Literature studies have identified a number of phenocopies producing the DGS phenotype resulting from administration of teratogenic agents. Retinoic acid, in the form of isotretinoin, an analogue of vitamin A, has been licensed as a treatment for severe cystic acne and other chronic dermatitis since 1982. Isotretinoin is a known teratogen and was contraindicated for use during pregnancy. Inadvertent usage however, was reported in a 1985 study (Lammer et al 1985). Among the high
incidence of malformed offspring was a characteristic pattern of abnormality including micrognathia, cleft palate, conotruncal heart defect and aortic arch abnormalities and thymic defects. Moreover, this paper summarises similar retinoic acid embryopathies in murine foetuses after exposure of pregnant mice to retinoids, and major craniofacial defects seen in mouse embryos treated with vitamin A. A deficiency of branchial arch mesenchyme, caused by abnormal activity and migration of a population of neural crest cells was proposed, due to the activity of retinoic acid as a candidate morphogen in embryogenesis.
A common mechanism was also attributed to cases of DGS bom to mothers with a history of alcohol abuse. Four cases have been described so far (Ammann et al 1982). The heart defects seen include toF and VSD. Again these defects have been
reproduced in mice (Daft et al 1984). toF, VSD and PTA were reported in 5-10% of patients with thalidomide induced phocomelia. Maternal diabetes has also been associated with a DGS phenotype (Pexieder T 1986).
Another animal study shows congenital defects resembling DGS in rat and hamster foetuses (Oster et al 1983). The fat soluble, zinc-chealating agent Win 18,446 (bis- dichloroacetylamine), was found to be a powerful teratogen when administed to pregnant rodents during days 9-21 of the gestation period. Day 11, in which the defects most closely resemble DGS, represents the fourth week of human
embryological development. The drug, which is non-toxic to adult animals, produced a triad of facial, conotruncal and thymic abnormalities. WIN 18,446 is known to complex zinc in a non-polar medium. Maternal zinc deficient diets in rats result in gross congenital abnormalities and a deficiency in zinc has been associated with alcoholism (reviewed in Oster et al 1983). The bis-dichloroacetylamine is highly lipid
soluble and could thus compete for available zinc with membrane bound zinc- dependent enzymes, or interfere with other zinc dependent cellular pathways.
L 3 3 Knockout Mouse Models and the Role o f Genetic Factors,
The mouse represents a system in which targeted mutations can be introduced into the genome and subsequently studied in heterozygous and homozygous mice.
Phenotypes seen in these knockout mouse models provide a detailed insight into the molecular and cellular basis of neural crest development.
Targeted disruption of the mouse Hoxa-3 gene resulted in mouse mutants with a spectrum of abnormalities in the branchial tissues (Chisaka & Capecchi 1991). These defects included deletions and malformations of the throat cartilage and bones of the jaw, disorganisation of the throat musculature, of the heart and great vessels, deletion
of the thymus and parathyroids, and thyroid hypoplasia; a phenotype reminiscent of DGS.
The Hox genes are a family of transcription factors that contains a highly conserved DNA binding motif, the Antennapedia homeodomain, and are thought to play a role in vertebrate pattern formation (for reviews see Hunt & Krumlauf 1991, McGinnis & Krumlauf 1992, Manley & Capecchi 1995). Humans and mice contain 38 Hox genes which are arranged in four linkage groups on four separate chromosomes, known as
Hox A, B, C and D. Of developmental importance was the finding that the observed cranial to caudal expression pattern, and timing of expression, of paralogous members of these groups could be correlated with their order along each chromosome.
Members of the most 3’ paralogous families have the most cranial limits of expression in the developing hindbrain spaced at approximately two rhombomeric intervals with the anterior limit of Hoxa-3 expression in r5. These Hox gene transcripts are also found in neural crest cells prior to, during, and subsequent to their migration into the branchial arches. The cranial limit of expression of each Hox gene was found to be within rhombomere boundaries. This Hox code suggests that Hox genes convey patterning information from the hindbrain into the target branchial arches, the distinct
anterior boundaries of expression resulting in specific rostro/caudal information within their expressed zones.
Further analysis of Hoxa3 mutants, following embryological development using labelled cells and specific antibodies to the Hox genes, showed that neither the amount nor the migration of the cardiac neural crest was grossly affected (Manley & Capecchi 1995). The authors suggested that the loss of Hoxa-3 affects the intrinsic capacity of this neural crest population to differentiate and/or to induce proper differentiation of the surrounding branchial arch and pouch tissue.
Mice deficient in the retinoic acid receptor {RAR^ exhibit conotruncal and aortic arch defects (Kastner et al 1994). As has been discussed, retinoic acid is a candidate morphogen, a substance thought to set up concentration gradients that can be interpreted by surrounding cell populations during development. During
segmentation of the hindbrain and subsequent development, retinoic acid acts by modulating the expression of various Hox genes containing specific retinoic acid binding sites. Ectopic expression of retinoic acid resulting in homeotic transformation of rhombomeres and leading to defective patterning of the branchial arches provided fijrther evidence for this (for review see Kirby & Waldo 1995). Thus a change in concentration of retinoic acid, seen in retinoic acid embryopathies and mutations in the RARy, can result in a similar phenotype to the Hoxa-3 knockout mice.
