Development of the adult form requires coordinated growth and patterning of multiple traits in response to local gene activity as well as to global endocrine and physiological effectors. An excellent example of such coordination is the skeleton. Skeletal development depends on the differentiation and morphogenesis of multiple cell types to generate elements with distinct forms and functions throughout the body. We show that zebrafish touchtone/nutria mutants exhibit severe growth retardation and gross alterations in skeletal development in addition to embryonic melanophore and touch-response defects. These alterations include accelerated endochondral ossification but delayed intramembranous ossification, as well as skeletal deformities. We show that the touchtone/nutria phenotype results from mutations in trpm7, which encodes a transient receptor potential (TRP) family member that functions as both a cation channel and kinase. We find trpm7 expression in the mesonephric kidney and show that mutants develop kidney stones, indicating renal dysfunction. These results identify a requirement for trpm7 in growth and skeletogenesis and highlight the potential of forward genetic approaches to uncover physiological mechanisms contributing to the development of adult form.

SUMMARY Early in the development of animal embryos, superficial cells of the blastula form a distinct lineage and adopt an epithelial morphology. In different animals, the fate of these primary superficial epithelial (PSE) cells varies, and it is unclear whether pathways governing segregation of blastomeres into the PSE lineage are conserved. Mutations in the gene encoding Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic forms of cleft lip and palate, consistent with a role for Irf6 in development of oral epithelia, and mouse Irf6 targeted null mutant embryos display abnormal differentiation of oral epithelia and skin. In Danio rerio (zebrafish) and Xenopus laevis (African clawed frog) embryos, zygotic irf6 transcripts are present in many epithelial tissues including the presumptive PSE cells and maternal irf6 transcripts are present throughout all cells at the blastula stage. Injection of antisense oligonucleotides with ability to disrupt translation of irf6 transcripts caused little or no effect on development. By contrast, injection of RNA encoding a putative dominant negative Irf6 caused epiboly arrest, loss of gene expression characteristic of the EVL, and rupture of the embryo at late gastrula stage. The dominant negative Irf6 disrupted EVL gene expression in a cell autonomous fashion. These results suggest Irf6 translated in the oocyte or unfertilized egg suffices for early development. Supporting the importance of maternal Irf6, we show that depletion of maternal irf6 transcripts in X. laevis embryos leads to gastrulation defects and rupture of the superficial epithelium. These experiments reveal a conserved role for maternally-encoded Irf6 in differentiation of a simple epithelium in X. laevis and D. rerio. This epithelium constitutes a novel model tissue in which to explore the Irf6 regulatory pathway.

A wide variety of modified oligonucleotides have been tested as antisense agents. Each chemical modification produces a distinct profile of potency, toxicity, and specificity. Novel cationic phosphoramidate-modified antisense oligonucleotides have been developed recently that have unique and interesting properties. We compared the relative potency and specificity of a variety of established antisense oligonucleotides, including phosphorothioates (PS), 2'-O-methyl (2'OMe) RNAs, locked nucleic acids (LNAs), and neutral methoxyethyl (MEA) phosphoramidates with new cationic N,N-dimethylethylenediamine (DMED) phosphoramidate-modified antisense oligonucleotides. A series of oligonucleotides was synthesized that targeted two sites in the Xenopus laevis survivin gene and were introduced into Xenopus embryos by microinjection. Effects on survivin gene expression were examined using quantitative real-time PCR. Of the various modified oligonucleotide designs tested, LNA/PS chimeras (which showed the highest melting temperature) and DMED/phosphodiester chimeras (which showed protection of neighboring phosphate bonds) were potent in reducing gene expression. At 40 nM, overall specificity was superior for the LNA/PS-modified compounds compared with the DMED-modified oligonucleotides. However, at 400 nM, both of these compounds led to significant degradation of survivin mRNA, even when up to three mismatches were present in the heteroduplex.

The experimental manipulation of early embryologic events, resulting in the misexpression of the homeobox transcription factor pitx2, is associated with subsequent defects of laterality in a number of vertebrate systems. To clarify the role of one pitx2 isoform, pitx2c, in determining the left-right axis of amphibian embryos, we examined the heart and gut morphology of Xenopus laevis embryos after attenuating pitx2c mRNA levels using chemically modified antisense oligonucleotides. We demonstrate that the partial depletion of pitx2c mRNA in these embryos results in alteration of both cardiac morphology and intestinal coiling. The most common cardiac abnormality seen was a failure of rightward migration of the outflow tract, while the most common intestinal laterality phenotype seen was a full reversal in the direction of coiling, each present in 23% of embryos injected with the pitx2c antisense oligonucleotide. An abnormality in either the heart or gut further predisposed to a malformation in the other. In addition, a number of other cardiac anomalies were observed after pitx2c mRNA attenuation, including abnormalities of atrial septation, extracellular matrix restriction, relative atrial-ventricular chamber positioning, and restriction of ventricular development. Many of these findings correlate with cardiac defects previously reported in pitx2 null and hypomorphic mice, but can now be assigned specifically to attenuation of the pitx2c isoform in Xenopus.

One of the hallmarks of early development is the rapid proliferation of cells immediately after fertilization. Many of the rules that govern cell division in normal somatic cells, such as contact inhibition and apoptosis, seem temporarily suspended in the early embryo. A similar suspension of mechanisms normally regulating cell division occurs in the development of cancer. Survivin, an inhibitor of apoptosis and a positive regulator of progression through the cell cycle, localizes to the mitotic spindle and interacts with several proapoptotic caspases. Survivin protein expression has been studied during the development of the salivary gland in mouse. However, the regulation of survivin during the critical transitions defining oocyte maturation and the early restriction of developmental potential are not easily examined in the mouse. We therefore studied survivin mRNA expression during oogenesis and early embryogenesis in Xenopus laevis. We found that survivin mRNA is present in the earliest stages of Xenopus oocytes and that it accumulates during oogenesis. Progesterone-induced maturation of Xenopus oocytes leads to polyadenylation of the survivin transcript. Survivin mRNA is also present in early Xenopus embryos. After the onset of zygotic transcription, however, the amount of survivin mRNA declines rapidly to undetectable levels. This decrease in survivin mRNA correlates temporally with both the slowing of the cell cycle and the onset of endogenous embryonic apoptosis. With the exception of the ovary, survivin mRNA was undetectable in all adult Xenopus tissues examined.

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Naïve CD8+ T cells express a predominantly CD62Lhigh phenotype, but CD62L expression is rapidly lost during the effector phase of the immune response and only slowly regained during the memory phase. We used adoptive transfer of TCR-transgenic T cells followed by Listeria infection to study the dynamics of cell division and differentiation in vivo. Adoptive transfer of larger quantities (400 000) of naïve TCR-transgenic T cells leads to diminished T cell growth following infection and a higher proportion of cells remaining CD62Lhigh than populations derived from adoptive transfer populations of smaller quantities (3200 naïve TCR-tg T cells). This suggests a process of 'division-linked differentiation', where a proportion of cells change phenotype (in this case CD62Lhigh => CD62Llow) upon division. A simple model of division-linked CD62L differentiation where 20% of CD62Lhigh cells differentiate to become CD62Llow on each division accurately predicts the phenotype of cells during acute infection. By contrast, expression of CD127 did not conform to this simple pattern of differentiation. These results suggests that CD62Lhigh and CD62Llow cells arise from the same precursors, and CD62Llow cells are formed from the CD62Lhigh population by a process of progressive, division-linked differentiation.