The Homeobox genes are a family of similar genes that contribute to organising the structures of the embryo during development. The structures include the limbs, bones organs and facial features. There are three specific Homeobox genes that are thought to participate in this process, Alx1, Alx3 and Alx4. It is not fully understood how these genes function during development, but it has been discovered that changes in these genes impact on facial development in the embryo. The changes within the genes can be inherited by parents who carry the defective genetic material, or it can happen randomly. A change in Alx1, Alx3 or Alx4 genes produces different types of a disorder called Frontonasal-Dysplasia. The changes in the Alx1 gene create the most severe type, Frontonasal Dysplasia Type 3. Such facial defects include an excessive broadening between the eyes, a lack of nasal tip and often a cleft palate.
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The three Alx Homeobox genes, Alx1, Alx3 and Alx4 and then belong to a subclass of Homeobox genes, called the PRD group. These genes are derived from two-round whole gene duplication. Therefore, Alx1 and Alx3 show more similarities to each other in comparison to Alx4, as both of those genes are stemmed from the same gene duplication. Due to the research conducted, it is evident that these three Alx genes play a fundamental role in craniofacial development. It is thought these genes are expressed in the Frontonasal mesenchyme and the first and second pharyngeal arches during embryogenesis. Mutations in these genes lead to the production of a defective homeodomain within the Alx1, Alx3 and Alx4 proteins. The mutations within these genes can be generated from the inheritance of two carrier parents. But the majority of the time the mutations are sporadic. Alx1 mutations are responsible for producing the most severe phenotype for facial disfigurement in Frontonasal Dysplasia (in comparison to mutations in the Alx3 and Alx4 gene). Also, Alx4 is the only gene out of the three that isn't only restricted to the facial region, it also causes disorders such as Alopecia and genital abnormalities. All types of Frontonasal dysplasia occur as autosomal recessive disorders.
1) An Introduction to Craniofacial development in humans.
Human craniofacial development is a complex and highly regulated process . It can be split into 5 stages, the formation of the pharyngeal apparatus, the tongue, the skull, face and palate.
Figure 1:- Image of the four pharyngeal pouches that lie in between the 5 pharyngeal arches. Image taken from 
Firstly the pharyngeal apparatus contains structures such as paired pharyngeal arches, pouches, membranes and clefts. There are five pharyngeal arches, named 1, 2, 3, 4, and 6 respectively, which are formed during the fourth week of embryonic development . All five of the pharyngeal have their own innervation by their specified cranial nerves. The bone, cartilage and connective tissue of the pharyngeal arches are originally formed from the mesenchyme of the neural crest, whilst the skeletal muscle and the pharyngeal arch artery develop from the somatic mesoderm.
There are four pharyngeal pouches that lie between the pharyngeal arches. They are formed from a layer of endoderm that lines the inner pharyngeal apparatus. Also, there is a layer of Ectoderm that lines the outer pharyngeal apparatus that then form the four pharyngeal clefts. The first pharyngeal cleft later develops as the external auditory meatus; this is the only pharyngeal cleft that remains into adult-hood.
Development of the Tongue is split into two stages, the formation of the anterior two-thirds of the tongue, followed by the fusion of the posterior third of the tongue. When the anterior and posterior parts fuse, it can be represented by the Terminal Sulcus. The anterior of the tongue is formed from the first pharyngeal arch, which then develops in the median and lateral tongue buds. The posterior of the tongue develops from the third and fourth pharyngeal arches. The muscles of tongue are derived from Occipital myoblasts .
The vertebrate skull can be subdivided into three parts which are the Chondocranium Neurocranium and the Viscerocranium  .There is also a Chordal skull, which is formed from the paraxial and cephalic mesoderm, alongside a Prechordal skull which is derived from the cephalic neural crest . There this supports that the vertebrate skull is derived from the neural crest tissues. The fusion of bones to form the human skull doesn't occur until after birth. Reason being, loose bones at this stage in the skull allows the baby's head to fit though the birth canal without causing damage to the brain of the newborn. The anterior skull bones and the hyoid bones are ossified from the mesenchyme of the neural crest whilst the bones on the base of the skull and the flat bones of the skull are ossified from the paraxial mesoderm.
