel Prize in Physiology or Medicine in 1995.
In 1983, the homeobox was discovered independently by researchers in two labs: Ernst Hafen, Michael Levine, and William McGinnis (in Walter Gehring's lab at the University of Basel, Switzerland) and Matthew P. Scott and Amy Weiner (in Thomas Kaufman's lab at Indiana University in Bloomington).