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HOX CLUSTERS

     Homeodomain genes are important transcription factors in eukaryotes and animals possess a number of homeodomain genes, many of which function in embryonic development.  Some of these genes are arranged in tandem clusters.  Although one set has received a great deal of study, often referred to simply as the Hox clusters, there is growing evidence of additional clusters which are related to the Hox cluster such as the NKL cluster, ParaHox cluster, EHGbox cluster, and the genes Evx and Mox (which, together with the Hox cluster, form the “extended Hox” cluster; Pollard, 2000.  While the most primitive animals lacked these clusters of Hox genes, they nevertheless possessed members of these groups.  Only 2 Hox genes known from the primitive group of animals called placozoans, including Trox-2, member of Hox/ParaHox family (Martinelli, 2004).

   Although sponges lack Hox/ParaHox cluster genes, they do possess Msx, NKL, and Tlx genes (Gauchat, 2000).   Sponges do show an axis during embryonic development and the differentiation of spicules.  Their Hox genes may be involved in these processes (Kruse, 1994).  Of the 13 families of non-Hox gene families, cnidarians possess family members of nine of them including Msx, Emx, Barx, Tlx, NK-2, Prh/Hex, Not, and Dlx.  Cnidarians possess gene members of two of the three classes of Hox cluster genes (anterior and posterior but not medial in addition to the two additional genes of the ‘extended Hox’ cluster, Evx and Mox.  They also possess two of the three genes of the ParaHox cluster (Gsx and Cdx but not Xlox) (Gauchat, 2000; Hill, 2003).  A simple cnidarian, Hydra,  is depicted below.

HYDRA

     Hox genes assumed a role in the development of body plans early in animal evolution.  Some sponge larvae (such as the amphiblastulae) have polarity which may involve homeodomain proteins (Muller, W.E.G., from Muller, 1998.)   Hox genes in cnidarians are involved in organizing the body plan(Yanze, 2001).

 

1)     HOX CLUSTERS, the Hox/HOM SUPERCLASS

      The embryonic development of higher vertebrates requires the complex body patterning which involves the expression of Hox genes arranged in Hox clusters.  Comparative genomics suggests that these multiple clusters evolved from one ancestral cluster and that the ancestral cluster evolved from tandem duplications of an ancestral orphan Hox gene (one which was not part of a cluster).  Many orphan Hox genes are involved in development.  Early in animal evolution, an orphan Hox gene (similar to EVX) was duplicated to produce a pair of Hox genes in tandem.  These genes were then duplicated and duplicated again to produce clusters of Hox genes.  A fascinating aspect of these clusters is that the order of the genes in the cluster is reflected by the order in which they are expressed in animals—the gene order in mammals corresponds to position along the anterior/posterior axis, the axis of limbs, and the regions of the intestines.

     There are 3 separate groups in a cluster which reflect the evolution of the cluster: anterior, medial, and posterior genes.  All members of each of the anterior, medial, and posterior groups resulted from tandem duplications of a single ancestral anterior, medial, and posterior genes.  In humans, the Hox genes numbered 1-3 belong to the anterior group, 4-8 to the medial group, and 9-13 to the posterior group.  In mammals, there are 4 such clusters; each gene is about 10 kilobases and each cluster about 100 kb.  Interestingly, there are 2 Hox genes Evx and Dlx which are not considered part of these clusters but are nevertheless located near some of the clusters on the same chromosomes (Ruddle, 1994).  

 

DIPLOBLASTIC ANIMALS

    The majority of families of Orphan Hox genes have homologs in diploblastic animals (such as the Hydra below).

HYDRA

  Cnidarians have a “proto-Hox” cluster resulting from a tandem duplication of the Hox gene found in sponges.  One of these genes (cnox-1) is similar to the anterior Hox genes of bilaterans, the other (cnox-9) is similar to the posterior bilateran genes.  There are at least 9 Hox genes known from cnidarians (as many as 7 can occur in a single species) and as many as 5 may be related to Hox cluster genes (such as a homolog of even-skipped) (Finnerty, 1997; Kuhn, 1996; Peterson, 2000).The expression of Hox genes in Cnidarians is specific to different developmental stages (Peterson, 2002) and one of the Homeobox genes known from cnidarians, cnox-Pc1 seems to be involved in both pattern formation and the differentiation of skeletal muscle (Aeme, 1995).  In Hydra, genes of the Hox/ParaHox cluster family are involved in the differentiation of head structures (Gauchat, 2000).  Hydrozoans involve Cnox-2 in polyp polymorphism and has a specific expression pattern along the body axis (Cartwright, 1999). 

   Ctenophores, which may represent the sister group of bilateran animals, have a three-gene Hox cluster, including a medial Hox gene (Peterson, 2000).  Similarities in Hox sequences between parasitic myxozoans and animals such as ctenophores and even chordates indicate that myxozoans have degenerated from their ancestral condition (Anderson, 1998).

The acoel Symsagittifera roscoffensis possesses a gene which seems to be the homolog of the ancestral gene which produced Hox4 (Dfd), Hox5 (Scr) and proto-subcentral genes. The proto-subcentral gene later was duplicated to form the Antp and proto-Ubx genes, the latter of which was duplicated to form the Ubx (Lox2) and Abd-A (Lox4) genes. The central genes are specific to bilaterans and were critical to the development of the bilateran body plan (Ogishima, 2007).

