Genetic regulation of mammalian diversity.

Mammals have evolved a variety of morphological adaptations that have allowed them to compete in their natural environments. The developmental genetic basis of this morphological diversity remains largely unknown. Bats are mammals that have the unique ability of powered flight. We have examined the molecular embryology of bats and investigated the developmental genetic basis for their highly derived limbs used for flight. Initially, we developed an embryo staging system for a model chiropteran, Carollia perspicillata, the short-tailed fruit bat that has subsequently been used for staging other bat species. Expression studies focusing on genes that regulate limb development indicate that there are similarities and differences between bats and mice. To determine the consequences of these expression differences, we have conducted an enhancer switch assay by gene targeting in mouse embryonic stem cells to create mice whose genes are regulated by bat sequences. Our studies indicate that cis-regulatory elements contribute to the morphological differences that have evolved among mammalian species.

Comparative studies on limb morphogenesis in mice and bats: a functional genetic approach towards a molecular understanding of diversity in organ formation.

The basis of species-specific morphogenesis has been a topic of fascination and speculation for centuries. In 1828, Karl Ernst von Baer noted that at the pharyngula stage of development all vertebrate embryos are morphologically very similar. Most subsequent hypotheses have proposed that the vertebrate body plan develops by a conserved mechanism, and that divergent forms develop by differential elaboration on this basic plan. Gene cloning and expression studies have largely confirmed that the genetic pathways of embryonic patterning are highly conserved. The finding that the proteins encoded by paralogous and orthologous genes within and between species can functionally replace each another is no longer novel; in most cases this is the expected result. How, then, does divergent morphology arise between species? One hypothesis that fits well with comparative data is that divergent morphogenesis arises from genetic differences in the timing, level and pattern of orthologous gene expression during development. This idea is being tested using a functional genetic approach comparing limb morphogenesis between the mouse and bat.

Maternal and embryonic expression of zebrafish lef1.

Transcription factors of the TCF/LEF family interact with the Wnt signaling pathway to control transcription of downstream genes (Clevers, H., van de Wetering, M., 1997. TCF/LEF factor earn their wings. Trends Genet. 13, 485-489). We were interested in cloning family members which were expressed in zebrafish neural crest, because Wnt signaling modulates specification of neural crest fate (Dorsky, R.I., Moon, R.T., Raible, D.W., 1998. Control of neural crest cell fate by the Wnt signalling pathway. Nature 396, 370-373). We cloned a zebrafish homolog of lef1 and localized its chromosomal position by radiation hybrid mapping. lef1 is expressed in the neural crest as well as the tailbud and developing mesoderm, and is maternally expressed in zebrafish, unlike mouse and Xenopus homologs. In addition, we cloned two tcf3 genes and a homolog of tcf4, neither of which were strongly expressed in premigratory neural crest.

alyron, an insertional mutation affecting early neural crest development in zebrafish.

alyronz12 (aln) is a recessive lethal mutation that affects early stages of neural crest development in the zebrafish. alyron appears to be an insertional mutation as the mutation was generated following microinjection of plasmid DNA into one-cell embryos and the stably integrated transgenic sequences are closely linked to the mutation. The insertion site harbors multiple copies of the plasmid sequence that have experienced complex rearrangements. Host-insert junction fragments have been molecularly cloned and host sequences adjacent to the transgene have been used to map the mutation to the distal arm of linkage group 15. alyron function is required cell-autonomously in the neural crest lineage. alyron mutants have a severe but not complete deficit of premigratory neural crest as judged by reduced expression of several markers associated with early stages of neural crest development. Lack of premigratory neural crest is likely to account for the two most conspicuous characteristics of alyron mutants: the absence of body pigmentation and the inability to affect blood circulation. The neural crest phenotype of alyron mutants resembles that observed in mouse mutants that lack Pax-3 or both Wnt-1 and Wnt-3a function, and expression of the zebrafish homologues of these genes is greatly reduced in the dorsal neural keels of alyron mutants. In contrast, ventral neural keel identity appears unaffected. Given our findings that the mutation is unlinked to pax or wnt genes that have been described in the zebrafish, we propose that alyron is a novel gene function required for the specification and/or proliferative expansion of neural crest progenitors.

Cell mixing during early epiboly in the zebrafish embryo.

Descendants of early blastomeres in the zebrafish come to populate distinctive regions of the fate map. We present a model suggesting that the distribution of cells in the early gastrula (the fate map stage) results from the passive response of cells to reproducible forces that change the overall shape of the blastoderm just prior to gastrulation. We suggest that one of the morphogenetic changes that accompanies epiboly, the upward doming of the yolk cell into the overlying blastoderm, could be responsible for cell mixing. In support of the model, we show that the timing, extent, and directions of cell mixing in the embryo accurately reflect the expectations of the model. Finally, we show that one portion of the gastrula, a marginal region that later gives rise to many of the mesendodermal derivatives, experiences little cell mixing during the doming process. As a result, this region in the gastrula is populated by the descendants of the subset of the early blastomeres that were originally at the margin. The finding that cytoplasm initially at the edge of the 1-celled blastodisc is transmitted specifically to mesendodermal precursors at the fate map stage raises the possibility that maternal determinants may contribute to initiation of embryonic patterning in the zebrafish embryo.

Contribution of early cells to the fate map of the zebrafish gastrula.

Previously, a tissue-specific fate map was compiled for the gastrula stage of the zebrafish embryo, indicating that development subsequent to this stage follows a reproducible pattern. Here it is shown that each early zebrafish blastomere normally contributes to a subset of the gastrula and thus gives rise to a limited array of tissues. However, the final contribution that any early blastomere makes to the fate map in the gastrula cannot be predicted because of variability in both the position of the future dorsoventral axis with respect to the early cleavage blastomeres and the scattering of daughter cells as the gastrula is formed. Therefore, early cell divisions of the zebrafish embryo cannot reproducibly segregate determinants of tissue fates.

Some techniques in rehabilitating the school phobic adolescent.

This paper has described several intervention strategies which may be of help to the school phobic adolescent. I have set forth the specifics of the attendance contract which is negotiated with the youngster and with the school. Principles of community mental health stress the importance of collaboration with other care givers to resolve psychological problems. Therefore, I have placed major emphasis on the role of the school in enhancing the early return of the school phobic youngster to school.