Search

ABSTRACTThis is the first report of a Cryptococcus neoformansvar. gattiistrain (serotype B) that switches reversibly between its parent mucoid (NP1-MC) colony morphology and a smooth (NP1-SM) colony morphology. Similar to C. neoformansvar. grubiiand C. neoformansvar. neoformansstrains, the switch is associated with changes in the polysaccharide capsule and virulence in animal models. In murine infection models, NP1-MC is significantly more virulent than NP1-SM (P< 0.021). In contrast to the serotype A and D strains, the serotype B strain switches in vivo reversibly between both colony morphologies. The polysaccharide of NP1-MC exhibits a thicker capsule, and thus NP1-MC exhibits enhanced intracellular survival in macrophages. Consistent with this finding, switching to the mucoid variant is observed in pulmonary infection with NP1-SM. In contrast, the thin polysaccharide capsule of NP1-SM permits better crossing of the blood-brain barrier. In this regard, only smooth colonies were grown from brain homogenates of NP1-MC-infected mice. Our findings have important implications for the pathogenesis of cryptococcosis and suggest that phenotypic switching affects host-pathogen interactions in the local microenvironment. This altered interaction then selects for specific colony variants to arise in a pathogen population.

Neural crest cells play a key role in craniofacial development. The endothelin family of secreted polypeptides regulates development of several neural crest sublineages, including the branchial arch neural crest. The basic helix-loop-helix transcription factor dHAND is also required for craniofacial development, and in endothelin-1 (ET-1) mutant embryos, dHAND expression in the branchial arches is down-regulated, implicating it as a transcriptional effector of ET-1 action. To determine the mechanism that links ET-1 signaling to dHAND transcription, we analyzed the dHAND gene for cis-regulatory elements that control transcription in the branchial arches. We describe an evolutionarily conserved dHAND enhancer that requires ET-1 signaling for activity. This enhancer contains four homeodomain binding sites that are required for branchial arch expression. By comparing protein binding to these sites in branchial arch extracts from endothelin receptor A (EdnrA) mutant and wild-type mouse embryos, we identified Dlx6, a member of the Distal-less family of homeodomain proteins, as an ET-1-dependent binding factor. Consistent with this conclusion, Dlx6 was down-regulated in branchial arches from EdnrA mutant mice. These results suggest that Dlx6 acts as an intermediary between ET-1 signaling and dHAND transcription during craniofacial morphogenesis.

Heart formation in vertebrates is believed to occur in a segmental fashion, with discreet populations of cardiac progenitors giving rise to different chambers of the heart. However, the mechanisms involved in specification of different chamber lineages are unclear. The basic helix-loop-helix transcription factor dHAND is expressed in cardiac precursors throughout the cardiac crescent and the linear heart tube, before becoming restricted to the right ventricular chamber at the onset of looping morphogenesis. dHAND is also expressed in the branchial arch neural crest, which contributes to craniofacial structures and the aortic arch arteries. Using a series of dHAND-lacZ reporter genes in transgenic mice, we show that cardiac and neural crest expression of dHAND are controlled by separate upstream enhancers and we describe a composite cardiac-specific enhancer that directs lacZ expression in a pattern that mimics that of the endogenous dHAND gene throughout heart development. Deletion analysis reduced this enhancer to a 1.5 kb region and identified subregions responsible for expression in the right ventricle and cardiac outflow tract. Comparison of mouse regulatory elements required for right ventricular expression to the human dHAND upstream sequence revealed two conserved consensus sites for binding of GATA transcription factors. Mutation of these sites abolished transgene expression in the right ventricle, identifying dHAND as a direct transcriptional target of GATA factors during right ventricle development. Since GATA factors are not chamber-restricted, these findings suggest the existence of positive and/or negative coregulators that cooperate with GATA factors to control right ventricular-specific gene expression in the developing heart.

Limb outgrowth and patterning of skeletal elements are dependent on complex tissue interactions involving the zone of polarizing activity (ZPA) in the posterior region of the limb bud and the apical ectodermal ridge. The peptide morphogen Sonic hedgehog (SHH) is expressed specifically in the ZPA and, when expressed ectopically, is sufficient to mimic its functions, inducing tissue growth and formation of posterior skeletal elements. We show that the basic helix-loop-helix transcription factor dHAND is expressed posteriorly in the developing limb prior to Shh and subsequently occupies a broad domain that encompasses the Shh expression domain. In mouse embryos homozygous for a dHAND null allele, limb buds are severely underdeveloped and Shh is not expressed. Conversely, misexpression of dHAND in the anterior region of the limb bud of transgenic mice results in formation of an additional ZPA, revealed by ectopic expression of Shh and its target genes, and resulting limb abnormalities that include preaxial polydactyly with duplication of posterior skeletal elements. Analysis of mouse mutants in which Hedgehog expression is altered also revealed a feedback mechanism in which Hedgehog signaling is required to maintain the full dHAND expression domain in the developing limb. Together, these findings identify dHAND as an upstream activator of Shh expression and important transcriptional regulator of limb development.