Cnidarian Cluster:

cluster 1

Ctenophore Cluster:

cluster 2

BILATERANS
planarian

At least 10 Hox genes (both orphan and Hox cluster genes) are known from platyhelminthes (planarians).  Three paralogs of the Hox cluster are known: paralogs of lab/Hox1, Dfd/Hox4, and Antp/Hox7.  Another gene might be the paralog of pb (Balavoine, 1995).  At least one of these genes is known to be involved in the development of the anteroposterior axis (Orli, 1999).  The Drosophila Hox cluster gene lab is homologous to the gene Pnox3 in planarians.   Pnox1a/b and Smox1 from the platyhelminths and lin39 and mab5 from C. elegans are members of the Atp/Dfd class.  There are no members of the Abd-B class known in animals without coeloms.  The ancestor of coelomates had at least 5 members in this cluster: lab, pb/Hox3, Dfd, Antp, and Abd-B.  The genes of this cluster are homologous to orphan Hox genes in more primitive organisms. 

 (Balavoine, 1995; 1998).  

antennipedia

Nematode (C.elegans) embryological development requires Hox genes for both anterior (ceh-13 homolog of lab/Hox1) and posterior (nob-1 and php-3 genes) patterning.  C.elegans has a cluster of 4 Hox genes with 2 additional Hox genes at other chromosomal locations.  No medial gene has yet been identified.  Hox genes in annelids are involved in both segmentation and organogenesis  (Van, Auken, 2000).  In flies, all Hox cluster genes are required for embryonic development but in C.elegans this is only true of most of them (Streit, 2002).

Hox genes vary among different species of nematodes.  Some orthologs (such as Hox3 and a central antennapedia-like gene) exist in some nematodes which are absent in Caenorhabditis genomes.  The genus Caenorhabditis possesses an extra posterior gene from a recent duplication (nob-1) (Aboobaker, 2003).

In both polychaete annelid Chaetopterus and sea urchins, Hox genes are used in those cells “Set-aside cells” which give rise to adult body plan.  (Peterson, 2000). 

 

COELOMATES

In a developing coelomate body, cells must know their positions.  There are several axes which may exist in an organisms which are regulated by Hox genes: There is a longitudinal axis, such as that which would occur along the length of this Amphio

lancelet
There is an axis along the brain, as evidenced by the development of segments in the hindbrain of the chick in the following image.
rhombomeres
Tetrapods also develop and axis along their developing limbs, as in the pid limb below.
limb

Hox genes were incorporated into the control of the developing longitudinal axis of the body and that of the posterior brain in the last common ancestor of protostomes and deuterostomes.  (Kammermeier, 2001).

     The ancestor of all coelomates had at least 5 Hox cluster genes: 2 anterior genes (lab/Hox1, pb/Hox2), 2 medial genes (dfd/Hox4, Ant/), and 1 posterior gene (AbdB/Hox9). The ribbonworm Lineus is thought to represent a lineage which split from those of other coelomates at about the point of this ancestral coelomate.  It has one cluster of at least 6 genes: 2 of the anterior class, three of the middle class, and 1 of the posterior class.  LsHox1 is homologous to Hox1 and labial; LsHox3 is homologous to Hox3 and proposcidea, LsHox4 is homologous to Hox4 and Dfd, LsHox7 is homologous to Hox7 and Antp, and LsHox9 is homologous to Hox9 and AbdB. Another medial gene, Lhox6 is homologous to vertebrate Hox6 genes.  Although Drosophila possesses additional medial genes in its cluster, such as Scr, LsHox6 is more similar to Antp than to Scr.  LsHox7 is also more similar to Antp than other Drosophila genes (Kmita-Cunisse, 1998; (Balavoine, 2002).  The number of ancestral protostome Hox genes may have been 9 or 10, instead of the 8 found in modern insects (Telford, 2000).  Mollusks have two posterior Hox genes (Barucca, 2003). 

 

 

ARTHROPODS

      After the split of the protostome and deuterostome lineages, some protostomes acquired additional Hox genes (such as Ubx, Scr, and AbdA) and vertebrate ancestors acquired additional Hox genes to result in a total of 13.  The Hox sequences of squid are more similar to annelid, brachiopod, and nemertean sequences than to arthropods and include two posterior group genes, as opposed to the single gene found in insects (Callaerts, 2002). The loss of Abd-A in barnacles may have been involved in the loss of abdominal segments. Significant Hox differences are observed when comparing Drosophila to Oncyphorans (a sister group of arthropods) such as the expression pattern of Ubx and its ability to repress Dll (Distalless); such modifications might have been crucial in the evolution of the arthropod or insect body plans. The Ultrabithorax gene of onynchohorans, when expressed in Drosophila, is capable of producing some, but not all, of the developmental changes induced by Drosophila Ultrabithorax.  During the course of insect evolution, modifications occurred in Ultrabithorax function, expanding its activity (Grenier, 2000).  While insects, onychophorans, and other arthropods possess the Ultrabithorax homeobox protein, there is a regulatory domain present in the carboxy end of the protein found in insects which is absent in onycophorans and other arthropods.      

     The onycophoran Ultrabithorax protein, when expressed in Drosophila, can induce some, but not all, of the developmental changes induced by the Drosophila Ultrabithorax protein.  The domain of the protein which was added in the insect lineage may have been involved in the specialization of the higher insect form.  The most primitive insects (such as collembolans) lack this addition to Ultrabithorax and differ from higher insects in that they possess limbs on their abdomens and the last thoracic and first abdominal segments are not well differentiated from each other.  (Galant, 2002). 