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The facial development occurs between weeks 5-10 of fetal development and is primarily formed from right and left Maxillary prominences and Mandibular prominences (in which they fuse together during fetal development), a Frontonasal prominence and paired nasomedial prominences . The Frontonasal prominence is formed from the mesenchymal neural crest . Numerous events occur at the stage, for example, the maxillary prominence fuses with the lateral nasal prominence at the midline to create a naslactrimal groove. Most of the groove disappears  aside from the superior portion of the groove where the nasolactrimal ducts and lacrimal sacs form. As the nasal prominences fuse, it forms two structures that include the philtrum of the upper lip, as well as the midline of the nose.
Finally, the palate undergoes two stages, firstly there's the primary palate, which is then followed by the formation of the secondary palate. The primary palate is formed from the fusion of the two medial nasal prominences. The secondary palate forms due to the further growth of the maxillary prominences, until they fuse at the midline from each side of the tongue.
2.0) Cranial Neural Crest Migration.
There four different types of Neural Crest Cells , and they are classified based on their regional role within the embryo. The four subclasses are; Cranial, Vagal, Trunk, and Lumbosacral. The migration of cranial neural crest cells are necessary for craniofacial development, these cells arise from the Forebrain and Midbrain and forms the parasympathetic ganglion of the third cranial nerve. It also contributes to the developing eyes, head mesoderm and mesenchyme. Cranial Neural crest cells from the Hindbrain and midbrain form parts of the pharyngeal arches of the neck and head that lead to the formation of several facial bones and cartilage. They also form the dermis, smooth muscle and odonoblasts of the developing teeth. The hindbrain neural crest cells form the neurons and glial cells of the sensory ganglia of cranial nerves five, seven, nine and ten. They also form parasympathetic portion of the autonomic nervous system. The cranial crest cells also give rise to other cells that arise in the head and neck of the embryo the inner and middle meninges of the occipital area. They also form melanocytes as they migrate away from the neural tube to occupy the ectodermal surface  .
2.1) Overview Of The Alx Homeobox Genes.
The Homeobox gene family are a group of similar genes which are involved in directing the arrangement of many structures of the body that takes place during embryonic development. They take part in many activities in this period such as arranging and developing the formation of the limbs and organs along the anterior-posterior axis. Some Homeobox genes function as tumour suppressers and they also regulate cellular differentiation. In every human genome there are clusters of Homeobox genes present. The Homeobox genes have a notable trait; that they have a specific DNA sequence which encodes a particular protein domain  ; a homeodomain. 
The ANTP and PRD classes are the two main types of groups of Homeobox genes and both groups have a fundamental participation in cellular differentiation  and patterning . The PRD (Paired Gene of Drosophila ) class is the second largest group of Homeobox genes, and within the PRD group are the three Alx Homeobox genes; Alx1, Alx3 and Alx4. Both humans and mice have these three notable Alx Homeobox genes in their genome as they are involved primarily involved in craniofacial development . They also participate in directing the formation of limbs  and neural tube closure .
It has been discovered  that the three Alx genes in mouse have related expression to the expression of that in the cranial region of the human embryo. Alx1 can be detected earlier in the head mesenchyme in comparison to Alx3 and Alx4  . Alx3 and Alx4 are primarily expressed in the mesenchyme (derived from the neural crest) in the developing medial face of vertebrates . During early human embryonic development, the Alx genes are expressed in the frontonasal mesenchyme followed by expression in the first and second pharyngeal arches. The causes of congenital craniofacial malformation are connected to mutations within the three Alx genes .
The three Alx Homeobox genes also have overlapping roles  .
In order to discover the functional roles of the Alx gene family, experiments have been conducted using Zebrafish. The reason behind this is because there are high similarities in craniofacial development of Zebrafish and mammals . For example the way the palate develops in both Zebrafish is very comparable to what occurs in mammals in cellular and genetic aspect  . Also the Frontonasal and maxillary aspects of the mesenchyme in Zebrafish are formed from the Cranial Neural Crest (CNC), which is the derivation in mammals .
2.2) The Three Alx Homeobox Genes; Alx1, Alx3 and Alx4
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The three Homeobox genes are a family that belonging to the Aristaless- like Homeobox genes. They have a "paired type homeodomain and a conserved C-terminal paired tail domain" (Meijlink et al 1999)  . Their main function is to express a distinct transcription factor during the development of the embryo. All three have a common source of being expressed in the mesenchyme of mammals during embryogenesis, especially during weeks 4-8 of development.
