Jeremy B. A. Green

Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor

The crystal structure of the tandem SH2 domains of human ZAP-70 in complex with a peptide derived from the ζ-subunit of the T-cell receptor reveals an unanticipated interaction between the two domains. A coiled coil of α-helices connects the two SH2 domains, producing an interface that constitutes one of the two critical phosphotyrosine binding sites. These and other unique features provide the molecular basis for highly selective association of ZAP-70 with the T-cell receptor.

BMP and Wnt Specify Hematopoietic Fate by Activation of the Cdx-Hox Pathway

The formation of blood in the embryo is dependent on bone morphogenetic protein (BMP), but how BMP signaling intersects with other regulators of hematopoietic development is unclear. Using embryonic stem (ES) cells, we show that BMP4 first induces ventral-posterior (V-P) mesoderm and subsequently directs mesodermal cells toward blood fate by activating Wnt3a and upregulating Cdx and Hox genes. When BMP signaling is blocked during this latter phase, enforced expression of either Cdx1 or Cdx4 rescues hematopoietic development, thereby placing BMP4 signaling upstream of the Cdx-Hox pathway. Wnt signaling cooperates in BMP-induced hemogenesis, and the Wnt effector LEF1 mediates BMP4 activation of Cdx genes. Our data suggest that BMP signaling plays two distinct and sequential roles during blood formation, initially as an inducer of mesoderm, and later to specify blood via activation of Wnt signaling and the Cdx-Hox pathway.

Association of valproate-induced teratogenesis with histone deacetylase inhibition in vivo

Chemically induced birth defects are an important public health and human problem. Here we use Xenopus and zebrafish as models to investigate the mechanism of action of a well‐known teratogen, valproic acid (VPA). VPA is a drug used in treatment of epilepsy and bipolar disorder but causes spina bifida if taken during pregnancy. VPA has several biochemical activities, including inhibition of histone deacetylases (HDACs). To investigate the mechanism of action of VPA, we compared its effects in Xenopus and zebrafish embryos with those of known HDAC inhibitors and noninhibitory VPA analogs. We found that VPA and other HDAC inhibitors cause very similar and characteristic developmental defects whereas VPA analogs with poor inhibitory activity in vivo have little teratogenic effect. Unbiased microarray analysis revealed that the effects of VPA and trichostatin A (TSA), a structurally unrelated HDAC inhibitor, are strikingly concordant. The concordance is apparent both by en masse correlation of fold‐changes and by detailed similarity of dose‐response profiles of individual genes. Together, the results demonstrate that the teratogenic effects of VPA are very likely mediated specifically by inhibition of HDACs.

LKB1 (XEEK1) regulates Wnt signalling in vertebrate development

Germline LKB1/STK11 mutations are associated with the cancer-prone Peutz–Jeghers syndrome (PJS) in humans, and nullizygosity provokes a poorly understood constellation of developmental perturbations in the mid-gestational mouse. To gain a better understanding of the processes regulated by LKB1, we have exploited the experimental merits of the developing Xenopus embryo. Here, specific inhibition of XEEK1, the Xenopus orthologue of LKB1, engendered developmental anomalies — shortened body axis and defective dorsoanterior patterning — associated previously with aberrant Wnt signalling. In line with this, LKB1/XEEK1 cooperates with the Wnt–β-catenin signalling in axis induction and modulates the expression of Wnt-responsive genes in both Xenopus embryos and mammalian cells. We establish that LKB1/XEEK1 acts upstream of β-catenin in the Wnt–β-catenin pathway in vivo. LKB1/XEEK1 regulates glycogen synthase kinase (GSK)3β phosphorylation and it is physically associated in vivo with GSK3β and protein kinase C (PKC)-ζ, a known GSK3 kinase. These studies show that LKB1/XEEK1 is required for Wnt–β-catenin signalling in frogs and mammals and provides novel insights into its role in vertebrate developmental patterning and carcinogenesis.

Functional communication between endogenous BRCA1 and its partner, BARD1, during Xenopus laevis development

The breast and ovarian susceptibility protein 1 (BRCA1) heterodimerizes with its structural relative, the BRCA1-associated RING domain protein (BARD1), which may have tumor suppressing function in its own right. Both proteins have evolved from a common evolutionary ancestor, and both exist in Xenopus laevis where, similar to their mammalian homologs, they form functional heterodimers. Depleting frog embryos of either BARD1 or BRCA1 led to similar and widely defective developmental phenotypes as well as depletion of the other polypeptide due to its decreased stability. Thus, each protein, in part, controls the abundance, stability, and function of the other, and these effects are heterodimerization-dependent. The interdependent nature of BRCA1 and BARD1 function supports the view that BARD1/BRCA1 heterodimers play a major role in breast and ovarian cancer suppression.