     Hox gene expression in all arthropods divides the heads into 6 segments, demonstrating a common ancestry (this is true even in spiders, which, until recently were thought to be missing the first antennal segment) (Damen 1998). Common genetic mechanisms, such as distal-less (Dll) and Ubx/abdA expression, direct the development of all arthropod limbs, suggesting that they evolved from a common ancestral form (Panganiban, 1995).

In insects, the expression pattern of Hox genes was modified to result in shorter, non-overlapping segments.  Although the same Hox genes are expressed in crustacean and insect heads, there are differences in the expression of Hox genes such as lab, pb, and Dfd.  Insect and crustacean heads possess modified versions of the same ancestral structures (Abzhanov, 1999).  Comparisons of Hox gene expression patterns in the heads of chelicerates and other arthropods show that divergent head structures are composed of equivalent head segments.  Chelicerates retain their deuterocerebral segment, which is not obvious from morphological studies (Telford, 1998).

    The difference in function of the first thoracic limbs (legs in insects; maxillipeds for feeding in crustaceans) may be the result of observed differences in Hox gene expression between these groups in these appendages (Ubx and AbdA).  In woodlice, the first thoracic appendage begins development as a leg and then tranforms to a maxilliped; this change is associated with the expression of Scr (Averof, 1997b).  Specializations in insect legs has occurred through changes in the temporal and spatial expression of Hox genes such as Ultrabithorax and abdominal-A (Mahfooz, 2004). A number of changes have been observed in invertebrates such as tandem duplications and the splitting of hox clusters through the insertion of other genes (Wagner, 2003)

     Epipodytes are gill-like appendages of crustacean legs which may have been the structures from which insect wings evolved. 

epipodite

These epipodytes (and crustacean legs in general) express several genes which insects during wing development such as apterous and the POU-domain Hox gene pdm1.  The use of pdm1 in the crustacean leg has no known function; the use of apterous appears to have no function.  Thus the development of wings from epipodytes seems to have involved signals which were already present in the crustacean leg (Averof, 1997a).  In the ancestral condition of winged insects (such as that observed in dragonflies), the forewings and hindwings are similar. Both butterflies and flies express Ubx in their hindwings, although the hindwing of flies is modified to form a balancing organ, the haltere.  In flies, Ubx is involved in the formation of the haltere and some of the downstream gene expression varies between flies and butterflies (Weatherbee, 1999).

     Flies have modified their second pair of wings (the primitive condition of insects is two pairs of wings) to stubby balancing organs known as halteres.  The Hox cluster gene Ultrabithorax (Ubx) represses genes required for normal wings in the development of halteres.  Misexpression of Ubx can turn what should be normal wings into halteres. 

     The Drosophila homeobox cluster includes a homolog of deuterostome Hox3, names zerknüllt.   A recent duplication of zerknüllt in insects has produced the bicoid gene of insects which is produced in the maternal nurse cell/oocyte syncytium to establish the anterior-posterior axis of the embryo (Stauber, 1999).

Two Hox genes in Drosophila, ftz and zen, are located in the Hox cluster but have been modified for other functions other than the establishment of the anterior-posterior axis (Wada, 1999). Ftz in primitive insects has a homeotic function and some role in segmentation.  In flies, the homeotic function is lost and ftz functions only in segmentation as a pair rule gene (Lohr, 2001).

 (Balavoine, 2002; (Akam, 1998).  Mollusks have a PG-3 gene homologous to zen2, zen, and bicoid of Drosophila (Barucca, 2003). 

      The Hox clusters of diverse coelomates are similar.

Ribbonworm:

cluster

Fly:

cluster

Vertebrate:

cluster

Sea urchins have a single Hox cluster of 10 genes which is essentially the same as that in chordates despite the differences in body plans such as the lack of many chordate head organs.  (There are some small differences: sea urchins have 1 gene of the PG4-5 group while hemichordates and cephalochordates have 2, PG4 and PG5; sea urchins have 3 genes of the PG 13 type while Amphioxus has 4 and vertebrates have 5.)  (Martinez, 1999; (Mito, 2000).    Echinoderms have modifications of the expression of their orphan Hox genes which are involved in the formation of their derived body plan (Lowe, 1997).  Crinoids, an early outgroup to all other modern echinoderms, possess at least 3 anterior Hox genes, 4 medial Hox genes, and 4 posterior Hox genes (Mito, 2000).  Echinoderms have a hox cluster which is equivalent to that of chordates even though their body plan is highly modified

 

 

 

SEA URCHIN CLUSTER

cluster
CHORDATE CLUSTER
cluster
     Most tunicates possess a single Hox cluster.  Some tunicates (such as Ciona intestinalis) lack Hox cluster genes known in all other chordates and the Hox cluster has been rearranged, breaking up the cluster (Spangnuolo, 2003).  A tunicate embryo is depicted below.
tunicate larva
Although lancelets lack the obvious rhombomeres evident in the hindbrains of vertebrates, expression of the Hox gene AmphiFoxB occurs in a repeated segments along the anterior-posterior axis of the hindbrain.  This seems that a cryptic segmentation induced by the surrounding somites had evolved before the origin of the vertebrates (Mazet, 2002; Wada, 1999).  The position of genes in the HOX clusters also correspond to the order of their expression in the gastrointestinal tract (Yahagi, 2001).  The brain of Amphioxus is depicted below.
head

In lampreys, the first pharyngeal arch develops into the muscular pumping velum with its single region cartilage while in gnathostomes the first arch (the mandibular arch) develops into separate dorsal and ventral regions of cartilage.  A Hox gene is expressed in the first arch of lampreys that is not expressed in the first arch of gnathostomes; the loss of this gene’s expression may have been involved in the evolution of vertebrate jaws (Cohn, 2002).  Lampreys have multiple clusters of hox genes but they appear to have arisen through a separate duplication event (Wagner, 2003).  