The location of the Alx1 gene in humans is at the position of 21.31 on the long arm of Chromosome 12.  The functional role of Alx1 in humans is not currently fully understood, but because there is information about the effects of homozygous mutations in the gene causes (such as Frontonasal dysplasia type 3), it is established that Alx1 must have a profound effect during facial development. It has been discovered that out of the three Alx genes, Alx-1 alone is expressed through Cranial Neural Crest (CNC) development . This then explains why that the Phenotype caused by depletion of Alx1 in zebrafish caused extreme malformation of the eyes, palate and lack of facial cartilage . Alx1 has a fundamental role in the function of the Cranial Neural crest, thus, defects in the gene can cause notable malformations such as cleft palate of oblique facial clefting.
Alx3 gene acts as a transcriptional regulator because it encodes a nuclear protein. This nuclear protein then becomes a binding site for the strands of homeobox DNA. Alx3 is important for cellular differentiation and development; it is also a possibility that Alx3 is involved in the patterning of mesoderm at early embryonic development . Defects in this gene can be associated with advanced-stage neuroblastoma tumours as well as Frontonasal dysplasia type 1 (FND1) and is associated with midfacial dysraphia. The Alx3 lies on the short arm of chromosome 1 at the position of 13.3 
The Alx4 gene is produces a transcription factor; Alx4 protein which is essential for complete development of the skull. The gene is located at the position of 11.2 on the short arm of chromosome 11  . There are two significant conditions associated with mutations within the Alx4 gene; Enlarged Parietal foramina and Potocki-Shaffer syndrome  It has been discovered that Alx4 has an important in the development of human hair follicles and the skin. 
It is thought that because Alx4 is expressed in the mesenchyme during embryonic development, it could also participate in the signalling between the mesenchyme and the epithelium of the skin. In mice defects in the Alx4 gene lead to dorsal alopecia and genital abnormalities which produces a similar effect of a mutated Alx4 gene in humans . Also as there would then be dysfunction of the hair follicle development, the little hair that was present was weak and fragile. It is therefore known that Alx4 has a role not only confined to the craniofacial region, but also in bone and limb development and as mentioned, hair follicle development.
Figure 2 :- The location of the three Alx genes, Alx1, Alx3, Alx4 within the human genome. Adapted from 
2.3) The evolution of the Homeobox genes.
Out of the three Alx Homeobox genes, Alx3 is the only one that is lost at different stages of evolution, for example, it is lost in three specific species of vertebrates; "The Amphibian X tropicallis, the Squamate reptile A.carolinensi, and the bird G.Gallus" (McGonnell et al 2011) . During early vertebrate evolution, the three alx genes underwent two complete genome duplications (2R-WGD) . Alx 1 and Alx3 where produced from the same genome duplication whilst Alx2 and Alx4 where also paired in the same duplication. Alx2 had been quickly lost from evolution, with no understanding yet of when and why. Since Alx1 and Alx3 share the same origin of duplication, it explains why these two genes share more similarities in comparison with Alx4. This is why, when mice lacked Alx3 expression, their health or physical stature was not deteriorated. This suggests that Alx3 can be compensated by Alx 1 as they share similar functions .
Both Alx1 and Alx3 are comparable in their role of neural tube closure  and because they're more similar to each other as they share the same whole gene duplication. If either Alx1 or Alx3 where to be lost, they can be compensated for by the other gene due to their functional similarities. Alx4 is the only member Alx Homeobox gene that participated in axial patterning of the limbs (Takahashi et al 1997). Therefore losing Alx4 from the genome would be more consequential in comparison to losing either Alx1 or Alx3. 
Figure 3; Two Round Whole Gene Duplication. Both Alx 1 and Alx3 are derived from the same duplication. Alx 2 and Alx4 are stemmed from a different duplication. Adapted from 
2.4 Alx Related Frontonasal Dysplasia.
Frontonasal Dysplasia due to mutations in the Alx genes.
During embryogenesis, the facial growth after week 4 of development is governed by mesenchymal - ectodermal communications. For this to occur, a number of genes that encode transcription factors are needed so that the epithelium to control the communication between both the mesenchyme and the ectoderm . It is thought that the three members of the Alx gene family are contributors to this process. Therefore, alterations in the signalling, such as mutations within the Alx genes, can cause severe disfigurement of frontofacionasal features, such as cleft palate .
Frontonasal dysplasia describes a series of facial malformations, consequently due to disruptive median facial development. Hypertelorism is common characteristic of this disorder due to improper migration of the neural crest . The only genetic cause of frontonasal dysplasia that has been proven, are mutations in a ligand of Ephrine receptors tyrosine kinases   Also, most cases of Frontonasal dysplasia are sporadic without any known reason .