However the human HOXA-3 gene has been mapped to chromosome 7pl3 and DGS in humans is a dominant genetic effect involving chromosome 22ql 1 (which will be discussed later). A common developmental pathway may be the key to explain the shared phenotype.
Krox20, a zinc finger transcription factor is expressed very early in embryogenesis in the developing hindbrain before segmentation occurs, and this expression continues into the newly forming r3 and r5. Krox20 directly regulates Hox gene expression (Sham et al 1993), and the phenotype of mice homozygous for a targeted disruption of the gene indicate that Krox20 is an important factor in the initial segmentation process (Schneider-Maunoury et al 1993). It has been suggested that Krox20 acts as an upstream regulator of transcripts involved in the DiGeorge developmental
Demczuk & Aurias 1995). The expression pattern of Krox20 in early and later development, and the targeted knockout mouse models show no significant evidence for this (Levi et al 1996).
The splotch mouse is a naturally occurring mouse mutant and has been shown to result from homozygous null mutations in the paired box containing gene, Pax-3. The human PAX-3 gene has been mapped to human chromosome 2q. Mutations in PAX-3
have been identified in two unrelated human diseases, Waardenburg syndrome type I (an autosomal dominant combination of pigmentary disturbances, lateral disturbance of the inner canthi, mental retardation and occasional deafness) and alveolar
rhabdomyosarcoma (a paediatric soft tissue tumour in the lungs) (for review see Macina et al 1995). The homozygous splotch (Sp^^) mouse mutant usually dies in utero and displays defects in neural tube closure, ex/encephalus and spina bifida. Other abnormalities include conotruncal or aortic arch defects. A sub-class of splotch mutants, Sp"^ homozygotes, only develop spina bifida and can survive until birth. Heterozygous mice display a white spotting of the abdomen, tail and feet (Epstein et al 1993). Expression of Pax-3 occurs along the entire dorsal portion of the neural tube and in migrating crest contributing to the dorsal root ganglia and the cephalic mesenchyme. Pax-3 has been proposed to be involved in establishing positional identity along the dorsoventral axis of the developing neural tube (Goulding et al
1991).
Pax-3 belongs to the family of paired box-containing genes, which function as transcription factors. Other Pax genes have been shown to play important roles in embryology. It is of interest that Pax-1 is essential for proper thymic development and maturation of T-cells, as shown by the undulated mouse (Wallin et al 1996).
Pax-1 is expressed first by precursor cells of the thymus epithelium in the branchial arches and may thus represent a transcript regulated by a gene or genes involved in DGS.
Neurofibromatosis type 1 is an autosomal dominant disease with phenotype
manifestations resulting from abnormalities of neural crest tissue. Targeted disruption of NF-1, a tumour suppressor gene, causes in utero death, postulated to be due to
heart defects resembling double-outlet right ventricle. Interestingly a consensus sequence in the 3’ untranslated region of NF-I is homologous to a consensus binding site for PAX-S, which can activate transcription. Expression of PAX-3 in neural crest cells may be important for expression of NF-1 and vice-versa (for review see Kirby & Waldo 1995).
The Endothelin-1 (Et-1) homozygous knockout mice manifest a phenotype that is strikingly similar to that of the Hoxa-3 mouse model. The gene Et-I, which encodes a signalling peptide, participates in regulation of cardiovascular homeostasis in the adult animal. Sites of expression in the developing embryo include, as may be expected, the vascular endothelium and branchial arch epithelium. The human ET-I, part of a developmental important family of signalling peptides, maps to chromosome 6 (Kurihara et al 1995).
Other mouse models include the Sox-4 homozygous knockout and the neurotrophin-3 deficient mice. Impaired development of the endocardial ridges into the outlet portion of the ventricular septum was observed in the Sox-4 knockout mouse (Schilham et al
1996). A block on B-cell maturation, resulting in the decreased numbers of B-cell progenitors in foetal liver, indicates that this transcriptional activator affects B-cell lineage. An identical finding also occurs in mice with mutations in the gene Pax-5.
Neurotrophin-3 {NT-3) belongs to the family of neurotrophic factors and acts as a mitogen for neural crest cell development. NT-3 is co-expressed with its tyrosine kinase C receptor (trkC), in many crest populations (for review see Srivastava & Olson 1996). Defects in the neural crest-derived peripheral nervous system were observed in the mutant mice, as well as cardiac defects that included PTA, toF, VSD and pulmonary stenosis (Donovan et al 1996).
A developmental field is a group of embryonic cells that develop in a co-ordinated manner and that, when in contact with different environmental insults or genetic causes, will react in the same way (Optiz J. 1985). Thus DGS is classified as a
developmental field defect, and the diverse teratogenic and genetic factors that induce the phenotype provide evidence for this.
It remains unclear how all of the above, unrelated factors could produce such distinct and similar abnormalities in the embryo. The common finding of expression in the developing and migrating neural crest populations suggests a simplistic model of overlapping pathways leading to a unique end point. This implicates the role of transcription factors, signalling molecules, and ligands and their receptors in the lineage of cardiac crest, a transcriptional pathway that may be analogous in other developmental systems, such as B-cell lineages. The establishment of hierarchy of genetic control during the cascade of neural crest development may provide
information about the varying phenotypes and the interactive role that these factors play in DGS.