     During the evolution of the vertebrates, a number of duplications of this ancestral HOX cluster of 13 genes occurred.  Thus higher vertebrates have at least 4 Hox clusters (HOXA, HOXB, HOXC, and HOXD) which are all descendants of a cluster homologous to that found in invertebrates.  Although the order is not certain, it seems that after the first cluster duplication, a duplication of one of the two clusters led to the HOXA and HOXB clusters while a duplication of the second cluster led to the HOXC and HOXD clusters.  HOXA1, B1, C1, and D1 are equivalent and experiments in mice have shown that mutants can often be rescued by the corresponding HOX gene from another cluster.

Some genes in the individual clusters have been lost: fish have lost the HOXA6, A7, D1, and D8 genes present in mammals while mammals have lost B10, C1A, and C3A present in fish.  In humans, each cluster spans about 200 kilobases of DNA and is composed of 9-11 genes (OMIM).

       Teleost fish (which compose over 99% of modern fish species and individuals) have additional duplications of HOX clusters for a total of 5 to 8 clusters.  This duplication of Hox chromosome segments occurred after the divergence of ray-finned and lobe-finned fishes and perhaps genetic events such as these had a role in the subsequent success of the teleosts (Chiu, 2002; .Amores, 1998).    Protosotomes possess only one Hox cluster while mammals possess four and zebrafish possess seven.  The zebrafish HoxAa and HoxAb clusters are duplications of the single HoxA cluster found in most vertebrates.  These teleost duplications of the HoxA cluster were modified with regard to their regulatory elements and lost several sequences conserved between sharks and humans (Chiu, 2001).  The existence of Hox14 genes in lancets, horn sharks, and coelocanths indicate that the ancestral chordate Hox cluster possessed 14 genes, although the last gene was lost independently in teleosts and tetrapods (Ferrier, 2004).

ANCESTRAL VERTEBRATE CLUSTER

cluster

DUPLICATION OF ANCESTRAL CLUSTER PRODUCES 2 CLUSTERS A/B AND C/D

cluster
cluster
DUPLICATION OF EACH OF THESE TWO CLUSTERS PRODUCES 4 VERTEBRATE HOX CLUSTERS
cluster
cluster
cluster
cluster

SUBSEQUENT GENE LOSS PRODUCES THE CLUSTERS AS THEY EXIST TODAY;

THE FOLLOWING CLUSTERS REPRESENT HOXA, HOXB, HOXC, AND HOXD CLUSTERS

HOXA
HOXB
HOXC
HOXD

While the vast majority of fish today are actinopterygians (such as the teleosts), amphibians evolved from the sarcopterygians (whose modern descendants are limited to lungfish and coelocanths). Lungfish possess 4 Hox clusters, as in tetrapods and unlike the additional sets found in teleosts (Longhurst, 1999).  Coelocanths possess 33 Hox cluster genes, of which  32 are homologs of those found in tetrapods (including HoxA6, D1, and D8 which are not known in actinopterygian fish).  HoxC1, present in coelocanths, was lost in the evolutionary line which led to mammals.  The Hox clusters of coelocanths are more similar to those of mammals than they are to those of actinopterygian fish (Koh, 2003).

Hoxa and Hoxb clusters involved in anteroposterior axis of digestive tract (Sakiyama, 2001).  There has been large modifications in the noncoding sequences In Hox clusters (Prohaska, 2004).

     Not only do vertebrates utilize the Hox cluster for positional signals along the anterior/posterior axis of the body, they also use them to delineate position along the limbs, with the last Hox genes, Hox 11, 12, and 13, involved in the formation of digits.  The initial formation of fins/arms is similar in fish and tetrapods.  In tetrapods, Hox genes continue to be expressed in the limb at the point where their expression stops in fish.  In humans and mice, HoxA13 and D13 are required for the normal development of fingers and mutations can either produce extra fingers or the absence of fingers (the absence of fingers being A13/D13 double mutants).  In experimental conditions in mice, an interesting correlation was seen: normal doses of HoxA13 and D13 produce normal fingers, a lower dose produced shorter fingers, a lower dose produced extra fingers, and no gene product produced a wrist without fingers.  This is interesting because the fossil record indicates that during tetrapod evolution the stages proceeded as a limb without fingers, a polydactyl hand with short fingers, a pentdactyl hand with short fingers, and then pentdactyl hands with longer fingers (Zakany, 1997; Zimmer, 1996).

 

     Hox D11, 12, 13, and Hox A13 are also involved in the formation of the baculum (penis/clitoral bone).  Altering Hox expression changes the size of the baculum and can cause its absence.  These mutations also affect the penis.  Perhaps the fact that organism-wide changes in these Hox genes affect reproductive structures led to the stabilization of the pentdacyl hand (Dickman, 1997).   Hox genes are involved in hematopoesis and mutations can be factors in leukemia (Pineault, 2002).  In whales, changes in the expression of HoxC8 seem to be correlated with changes in axial development (Shashikant, 1998).

