Induction of pancreatic differentiation by signals from blood vessels.

Blood vessels supply developing organs with metabolic sustenance. Here, we demonstrate a role for blood vessels as a source of developmental signals during pancreatic organogenesis. In vitro experiments with embryonic mouse tissues demonstrate that blood vessel endothelium induces insulin expression in isolated endoderm. Removal of the dorsal aorta in Xenopus laevis embryos results in the failure of insulin expression in vivo. Furthermore, using transgenic mice, we show that ectopic vascularization in the posterior foregut leads to ectopic insulin expression and islet hyperplasia. These results indicate that vessels not only provide metabolic sustenance, but also provide inductive signals for organ development.

Notochord patterning of the endoderm.

Endodermally derived organs of the gastrointestinal and respiratory system form at distinct anterioposterior and dorsoventral locations along the vertebrate body axis. This stereotyped program of organ formation depends on the correct patterning of the endodermal epithelium so that organ differentiation and morphogenesis occur at appropriate positions along the gut tube. Whereas some initial patterning of the endoderm is known to occur early, during germ-layer formation and gastrulation, later signaling events, originating from a number of adjacent tissue layers, are essential for the development of endodermal organs. Previous studies have shown that signals arising from the notochord are important for patterning of the ectodermally derived floor plate of the neural tube and the mesodermally derived somites. This review will discuss recent evidence indicating that signals arising from the notochord also play a role in regulating endoderm development.

VEGF mediates angioblast migration during development of the dorsal aorta in Xenopus.

Angioblasts are precursor cells of the vascular endothelium which organize into the primitive blood vessels during embryogenesis. The molecular mechanisms underlying patterning of the embryonic vasculature remain unclear. Mutational analyses of the receptor tyrosine kinase flk-1 and its ligand vascular endothelial growth factor, VEGF, indicate that these molecules are critical for vascular development. Targeted ablation of the flk-1 gene results in complete failure of blood and vascular development (F. Shalaby et al. (1995) Nature 376, 62-66), while targeted ablation of the VEGF gene results in gross abnormalities in vascular patterning (P. Carmeliet et al. (1996) Nature 380, 435-439; N. Ferrara et al. (1996) Nature 380, 439-442). Here we report a role for VEGF in patterning the dorsal aorta of the Xenopus embryo. We show that the diffusible form of VEGF is expressed by the hypochord, which lies at the embryonic midline immediately dorsal to the location of the future dorsal aorta. We find that, initially, no flk-1-expressing angioblasts are present at this location, but that during subsequent development, angioblasts migrate from the lateral plate mesoderm to the midline where they form a single dorsal aorta. We have demonstrated that VEGF can act as a chemoattractant for angioblasts by ectopic expression of VEGF in the embryo. These results strongly suggest that localized sources of VEGF play a role in patterning the embryonic vasculature.

Endoderm patterning by the notochord: development of the hypochord in Xenopus.

The patterning and differentiation of the vertebrate endoderm requires signaling from adjacent tissues. In this report, we demonstrate that signals from the notochord are critical for the development of the hypochord, which is a transient, endodermally derived structure that lies immediately ventral to the notochord in the amphibian and fish embryo. It appears likely that the hypochord is required for the formation of the dorsal aorta in these organisms. We show that removal of the notochord during early neurulation leads to the complete failure of hypochord development and to the elimination of expression of the hypochord marker, VEGF. Removal of the notochord during late neurulation, however, does not interfere with hypochord formation. These results suggest that signals arising in the notochord instruct cells in the underlying endoderm to take on a hypochord fate during early neural stages, and that the hypochord does not depend on further notochord signals for maintenance. In reciprocal experiments, when the endoderm receives excess notochord signaling, a significantly enlarged hypochord develops. Overall, these results demonstrate that, in addition to patterning neural and mesodermal tissues, the notochord plays an important role in patterning of the endoderm.

Overexpression of the tinman-related genes XNkx-2.5 and XNkx-2.3 in Xenopus embryos results in myocardial hyperplasia.

Drosophila tinman is an NK-class homeobox gene required for formation of the dorsal vessel, the insect equivalent of the vertebrate heart. Vertebrate sequences related to tinman, such as mouse Nkx-2.5, chicken cNkx-2.5, Xenopus XNkx-2.5 and XNkx-2.3 are expressed in cardiac precursors and in tissues involved in induction of cardiac mesoderm. Mice which lack a functional Nkx-2.5 gene die due to cardiac defects. To determine the role of tinman-related sequences in heart development, we have overexpressed both XNkx-2.3 and XNkx-2.5 in Xenopus laevis embryos. The resulting embryos are morphologically normal except that they have enlarged hearts. The enlarged heart phenotype is due to a thickening of the myocardium caused by an increase in the overall number of myocardial cells (hyperplasia). Neither ectopic nor precocious expression of cardiac differentiation markers is detectable in overexpressing embryos. These results suggest that both XNkx-2.3 and XNkx-2.5 are functional homologues of tinman, responsible for maintenance of the heart field.