The Alx genes encode a specific DNA sequence to produce homeodomain proteins. Severe frontonasal dysplasia phenotype occurs when homozygous mutations are present within the Homeobox transcription factors of either one of the three Alx Homeobox genes. Various mutations lead consequently to a defective homeodomain in the Alx proteins.
Alx1 related frontonasal dysplasia
Mutations in the Alx1 gene are known to cause Frontonasal Dysplaisa type 3 (FND3)  , which is the most severe form of Frontonasal Dysplasia . Regardless of its severity it is possible for the individual with FND3 to survive into childhood and sometimes even longer than that . In comparison to Alx3 and Alx4, the mutations within the Alx1 gene create a far worse impact on the frontonasal area , Frontonasal dysplasia is inherited as an autosomal recessive disorder. Micro deletions as well as point mutations of the Alx1 gene produce phenotypes for Frontonasal dysplasia type 3 . Thus, when both parents are normal, the child will have a 25% percent chance of developing alx-related dysplasia from parents who are both carriers. It affects the face by causing frontonasal dysplasia, bilateral oblique facial cleft, hypertelorism and extreme bilateral microphthalmia. The ears will be low set and posteriorly rotated. There could be lack of upper lip, macrostomia and a complete cleft palate. A wide nasal bridge would be seen alongside hypoplasia of the alae nasi. Other affects could be mild mental retardation and caudal appendage .
Alx3 related Fronotnasal Dysplasia.
Mutations such as splice-site, frame-shift and missense within the Alx3 gene can cause the gene to partially lose its function or even lead to complete loss of function. Frontonasal Dysplasia type 1 (FND1) (also known as Frontorhiny) is a major disorder arising from mutations within this gene . This FND1 phenotype is a milder form in comparison to Alx1 and Alx3 related FND . Similar to Alx1 FND, it is inherited as an autosomal recessive disorder, which causes frontonasal dysplasia to the face. It will cause hypertelorism in the eyes and sometimes it can cause ptosis, iris coloboma and oribital dystopia. The mouth will generally have a long philtrum with bilateral swellings. Prominent philtral ridges can be a physical diagnosis for Alx3- related Frontonasal Dysplasia. Also FND1 can cause columella of the nose, a bifid nasal tip, a wide nasal bridge and a short nasal ridge . During FND1, it is known that during fetal development, the fusion between the nasomedial and frontonasal prominences do not finish completion and therefore accounting for facial disfigurements such as the distinctive bifid nasal tip and a broad nasal bridge .
Alx4-related Frontonasal dysplasia.
Mutations in the Alx4 gene is the cause Frontonasal Dysplasia type 2 (FND2). In comparison to Alx1 and Alx3 - related FND, the Alx4-related FND has affects that are not confined to that facial area. For example, the individual can have total alopecia, and also genital abnormalities  such as Hypogonadism.
The mutations within the Alx4 gene can either be Homozygous or heterozygous each of which produces different phenotypes. Point mutations are likely to occur if the mutations were heterozygous; an example of this would be in the Potocki-Shaffer syndrome , where there deletions are present on chromosome 11 in the location where the Alx4 gene is located (short arm of at point 11.2) the chromosome where the Alx4 gene is present (Chromosome 11p11.2). Also in heterozygous mutations, frame-shift and missense mutations can occur that result in a defective alx4 protein. In contrast homozygous mutations normally present with nonsense mutation a truncating affect resulting in a defective protein thus, changing its homeodomain .
The defects in the Alx4 gene have a more severe impact on the eyes. It can cause extreme hypertelorism  as well as blepharopmiosis and rotatory nystagmus. The nose in Type 2 frontonasal dysplasia can cause a depressed nasal bridge and nasal ridge, and a bifid nasal tip which can be compared with that of Alx3-related Frontonasal dysplasia. FND2 has a profound impact on the development of the skull which can lead to disorders such as Brachycephaly.
Figure 4:- Physical appearances due to mutations within the Alx gene family, Alx1, Alx3 and Alx4. Each of which causing a type of Frontonasal Dysplasia (FND)
Image taken from 
To conclude, the Alx Homeobox genes are known to have a participation in human craniofacial development during embryogenesis. After experiments conducted of Zebrafish for example, Alx1 had been expressed in the cranial neural crest and the frontonasal mesenchyme. Although it is not fully understood how these three Homeobox genes function in humans yet, the mutations within the genes cause severe facial disfigurement such as Frontonasal dysplasia. Mutations in the genes cause a faulty homeodomain in the Alx proteins produced leading to loss of function in the protein and disrupting the craniofacial developmental process