 

HOXA—chromosome 7

HOXA1

--Fetuses which are homozygous for mutations are stillborn due to abnormalities such as hindbrain problems, a neural tube that doesn’t close, missing cranial nerves and ganglia, 5 rhombomeres composing the brain instead of 7, and absence of some middle ear structures.  One allele (histidine73 changed to arginine) may have a role in autism. The proper development of the brain respiratory centers require the expression of homeobox transcription factors such as Krox, HoxA1, and kreisler (Chatonnet, 2006).

 

HOXA2

      This is the most anteriorly expressed Hox gene.  Mutations affect the paths of motor neuron axons and cause abnormalities in brain rhombomeres 2 and 3.Mutations in HOXA2 can cause microtia, the malformation of absence of the external ear (Alasti, 2008).

HOXA3

     Mutations in this gene cause cartilage abnormalities.  Cartilage is depicted below.

CARTILAGE

 

HOXA4

Hoxa-4 seems to function in the development of vertebrae C3 and C7.  Some Hoxa-4 mutant mice developed ribs on their last cervical vertebra (Horan, 1994).

 

HOXA5

      This gene is found mutated in many breast cancers.  Decreased activity of HOXA5 decreases the activity of the tumor suppressor protein p53.

Hoxa5 and Pax1 are involved in the formation of the acromion (Aubin, 2002).

 

HOXA9

     One mutation (a fusion with a nucleoporin gene) is a cause of leukemia.

Hoxa9, Hoxb9, Hoxc9, and Hoxd9 are all expressed in mammary gland development and the offspring of mutant mice died because of the inability to make milk (Duboule, 1999).

 

HOXA10

     This gene is expressed in the developing urinary and reproductive tracts.  Mutations cause fertility problems (such as female infertility if the maternal copy of HOXA10 is lost) and abnormalities of the lumbar region of the back.  Overexpression of this gene can increase risk for leukemia.  This gene is also expressed in the midsecretory phase of the menstrual cycle.  Other Hox genes (Hoxa10, Hoxa11, and Hoxd11) are expressed in the development of the uterus and during the menstrual cycle (Duboule, 1999).   The endometrium is pictured below.

ENDOMETRIUM

Hoxa-10 mutations in mice cause homeotic changes in vertebrae and lumbar spinal nerves, in addition to cryptorchidism and sterility (Ruli, 1995).

 

 

HOXA11

     This gene is involved in the development of the forearm.  One mutation in humans causes radioulnar synostosis.

HoxA11 down regulation is required for the differentiation of the trophoblast (Zhang, 2002).

In mice, Hoxa11 and Hoxd11 mutations can cause a number of abnormalities of forearm, lower leg, and wrist including double mutants which possess relatively normal digits but in which the radius and ulna are almost completely absent Albrecht, 2002;(Davis, 1995)

 

 

HOXA13

Hoxa13 mutations can cause the hand-foot-genital syndrome which results in a number of abnormalities including short thumbs and big toes, a double uterus in females, a bicornuate uterus, and a double vagina (caused by the formation of a vaginal septum (Goodman, 2003; Manouvrier-Hanu, 1999).

 

 

 

 

HOXB—chromosome 17

HOX B1

HOXB2

    This gene is expressed in the hindbrain (primarily in the ventral part of rhombomere 4).

 

HOXB5

     Mice which are homozygous for mutations in this gene die.  Mice which are heterozygous for mutations can develop short, kinky tails and have abnormalities of the vertebral column (fused vertebrae, undeveloped vertebrae, missing vertebrae, and extra vertebrae). 

Hoxb-5 is involved in the branching of bronchi in the developing lung.  Other Hox genes also have distinct temporal and spatial expression patterns in the developing lung (Volpe, 2000).

 

 

HOXB6

     Mice with mutations in this gene typically die after birth with abnormalities of the vertebral column, the exoccipital and basioccipital bones, complete or partial absence of the supraoccipital, and cleft palate.

 

HOXB7

     In mice, the only cells known to express this gene are macrophages.

 

HOXB13

     In mice, this gene is expressed in the developing tail bud, hindgut, urogenital system, and posterior spinal cord.  It is expressed in fetal fibroblasts and the fetal dermis and may be one of the genes which allow fetuses to heal without scarring.

 

Hoxb13 seems to regulate the development of caudal vertebrae and the caudal spinal cord.  In mice, Hoxb13 mutations cause an overgrowth of tail vertebrae, spinal cord, and related structures (such as ganglia).

In mice, the spinal cord develops in the tail region only to degenerate (Economides, 2003).

 

 

HOXC—chromosome 12

 

HOXC6

       Mutations in this gene (or changes in its expression) are involved in some breast and ovarian cancers.

 

HOXC8

     This gene is expressed in cartilage and mutations cause cartilage disorders.

 

HOXC13

      In mice, this gene is expressed in nails, the tail, whiskers, the tongue, and hair follicles.  Mutant mice may have hair which is so brittle that the mice are hairless (OMIM).

 

 

HOXD—chromosome 2

HOXD3

Hox3 (Hoxa3, Hoxb3, Hoxd3) mutations can prevent the thymus and parathyroid glands from migrating normally or prevent these from forming.