Distinct expression patterns for two Xenopus Bar homeobox genes.

The 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 Drosophila Bar 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.

Xbap, a vertebrate gene related to bagpipe, is expressed in developing craniofacial structures and in anterior gut muscle.

The Drosophila bagpipe (bap) gene is involved in the specification of the musculature of the embryonic midgut. We report the isolation and characterization of a Xenopus sequence, Xbap, which is closely related to bap. Xbap is also expressed in the developing musculature of the midgut, suggesting that this developmental role of bagpipe is evolutionarily conserved. However, a second, novel role in development is suggested by the observation that Xbap is also expressed in a region of the developing facial cartilage. Using a combination of cartilage staining and comparison to the goosecoid head expression pattern, we show that Xbap expression marks the precursors to the basihyobranchial, palatoquadrate, and possibly Meckel's cartilages. This vertebrate bagpipe sequence therefore is expressed in both mesodermally and neural crest-derived tissues.

Neovascularization of the Xenopus embryo.

The receptor tyrosine kinase, Flk-1 or VEGFR-2, and its ligand, vascular endothelial growth factor (VEGF) are required for the development of the embryonic vasculature. Targeted disruption of either gene in mice results in the failure of vascular system formation. The Xenopus homologues of flk-1 and VEGF have been cloned and their expression has been examined throughout early embryonic development. These studies indicate that flk-1 is expressed in groups of endothelial precursor cells which will form the major blood vessels of the embryo, including the posterior cardinal veins, the dorsal aorta, the vitelline veins, and the endocardium. VEGF expression is found in tissues adjacent to the mesenchyme containing the flk-1-expressing endothelial precursors. Expression of both flk-1 and VEGF is transient, appearing as the primary vascular plexus is forming and declining steadily after the onset of functional embryonic circulation. After establishment of the primary vascular structures, flk-1 expression is also observed in the intersegmental veins which form by an angiogenic mechanism. Overall, these results support a role for VEGF/flk-1 signaling in both vasculogenesis and angiogenesis in the Xenopus embryo. When VEGF is expressed ectopically in Xenopus embryos by microinjection of either plasmid DNA or synthetic mRNA, large, disorganized vascular structures are produced. This result indicates that ectopic VEGF is capable of altering the architecture of the developing vascular network.

Homeobox genes in cardiovascular development.

As summarized earlier, a surprisingly large number of different homeobox genes are expressed in the developing heart. Some are clearly important, as demonstrated by mouse gene ablation studies. For example, knockout of Nkx2-5 or Hoxa-3 function is embryonic lethal due to defects in cardiovascular development. However, gene ablation studies indicate that other homeobox genes that show cardiovascular expression are either not required for heart development or their function is effectively complemented by a redundant gene activity. Given the number of closely related homeobox genes that are expressed in the heart (and the rate at which new genes are being discovered), this is very likely to be the case for at least some homeobox gene activities. At present little is known of the precise mechanism of action of homeobox genes in embryonic development. This statement applies to homeobox genes in general, not just to genes involved in cardiovascular development. There is a popular view that homeobox genes are master regulators that control expression of a large number of downstream genes. In at least some cases, e.g., the eyeless gene of Drosophila (Holder et al., 1995), homeobox genes appear to be capable of activating and maintaining a very complex developmental program. Significantly, the eyeless gene is able to initiate eye development at numerous ectopic locations. Increasing evidence, however, suggests that genes of this type may be rather rare. Certainly there is no evidence to date that any of the homeobox genes expressed in the heart are able to initiate the complete heart development pathway. This is probably best understood in the case of the tinman gene in Drosophila, which, although absolutely required for heart development, is not capable of initiating the cardiac development pathway in ectopic locations (Bodmer, 1993). This conclusion is supported by studies of the vertebrate tinman-related gene Nkx2-5. Gene ablation studies show that Nkx2-5 is essential for correct cardiac development (Lyons et al., 1995) but is not able to initiate the regulatory pathway leading to cardiac development when expressed ectopically (Cleaver et al., 1996; Chen and Fishman, 1996). If most homeodomain proteins are not direct regulators of a differentiation pathway, what is their role during organogenesis? The cardiovascular homeobox gene about which most is known at the mechanistic level is gax (Smith et al., 1997). A number of experiments indicate that the Gax protein is involved in the regulation of cell proliferation and that it interacts with components of the cell cycle regulation machinery. Indeed, over recent years, the idea that at least some homeobox genes play their role in organogenesis through regulation of proliferation has been developed in some detail by Duboule (1995). Further evidence that this mechanism of homeobox activity is important, especially during organogenesis, comes from studies of the Hox11 homeobox gene, which is absolutely required for development of the spleen in mouse (Roberts et al., 1994). Studies indicate that Hox11 is able to interact with at least two different protein phosphatases, PP2A and PP1, which in turn, are involved in cell cycle regulation (Kawabe et al., 1997). It is quite clear that research in future years will need to focus on the precise mode of action of the different homeodomain proteins if we are to understand their role in the development of the cardiovascular system.