Microarray-based analysis of early development in Xenopus laevis.

In order to examine transcriptional regulation globally, during early vertebrate embryonic development, we have prepared Xenopus laevis cDNA microarrays. These prototype embryonic arrays contain 864 sequenced gastrula cDNA. In order to analyze and store array data, a microarray analysis pipeline was developed and integrated with sequence analysis and annotation tools. In three independent experimental settings, we demonstrate the power of these global approaches and provide optimized protocols for their application to molecular embryology. In the first set, by comparing maternal versus zygotic transcription, we document groups of genes that are temporally regulated. This analytical approach resulted in the discovery of novel temporally regulated genes. In the second, we examine changes in gene expression spatially during development by comparing dorsal and ventral mesoderm dissected from early gastrula embryos. We have discovered novel genes with spatial enrichment from these experiments. Finally, we use the prototype microarray to examine transcriptional responses from embryonic explants treated with activin. We selected genes (two of which are novel) regulated by activin for further characterization. All results obtained by the arrays were independently tested by RT-PCR or by in situ hybridization to provide a direct assessment of the accuracy and reproducibility of these approaches in the context of molecular embryology.

Neural patterning in the vertebrate embryo.

The embryonic central nervous system (CNS) is patterned along its antero-posterior, dorsal-ventral, and left-right axes. Along the dorsal-ventral axis, cell fate determination occurs during and following neural tube closure and involves the action of two opposing signaling pathways: SHH ventrally from the notochord and BMP/GDF dorsally from the boundary of neural and nonneural ectoderm and later from the roof plate. In addition, Wnt and retinoic acid signaling have been shown to act in dorsal-ventral patterning; however, their roles are understood in less detail. Along the antero-posterior axis, signals divide the neural tube into four major divisions: forebrain, midbrain, hindbrain, and spinal cord, and these differences can be detected soon after the formation of the neural plate. The FGF, Wnt, and retinoic acid signaling pathways have been implicated in the caudalization of neural tissue. Boundaries of Hox gene expression are observed along the anteroposterior axis and have been suggested to be involved in establishing different identities in the hindbrain and spinal cord. Complex gene expression patterns in the brain suggest the development of neuromeres dividing the brain into different regions that are elaborated further during development. Patterning along the left-right axis occurs concurrently with antero-posterior and dorsal-ventral patterning during gastrulation. A leading candidate for initiating asymmetry is activin, which acts through Nodal and Lefty before any morphological differences are observed. The big challenge will be understanding how these diverse signaling pathways interact both temporally and spatially to generate the complex adult nervous system.

Pax6 induces ectopic eyes in a vertebrate.

We report here that misexpression of the transcription factor Pax6 in the vertebrate Xenopus laevis leads to the formation of differentiated ectopic eyes. Multiple molecular markers indicated the presence of mature lens fiber cells, ganglion cells, Müller cells, photoreceptors and retinal pigment epithelial cells in a spatial arrangement similar to that of endogenous eyes. Lineage tracing experiments showed that lens, retina and retinal pigment epithelium arose as a consequence of the cell-autonomous function of Pax6. These experiments also reveal that the cell autonomous activity of misexpressed Pax6 causes the ectopic expression of a number of genes including Rx, Otx2, Six3 and endogenous Pax6, each of which has been implicated in eye development. The formation of ectopic and endogenous eyes could be suppressed by coexpression of a dominant-negative form of Pax6. These data show that in vertebrates, as in the invertebrate Drosophila melanogaster, Pax6 is both necessary and sufficient to trigger the cascade of events required for eye formation.

A highly conserved lens transcriptional control element from the Pax-6 gene.

We have identified a short segment of the mouse Pax-6 gene 5' flanking region that is necessary and sufficient for reporter construct expression in components of the eye derived from non-neural ectoderm. This transcriptional control element has a highly conserved nucleotide sequence over 341 bp and is located approximately 3.5 kb upstream of the start-point for transcription from the most proximal promoter (PO) of the Pax-6 gene. The level of identity between the human and mouse Pax-6 genes in this region is 93%. When combined either with its natural promoter or a heterologous minimal promoter, this element directs reporter construct expression to a region of surface ectoderm overlying the optic cup beginning at E8.5-9.0 (12-14 somites). Subsequently, expression is restricted to the lens (primarily the lens epithelium) and the corneal epithelium. This element will provide an important tool in future transgenic analyses of lens formation and will allow identification of transcription factors with a central function in lens development.

Lens induction by Pax-6 in Xenopus laevis.

Despite extensive study following the pioneering work of Spemann on lens development (Spemann, H. (1901) Verh. Anat. Ges. 15, 61-79) and the subsequent establishment of the concept of embryonic induction, the molecular mechanism of vertebrate lens induction remains largely unknown. Here we report that in Xenopus expression of Pax-6 results in lens formation in a cell autonomous manner. In animal cap experiments, Pax-6 induced expression of the lens-specific marker beta B1-crystallin without inducing the general neural marker NCAM. Ectopic Pax-6 expression also resulted in the formation of ectopic lenses in whole embryos as well as in animal cap explants indicating that in vertebrates, as well as Drosophila (Halder, G., Callaerts, P., and Gehring, W.J. (1995) Science 267, 1788-1792), Pax-6 can direct the development of major components of the eye. Interestingly, ectopic lenses formed in whole embryos without association with neural tissue. Treatments giving rise to anterior neural tissue in animal cap explants resulted in the expression of both beta B1-crystallin and Pax-6. Given the ability of Pax-6 to direct lens formation, we propose that the establishment of Pax-6 expression in the presumptive lens ectoderm during normal development is likely to be a critical response of lens-competent ectoderm to early lens inducers.

RNA cleavage and chain elongation by Escherichia coli DNA-dependent RNA polymerase in a binary enzyme.RNA complex.

In the absence of DNA, Escherichia coli RNA polymerase (EC 2.7.7.6) can bind RNA to form an equimolar binary complex with the concomitant release of the sigma factor. We show now that E. coli RNA polymerase binds at a region near the 3' terminus of the RNA and that an RNA in such RNA.RNA polymerase complexes undergoes reactions previously thought to be unique to nascent RNA in ternary complexes with DNA. These include GreA/GreB-dependent cleavage of the RNA and elongation by 3'-terminal addition of NMP from NTP. Both of these reactions are inhibited by rifampicin. Hence, by several criteria, the RNA in binary complexes is bound to the polymerase in a manner quite similar to that in ternary complexes. These findings can be explained by a model for the RNA polymerase ternary complex in which the RNA is bound at the 3' terminus through two protein binding sites located up to 10 nt apart. In this model, the stability of RNA binding to the polymerase in the ternary complex is due primarily to its interaction with the protein.

Melanoma recurrence after excision. Is a wide margin justified?

Through retrospective analysis of patient records for 187 patients with melanoma seen between 1975 and 1989, the aim of this study was to determine whether outcome varied according to degrees of surgical intervention in the primary treatment of stage I disease for thin, intermediate, and thick lesions. There were no significant differences in recurrence rate associated with an excision margin of 15 mm or less compared with wider excision margins; with initial excision compared with wider re-excision after excision biopsy; or for primary closure as compared with closure with a graft. There was, however, a significant difference in wound complication rate between wounds closed primarily (6%) and those closed by grafting (31%) (p < 0.01). The authors advocate the more conservative excision margin of 1.00 cm to 1.50 cm in the treatment of stage I melanoma with primary closure of the wound where possible.