In chickens and zebrafish, the thymus, ultimobranchial body, and parathyroids develop as bilateral structures which do not migrate from their original pharyngeal site of development.

Hoxd3 mutants suffer abnormalities of the atlas and axis (Manley, 1998).

 

HOXD4

     This gene is expressed in the developing spinal cord, brain, vertebrae, and limb buds.  The brain of a developing pig is pictured

BRAIN

HOXD8

      This gene is expressed in the developing urogenital tract, especially in the uterus.  It is also expressed in adults (OMIM).

 HOX D12

The loss of HoxD12 in frogs may be related to the loss of a finger (Mannaert, 2006).

HOXD13

      In both mice and humans, mutants can possess extra digits. The triphalangeal thumb-brachyectrodactyly syndrome causes the formation of triphalangeal thumbs in addition to other abnormalities (ectrodactyly of the feet and brachyectrodactyly of the hands) (Perez-Cabrera, 2002).

A modification in the expression pattern of Hoxd13 seems to have been an important prerequisite to the formation of fingers and toes (Carroll, 2001).Hoxd13 mutations can cause synpolydactyly in which the third and fourth digits are fused with an extra digit existing between them in addition to syndactyly of the toes (Albrecht, 2002).  In humans and other verterbrates, HOXD13 and HOXA13 are important for distal part of limb.  HOXD9 more proximal (Manouvrier-Hanu, 1999).  The proximal portion of the os penis is composed of hyaline cartilage and bone while the distal portion is composed of fibrocartilage.  Hoxd13 and Hoxa13 are required for the formation of both digits and external genetalia (Perriton, 2002). Mutant mice which lacked any HoxA/HoxD expression in their limbs did not produce the Sonic Hedgehog signal needed for the development of the forearms and digits. The arm consisted only of a humerus which interestingly had an “L”-like shape. Although there were cartilaginous rays which formed distal to the humerus, these were the products of an “abnormal cartilaginous plate” and not homologues of any normal forelimb bones (Kmita, 2005). It is the non-homeodomain portion of the Hox13 genes which are critical for the development of the posterior portions of limbs (Williams, 2006).

 

 

 

HOX GENES ASSOCIATED WITH HOX CLUSTERS BUT NOT MEMBERS OF THE CLUSTER

It is useful to consider the Hox cluster as a cluster since the genes are expressed in order to establish positional information in axis formation.  The duplication of this cluster provided a diversity of orthologous genes.  However, there are other Hox genes which are linked to the Hox clusters, indicating that the tandem array of ancestral Hox genes, the original ‘cluster’, contained more Hox genes than those which gave rise to what are now considered the Hox clusters.  Two genes, are very closely linked to the modern clusters, Evx and Mox, and the term ‘extended Hox cluster’ has been used to describe these genes plus the traditional Hox clusters.  Duplications in the Hox cluster resulted in duplications in these ‘extended Hox cluster’ genes as well.

CLUSTER

Both Evx and Mox are known in cnidarians, the post primitive animal group which possesses a Hox cluster (Gauchat, 2000; Hill, 2003). 

 

EVX1 and EVX2

Cnidarians possess an Evx homolog linked to its Hox gene cluster.  In vertebrates, Evx was moved from the cluster but is still linked to it.  In Drosophila, the even-skipped gene was shown to control a number of steps in development.  Vertebrate homologs Evx1 and Evx2 resulting from a gene duplication in early vertebrate ancestry also function in development.  In all bilateral animals studied to date, Evx functions during gastrulation, which may the original role for the gene.  In coelomate animals, Evx has also been recruited into the development of the nervous system.  In vertebrates (but not Amphioxus), the role of Evx in the nervous system is augmented to include expression in the midbrain-hindbrain boundary (MHB) which is an important organizing region for the vertebrate brain.  In Amphioxus, Evx plays a role in the development of the post-anal tail, one of the major characteristics shared by all chordates.  Later vertebrates recruited Evx in limb development as well (Ferrier, 2001).

 

MOX

Somites are a feature possessed by vertebrates and cephalochordates.   Mox genes (Mox1 and 2 in vertebrates; AmphiMox in Amphioxus) function in the formation of somites in both vertebrates and Amphioxus.  In protostomes, Mox homologs function in the expressed during mesoderm differentiation in embryonic development (Minguillon, 2002).  Placental mammals use Mox2 in the formation of the placenta (Morasso, 1999). 

 

MORE HOX CLUSTERS LINKED TO THE ‘EXTENDED HOX’ CLUSTER

Evx and Mox are not the only Hox genes which are linked to the Hox cluster and seem to have been duplicated along with the duplications of the ancestral Hox cluster.  In fact, it seems that there were originally 4 groups of Hox genes in ancestral organisms: the ‘extended Hox’ cluster (Hox genes plus Evx and Mox), the NKL cluster (Nk1, NK3, NK4, Lbx, Tlx, Emx, Vax, Hmx, Nk6, Msx, and perhaps Dlx and Not), the ParaHox cluster (Cdx, Xlox, Gsx), and the Ehgbox cluster (En, HB9, Gbx).  Given the linkages between some of these genes and at least some of the mammalian Hox clusters, it seems that the original Hox cluster was linked to Mox, Evx, Ehgblox, Parahox, and NKL genes.  These seem to have existed in one ancestral tandem array which was duplicated in ancestral vertebrates.  These duplications not only created the 4 ‘extended Hox’ clusters known in higher vertebrates, but also at least 10 additional clusters of NK, ParaHox, and Ehgbox genes as well (Pollard, 2000).

