Paul A. Krieg

The XHex homeobox gene is expressed during development of the vascular endothelium: overexpression leads to an increase in vascular endothelial cell number

The Hex/Prh homeobox gene is expressed in a subset of adult blood cell types and may play a role in the differentiation of the myeloid and B-cell lineages. In a search for homeobox genes involved in cardiovascular development, we have independently isolated a Xenopus laevis cDNA which appears to be the amphibian orthologue of Hex/Prh. Based on high sequence similarity in a number of regions, particularly the critical homeobox, we have named this gene XHex. This developmentally regulated gene is first expressed in the dorsal endomesoderm of the gastrula stage embryo. This tissue goes on to contribute to the structures of the embryonic liver and XHex continues to be expressed in the liver throughout development. From the tailbud stage, XHex is expressed in vascular endothelial cells throughout the developing vascular network. Vascular expression of XHex is transient and commences slightly after expression of the receptor tyrosine kinase gene, flk-1, which is known to be essential for vascular development. This observation raises the possibility that XHex is one of the transcription factors that responds to the VEGF/Flk-1 signal transduction pathway leading to differentiation of vascular endothelial cells. XHex is unique amongst homeobox genes in displaying expression in the endothelial layer throughout the developing vasculature. Overexpression of XHex sequences in the frog embryo causes disruption to developing vascular structures and an increase in the number of vascular endothelial cells, suggesting a possible role in regulation of cell proliferation.

Sfrp5 coordinates foregut specification and morphogenesis by antagonizing both canonical and noncanonical Wnt11 signaling

Cell identity and tissue morphogenesis are tightly orchestrated during organogenesis, but the mechanisms regulating this are poorly understood. We show that interactions between Wnt11 and the secreted Wnt antagonist secreted frizzled-related protein 5 (Sfrp5) coordinate cell fate and morphogenesis during Xenopus foregut development. sfrp5 is expressed in the surface cells of the foregut epithelium, whereas wnt11 is expressed in the underlying deep endoderm. Depletion of Sfrp5 results in reduced foregut gene expression and hypoplastic liver and ventral pancreatic buds. In addition, the ventral foregut cells lose adhesion and fail to form a polarized epithelium. We show that the cell fate and epithelial defects are due to inappropriate Wnt/beta-catenin and Wnt/PCP signaling, respectively, both mediated by Wnt11. We provide evidence that Sfrp5 locally inhibits Wnt11 to maintain early foregut identity and to allow an epithelium to form over a mass of tissue undergoing Wnt-mediated cell movements. This novel mechanism coordinating canonical and noncanonical Wnt signaling may have broad implications for organogenesis and cancer.

pXeX, a vector for efficient expression of cloned sequences in Xenopus embryos

We describe the construction of pXeX, a plasmid vector that efficiently expresses cloned sequences in Xenopus embryos. This plasmid contains the transcription regulatory regions from the Xenopus laevis elongation factor-1 alpha-encoding gene (EF-1 alpha). Expression of cloned sequences commences in blastula-stage embryos, coincident with transcriptional activation of the embryonic genome, and transcripts may persist until the tadpole stage of development.

The RNA-binding protein gene, hermes, is expressed at high levels in the developing heart

In a screen for novel sequences expressed during embryonic heart development we have isolated a gene which encodes a putative RNA-binding protein. This protein is a member of one of the largest families of RNA-binding proteins, the RRM (RNA Recognition Motif) family. The gene has been named hermes (for HEart, RRM Expressed Sequence). The hermes protein is 197-amino acids long and contains a single RRM domain. In situ hybridization analysis indicates that hermes is expressed at highest levels in the myocardium of the heart and to a lesser extent in the ganglion layer of the retina, the pronephros and epiphysis. Expression of hermes in each of these tissues begins at approximately the time of differentiation and is maintained throughout development. Analysis of the RNA expression of the hermes orthologues from chicken and mouse reveals that, like Xenopus, the most prominent tissue of expression is the developing heart. The sequence and expression pattern of hermes suggests a role in post-transcriptional regulation of heart development.

tinman-related genes expressed during heart development in Xenopus

The tinman homeobox gene of Drosophila is absolutely required for development of the insect heart. This observation prompted the isolation of tinman-related genes from vertebrates, in the hope that the developmental function of the gene would be conserved between evolutionarily distinct species. The first vertebrate tinman gene, Nkx2-5, was isolated from mouse and subsequently, orthologues of Nkx2-5 have been isolated from a number of different species. In all cases, a conserved pattern of Nkx2-5 expression is observed in the developing heart, commencing prior to differentiation. Genetic ablation of Nkx2-5 in the mouse results in embryonic lethality due to heart defects, but most myocardial genes are expressed normally and a beating heart tube forms. This observation raises the possibility that additional genes related to Nkx2-5 are partially rescuing Nkx2-5 function in the null mouse. Recently, additional members of the tinman-related gene family have been discovered and characterized in a number of different species. Somewhat surprisingly, orthologous genes in different organisms can be rather divergent in sequence and may show completely different expression patterns. In at least some organisms, expression of the tinman-related genes is not observed in the heart. Due to the increasing number of family members and the somewhat divergent expression patterns, the precise role of the tinman-related genes in cardiac development remains an open question. In a search for additional tinman-related genes in the frog, Xenopus laevis, we have identified Nkx2-9, a novel member of the tinman-related gene family. Preliminary characterization reveals that Nkx2-9 is expressed in the cardiogenic region of the embryo prior to differentiation, but transcript levels decrease rapidly, in the heart, at about the time that differentiation commences.