Periodic stripe formation by a Turing mechanism operating at growth zones in the mammalian palate

We present direct evidence of an activator-inhibitor system in the generation of the regularly spaced transverse ridges of the palate. We show that new ridges, called rugae, that are marked by stripes of expression of Shh (encoding Sonic hedgehog), appear at two growth zones where the space between previously laid rugae increases. However, inter-rugal growth is not absolutely required: new stripes of Shh expression still appeared when growth was inhibited. Furthermore, when a ruga was excised, new Shh expression appeared not at the cut edge but as bifurcating stripes branching from the neighboring stripe of Shh expression, diagnostic of a Turing-type reaction-diffusion mechanism. Genetic and inhibitor experiments identified fibroblast growth factor (FGF) and Shh as components of an activator-inhibitor pair in this system. These findings demonstrate a reaction-diffusion mechanism that is likely to be widely relevant in vertebrate development.

Morphogen gradients, positional information, and Xenopus: interplay of theory and experiment

The idea of morphogen gradients has long been an important one in developmental biology. Studies with amphibians and with Xenopus in particular have made significant contributions to demonstrating the existence, identity, and mechanisms of action of morphogens. Mesoderm induction and patterning by activin, nodals, bone morphogenetic proteins, and fibroblast growth factors have been analyzed thoroughly and reveal recurrent and combinatorial roles for these protein growth factor morphogens and their antagonists. The dynamics of nodal‐type signaling and the intersection of VegT and β‐catenin intracellular gradients reveal detailed steps in early long‐range patterning. Interpretation of gradients requires sophisticated mechanisms for sharpening thresholds, and the activin‐Xbra‐Gsc system provides an example of this. The understanding of growth factor signal transduction has elucidated growth factor morphogen action and provided tools for dissecting their direct long‐range action and distribution. The physical mechanisms of morphogen gradient establishment are the focus of new interest at both the experimental and theoretical level. General themes and emerging trends in morphogen gradient studies are discussed.

Positional information and reaction-diffusion: two big ideas in developmental biology combine

One of the most fundamental questions in biology is that of biological pattern: how do the structures and shapes of organisms arise? Undoubtedly, the two most influential ideas in this area are those of Alan Turings ‘reaction-diffusion’ and Lewis Wolperts ‘positional information’. Much has been written about these two concepts but some confusion still remains, in particular about the relationship between them. Here, we address this relationship and propose a scheme of three distinct ways in which these two ideas work together to shape biological form. Summary: This Hypothesis discusses the relationship between positional information and reaction-diffusion, two fundamental principles governing the arisal of structures and shapes in organisms.

PAR1 specifies ciliated cells in vertebrate ectoderm downstream of aPKC

Partitioning-defective 1 (PAR1) and atypical protein kinase C (aPKC) are conserved serine/threonine protein kinases implicated in the establishment of cell polarity in many species from yeast to humans. Here we investigate the roles of these protein kinases in cell fate determination in Xenopus epidermis. Early asymmetric cell divisions at blastula and gastrula stages give rise to the superficial (apical) and the deep (basal) cell layers of epidermal ectoderm. These two layers consist of cells with different intrinsic developmental potential, including superficial epidermal cells and deep ciliated cells. Our gain- and loss-of-function studies demonstrate that aPKC inhibits ciliated cell differentiation in Xenopus ectoderm and promotes superficial cell fates. We find that the crucial molecular substrate for aPKC is PAR1, which is localized in a complementary domain in superficial ectoderm cells. We show that PAR1 acts downstream of aPKC and is sufficient to stimulate ciliated cell differentiation and inhibit superficial epidermal cell fates. Our results suggest that aPKC and PAR1 function sequentially in a conserved molecular pathway that links apical-basal cell polarity to Notch signaling and cell fate determination. The observed patterning mechanism may operate in a wide range of epithelial tissues in many species.

Differential effects on Xenopus development of interference with type IIA and type IIB activin receptors

One candidate for a mesoderm-inducing factor in early amphibian development is activin, a member of the TGF beta family. Overexpression of a truncated form of an activin receptor Type IIB abolishes activin responsiveness and mesoderm formation in vivo. The Xenopus Type IIA activin receptor XSTK9 differs from the Type IIB receptor by 43 and 25% in extracellular and intracellular domains respectively, suggesting the possibility of different functions in vivo. In this paper, we compare the Type IIA receptor with the Type IIB to test such a possibility. Simple overexpression of the wild-type receptors reveals minimal differences, but experiments with dominant negative mutants of each receptor show qualitatively distinct effects. We show that while truncated (kinase domain-deleted) Type IIB receptors cause axial defects as previously described, truncated type IIA receptors cause formation of secondary axes, similar to those seen by overexpression of truncated receptors for BMP-4, another TGF beta family member. Furthermore, in animal cap assays, truncated type IIB receptors inhibit induction of all mesodermal markers tested, while truncated type IIA receptors suppress induction only of ventral markers; the anterior/dorsal marker goosecoid is virtually unaffected. The suppression of ventral development by the type IIA truncated receptor suggests either that the truncated Type IIA receptor interferes with ventral BMP pathways, or that activin signaling through the Type IIA receptor is necessary for ventral patterning.