 

CLUSTER

NK CLUSTER

NK Family

Hox genes of the NK family are known in animals as primitive as sponges and gene duplications early in animal evolution produced a diversity of genes, with paralogues existing in animals as diverse as humans and Drosophila.  In the human genome, NK genes exist in duplicated tandem pairs.  Sponges possess Msx, NK, and Tlx families (Gauchat, 2000). 

     Two NK2-type homeobox genes in Drosophila, tinman and bagpipe, are closely linked and are involved in the differentiation of the embryonic visceral mesoderm.  The last common ancestor of protostomes and deuterostomes probably possessed some type of primitive heart since homologous homeodomain genes are involved in the earliest formation of the heart (Msh-2 in Drosophila and csx in mammals) (Komuro, 1993).  tinman/csk is expressed in heart progenitor cells in protostomes and deuterostomes but on opposite sides of the body (Gerhart, 2000).   Several vertebrate proteins related to tinman are also involved in the development of the embryonic vertebrate heart.  These vertebrate homologs can actually be substituted for tinman in Drosophila to promote the differentiation of the visceral mesoderm  (but they do not replace all of the functions of Drosophila tinman, such as the function in promoting the development of the heart, Park, 1998).   Mutations in tinman result in abnormal development of fly hearts and mutations in NKX2-5 cause abnormalities in the division and conduction system of vertebrate hearts (Goodman, 2003).  Although the amphioxus heart has no chambers, valves, endocardium, or epicardium, it does express amphiNk2-tin, a homolog of vertebrate NK2  and Drosophila tinman genes which are expressed in the developing heart.  (Holland, 2003).

 

--NK-2 (CSX—Cardiac Specific HOX)

--In Drosophila embryos, mutations of this gene can lead to the absence of a heart (the “tinman” condition).  In humans and mice, this gene is only expressed in the developing heart.  Mutations can cause such things as atrial and ventricular septal defects, AV block, and tricuspid valve abnormalities.

--NK-3 is expressed in the skeleton of the limb; the paralog of bagpipe in Drosophila.

--Nkx2.1 homeobox for lung-specific genes and is expressed in lung bud.  Mutations in mice cause abnormal lung formation and the failure of the trachea and esophagus to separate (Warburton, 1998).

--TTTF1 is expressed in the thyroid (OMIM).

 

--empty spiracles Homologs (Drosophila)

--Emx1

--Emx2, when mutated, can cause schizencephaly, the absence of large portions of the cerebral hemispheres (replaced by CSF), seizures, and retardation.

--VAX1 exists in tandem with Emx1 as a result of a duplication.

--VAX2 is expressed in the ventral part of the retina.

The gene empty spiracles (ems) and its homologs in vertebrates (Emx) are homeodomain-containing genes expressed in, and function in the development of, the head. They are also known in cnidarians which lack a head (Mokady, 1998; Tallafuβ, 2002). 

 

--MSX1 (HOX7)

Sponges possess a Msx gene (Gauchat, 2000). 

--This gene is homologous to the Drosophila gene msh.  In humans, it is expressed in the developing heart valves, limb buds, jaws, and hyoid arches.  Mutations can result in cleft palate, the absence of teeth (specifically the 2nd premolar and 3rd molar), heart defects and retardation.  It is deleted in Wolf-Hirschhorn syndrome which results in retardation, facial clefts, and heart defects.  Some mutations result in 11 to 28 missing teeth.  This was the first HOX gene identified as the cause of a human genetic disorder (OMIM).

While the development of the intervetebral disc involves Pax1, that of the vertebrae involve Hox genes of the Antennapedia type.  Msx2 is important for the dorsal part of vertebrae: loss of Msx2 expression results in the absence of the dorsal part of the vertebrae while inappropriate expression of Msx2 can cause ectopic spinous processes.  Msx1 and 2 are expressed in the neural crest cells of the developing face, teeth, and limbs. The ablation of the notochord results in the absence of the vertebral column (Monsoro-Burq, 1994).

NOT

Not may also be part of the NK cluster (Pollard, 2000).  Only 2 Hox genes known from placozoans: a Not homolog and Trox-2, a member of Hox/ParaHox family (Martinelli, 2004).  Not functions in notochord formation in frogs, fish, and birds.  It is also known in animals without notochords such as echinoderms, flies, hydra, and placozoans (Martinelli, 2004; Gauchat, 2000).     Xnot and Xnot2 were named for their important role in the development of the notochord in Xenopus.  Not homologs function in the development of the notochord, mesoderm, and somites of fish, amphibians, and birds.  (The function of a Not-like gene in mammals is not yet clear).  Not is expressed in animals which possess mesoderm but do not possess notochords (such as flies and sea urchins) and is even expressed in animals which lack mesoderm (such as Hydra and placozoans).  Not is one of only two homeobox genes known from placozoans (Martinelli, 2004).