Molecular Mechanisms of Vascular Development

Publisher Summary This chapter focuses on the molecules involved in regulation of embryonic vascular development. Molecule involved in early mesodermal differentiation of endothelial cells include fibroblast growth factor (FGF), which plays a critical role in the induction of the mesodermal germ layer during the earliest stages of embryogenesis. FGFs are also required for the development of certain dorsal types of mesoderm, suggesting a synergism between FGF and the transforming growth factor-β family of growth factors, which are required for dorsal mesoderm formation. Angioblast differentiation in the mesoderm requires the activity of vascular endothelial growth factor (VEGF) and its receptor, Flk-1. It seems possible that the directed migration of flk-1 expressing endothelial cells from the lateral plate mesoderm toward the midline of the frog embryo occurs in response to a VEGF signal gradient. In vitro experiments have demonstrated that fibronectin can stimulate the migration of vascular endothelial cells. Second, when the distribution of fibronectin is analyzed in the chick embryo or the chick chorioallantoic membrane, it is associated with both migrating endothelial cells, prior to their coalescence into vessels, and with the early steps of angiogenesis, when capillaries are extending and invading avascular tissue. Further, platelet-derived growth factor is important for the recruitment of vascular wall components. It is found that Tie-2 is required for vascular remodeling. In addition to a role in vasculogenesis, angiogenesis, and endothelial cell migration, in vitro studies suggest that the extracellular matrix also influences the differentiation of endothelial cells.

Remarkable sequence conservation of transcripts encoding amphibian and mammalian homologues of quaking, a KH domain RNA-binding protein

Mutations in the mouse quaking locus can result in two different types of developmental phenotypes: (1) a deficiency of myelin in the central nervous system that is accompanied by a characteristic tremor, or (2) embryonic lethality around day 9 of gestation. A quaking candidate gene (qkI) that encodes a KH motif protein has recently been identified. We have isolated and characterized cDNAs encoding the Xenopus quaking homologue (Xqua) and also assembled an almost complete human quaking sequence from expressed sequence tags. Sequence comparisons show that the amphibian and mammalian quaking transcripts exhibit striking conservation, both within the coding region and, unexpectedly, in the 3 UTR. Two Xqua transcripts 5 kb and 5.5 kb in length are differentially expressed in the Xenopus embryo, with the 5 kb transcript being detected as early as the gastrula stage of development. Using an in vitro assay, we have demonstrated RNA-binding activity for quaking protein encoded by the 5 kb transcript. Overall, the high sequence conservation of quaking sequences suggests an important conserved function in vertebrate development, probably in the regulation of RNA metabolism.

Hox11-family genes XHox11 and XHox11L2 in Xenopus: XHox11L2 expression is restricted to a subset of the primary sensory neurons

The mouse genome contains a small family of homeobox genes related to Hox11, but relatively little is known about the expression of these genes during early development. Hox11 itself is expressed in the embryonic spleen, among other tissues, and is required for its formation. No description of Hox11L2 expression has been presented previously. We have isolated the Xenopus orthologs of Hox11 and Hox11L2 and have carefully compared their expression patterns during embryogenesis. The localization of Xhox11 transcripts in the branchial arches, cranial sensory ganglia and spinal cord is similar, but not identical, to that of mouse Hox11. Xhox11 expression is not detected in the developing spleen. XHox11L2 is expressed exclusively in a portion of the primary sensory system in the frog embryo, including the cranial sensory ganglia and the Rohon‐Beard sensory neurons. There is significant overlap in the patterns of Xhox11 and XHox11L2 expression in the spinal cord and cranial sensory ganglia during early development, suggesting that they may function redundantly in these tissues. The timing of Xhox11 and Xhox11L2 expression indicates that Hox11‐family members may participate in the final stages of the differentiation process. Dev Dyn 1999;214:34–43.

Medium weight neurofilament mRNA in goldfish Mauthner axoplasm

Although axons are generally considered to lack the ability to synthesize proteins, the Mauthner axon (M-axon) of the goldfish has been reported to contain some of the basic components of the translational machinery, such as transfer RNA (tRNA), ribosomal RNA (rRNA), and ribosomes. To determine if the M-axon also contains mRNA, we isolated samples of M-axoplasm free of glial contamination as demonstrated by the absence of glial-specific mRNA and protein. Reverse transcription-polymerase chain reaction (RT-PCR) of M-axoplasmic cDNA in the presence of primers for the goldfish medium-weight neurofilament (NF-M) gene produced a single product of the expected length for RT-PCR amplification of goldfish NF-M mRNA. This mRNA might direct protein synthesis of NF-M within the M-axoplasm.

Distinct expression patterns for two Xenopus Bar homeobox genes.

Abstractu2002The BarH1 and BarH2 homeobox genes are coexpressed in cells of the fly retina and in the central and peripheral nervous systems. The fly Bar genes are required for normal development of the eye and external sensory organs. In Xenopus we have identified two distinct vertebrate Bar-related homeobox genes, XBH1 and XBH2. XBH1 is highly related in sequence and expression pattern to a mammalian gene, MBH1, suggesting that they are orthologues. XBH2 has not previously been identified but is clearly related to the DrosophilaBar genes. During early Xenopus embryogenesis XBH1 and XBH2 are expressed in overlapping regions of the central nervous system. XBH1, but not XBH2, is expressed in the developing retina. By comparing the expression of XBH1 with that of hermes, a marker of differentiated retinal ganglion cells, we show that XBH1 is expressed in retinal ganglion cells during the differentiation process, but is down-regulated as cells become terminally differentiated.