A Practical Approach, Second Edition=Ronald D. Ho.pdf

PATHOGENESIS OF ABNORMAL DEVELOPMENT 73those that are receptor mediated, may alter signaling pathways more specifically. This class ofteratogens includes the Veratrum alkaloids, plant toxins long known to cause cyclopia in sheep,that inhibit tissue responsiveness to Shh. 107,108 In studies with chicks, jervine, an especially toxicVeratrum alkaloid, also caused defects that were characteristic of holoprosencephaly. 108Another developmental toxicant that causes changes in cell signaling pathways is retinoic acid(RA). RA is a natural morphogen that is essential to many normal developmental processes. 109Investigators using animal models have shown that both a deficiency and an excess of RA can leadto malformations, including limb defects such as oligodactyly, polydactyly, and reduction defects,and in some cases, limb duplication. Differences in observed abnormalities depend on the timingof administration, dose, route, and species employed. 109–111 RA deficiency prevents normalAER–FGF-4–ZPA signaling in both chick and mouse embryos. 112,113 Studies using RA-deficientmice have demonstrated that RA is required for cells of the ZPA to acquire competence for Shhsecretion, most likely via induction of Hox genes in the mesenchyme and by secretion of FGF-4by the AER. 85,100 Mice deficient in RA lack normal ZPA patterning and exhibit oligodactyly. 109 Incontrast to RA deficiency, direct application of RA to the anterior domain of the chick limb budresults in extra digits or in mirror image limb duplication via the formation of a second ZPA. 93,114–116At higher doses of RA, skeletal elements are reduced or do not form.110, 117Although RA is teratogenic in humans 118,119 and in animals, 120–123 there are notable speciesdifferences in the effects observed. While similar defects exist in humans and rodents for craniofacial,CNS, cardiac, and thymic development, limb defects are seldom reported in humans exposedto RA in utero. 124 Like other teratogens, the effects of exogenous RA on development are highlystage dependent. Late administration during limb development in the mouse causes skeletaldefects, 125 whereas administration during preorganogenesis can induce the development of supernumeraryhind limbs. 126,127 Differences also occur with different methods of administration. Directapplication of RA leads to digit duplication, while systemic administration more often results inoligodactyly. Polydactyly can also occur, particularly at lower doses. 109,128,129 As is the case withseveral other teratogens, there appears to be a preferential loss of postaxial digits of the rightforelimb, 116 a phenotype also prevalent in Wnt7a null mice that display reduced Shh expression. 91Studies have also implicated alterations in the underlying mesenchyme and interference withectodermal-mesenchymal interactions in loss of AER function and signaling in RA pathogenesis,and it is of no small interest that cells of the underlying mesenchyme express receptors forRA. 116,130–133It is important to keep in mind that the preceding overview of signaling pathways in the limbis necessarily greatly simplified for the present discussion. Although discrete morphogenetic functionscan be assigned to regions such as the AER and ZPA, these regions are codependent for limbdevelopment, adding an additional layer of complexity to the system. For instance, maintenanceof the AER requires a functional progress zone and ZPA, while signaling from the AER and theZPA is required to maintain proliferation in the underlying mesenchyme and progress zone(reviewed by Johnson et al. 103 ). The complexity of this system, as well as species differences andeven differences between fore- and hind limb development within a species, will often makedetermining the initial pathological deviation from normal development quite difficult.Perhaps the preferred technique for detecting alterations in cell-signaling patterns followingtoxicant exposure is in situ hybridization using labeled probes to the mRNAs of the molecules ofinterest. There are several good reviews covering this highly valuable technique. 134–137 Patterns ofsignaling molecules in tissues can also be detected by whole mount immunostaining or immunohistochemistrywhen appropriate antibodies are available. For instance, Shh is a protein thatundergoes autocatalytic cleavage to yield a secreted amino (N) peptide and a carboxy (C) peptidethat remains cell associated. Specific antibodies are available to both peptides for several species.Antibodies to cellular receptors, such as cellular retinoic acid binding proteins (CRABPs), are alsouseful in identifying changes to populations of cells responsive to signaling molecules.© 2006 by Taylor & Francis Group, LLC