 

 

--Dlx (homologues of arthropod distal-less)

    In arthropods, distal-less is involved in the development of the distal parts of antennae, legs, and mouthparts and its expression pattern is involved in whether arthropod limbs branch (as in crustaceans) or do not branch (as in insects) (Rousch, 1995).       Tunicates have one Hox cluster but have a pair of Dlx genes, indicating that the initial duplication of Dlx genes preceded the evolution of the first chordates.   When the Hox clusters duplicated to produce 4 clusters in ancestral vertebrates, these duplications included the Dlx genes, to produce 3 pairs (6 genes) in mammals (it is thought that ancestral vertebrates had 4 pairs but subsequently lost a pair of Dlx genes).  Just as there were additional duplications of the Hox clusters in teleost fish, teleosts have additional copies of Dlx genes.

     Amphioxus has one Dlx gene (2 have been identified in urochordates but require more study).  At least one duplication at the base of the vertebrate tree and the resulting vertebrate Dlx genes can be grouped into one of two families (lampreys have 4 genes, zebrafish have 8, humans and mice have 6).  This addition of Dlx genes is significant: they were duplicated in vertebrate ancestry at the same time in which many of the tissues they are expressed in (cranial neural crest, sensory placodes, pharygeal arches, and forebrain) were being modified (Stock, 1996; Holland, from Ahlberg, 2001. 

     In vertebrates, Dlx genes occur in pairs on the same chromosomes which contain Hox clusters and are involved in the development of a variety of tissues including the brain, branchial arches, sensory structures, limb buds, hair, and teeth.  Gnathostomes and lamprey have 3 copies of this tandem (although lampreys have lost 2 genes of 6 genes).  In lampreys, Dlx is also expressed in the brain and some sensory structures but there was an interesting difference of expression in the branchial arches. Evolution of jaws preceded by duplications of Dlx genes.  In lampreys, Dlx is expressed in the pharyngeal arches where cartilage forms but in jawed vertebrates, there are two different regions of expression in the dorsal and ventral regions of these arches.  In gnathostomes, Dlx 1and 2 are expressed throughout the region of the developing jaws, Dlx 5 and 6 are expressed in the lower jaw and hyoid, and  Dlx3 and 7 are expressed in the ventral lower jaw and hyoid.  This change in expression pattern might underlie the ability of the ventral arches and the lower jaws to move.  In mice with Dlx 1 and 2 mutations, the distal pharyngeal arch assumes identity of the proximal arch. (Neidert, 2001; Stock, 1996; Graham, 2002).  Dlx3 and Dlx4 are involved in the formation of the placenta (Morasso, 1999). 

.

 

The ParaHox cluster

     It seems that before the evolution of the metazoans, a duplication of the ancient Hox cluster occurred to produce a ParaHox cluster.    Cnidarians possess Gsx and Mox homologs of the ParaHox cluster, but not Xlox (Hill, 2003). Mollusks have two ParaHox genes and one Gbx Hox gene (Barucca, 2003).

Echinoderms possess possess ParaHox genes (Mito, 2000).  

NOTOCHORD ENDOSTYLE
  Amphioxus has a second Hox cluster of 3 genes which are expressed in order along the anterior-posterior axis (in the neural tube; at least two of the three are also expressed in order in the gut; the neurual tube and gut of Amphioxus are pictured above).  The first of these genes belongs to the family of anterior Hox genes on the main Hox cluster, the second to the medial gene group, and the third to the posterior gene group.  These three genes have homologues in other organisms: the anterior gene Gsx is homologous to vertebrate Gsh genes which are also expressed in the brain,  the medial gene Xlox is homologous to Xlox genes in leeches and vertebrates (the mammalian copy is named pdx-1) which are also expressed in the midgut, and the posterior gene Cdx is homologous to both arthropod and vertebrate Cdx genes which are also expressed in the hindgut.  That is not yet certain whether how typical these clusters are in organisms, but both in humans, medial (Pdx1) and posterior (Cdx) ParaHox genes map to the same region of chromosome 13 (13q12.1) and the anterior gene Gsh-2 maps to a mouse chromosome region equivalent to region 13q12.1 in humans.
CLUSTER

a)     PDX1 (IPF1)

This ParaHox gene regulates the development of the pancreas and the expression of pancreatic hormones.  Mutations in this gene in both mice and humans can cause the absence of a pancreas, diabetes, and other pancreatic problems (Takatori, 2002).

 

 

b)     Cdx Genes (homologs of caudal in Drosophila)

--Cdx1 is expressed in the developing brain, especially in the hindbrain.  Mutations in mice cause abnormalities of the vertebral column.

--Mice which are homozygous for Cdx2 mutations die early with vertebral, rib, and tail abnormalities.  In heterozygotes, the mutation causes the formation of intestinal polyps.

--Cdx4 is located on the X chromosome.

 

 

 

EHGBOX FAMILY

engrailed

The orphan Hox gene engrailed is a determinant of segmentation.  Engrailed is involved in the segmentation of annelids (Shankland, 2000).In Drosophila, this gene is involved in a number of developmental processes such as segmentation and the pattern of engrailed expression during segmentation is similar in both protostomes and chordates, indicating an ancient role of engrailed in segmentation (DeRobertis, 1997).  In vertebrates, en1 is expressed in the midbrain (mice which have muations in en1 die with deletions of the midbrain).  Mutations in en2 cause problems of the cerebellum.

 

 

GBX2—Gastulation Brain Homeobox

--This gene is expressed in the early embryo in the inner mass cells before implantation and the borders of Otx2 and GBX2 expression define the midbrain/hindbrain border.  Mutations in GBX2 result in brain abnormalities such as a large inferior colliclus and the absence of the cerebellar vermis.