Henk W.J. Hakvoort

Lysyl oxidase-like 3b is Critical for Cartilage Maturation During Zebrafish Craniofacial Development

Vertebrate craniofacial development requires coordinated morphogenetic interactions between the extracellular matrix (ECM) and the differentiating chondrocytes essential for cartilage formation. Recent studies reveal a critical role for specific lysyl oxidases in ECM integrity required for embryonic development. We now demonstrate that loxl3b is abundantly expressed within the head mesenchyme of the zebrafish and is critically important for maturation of neural crest derived cartilage elements. Histological and ultrastructural analyses of cartilage elements in loxl3b morphant embryos reveal abnormal maturation of cartilage and altered chondrocyte morphology. Spatiotemporal analysis of craniofacial markers in loxl3b morphant embryos shows that cranial neural crest cells migrate normally into the developing pharyngeal arches but that differentiation and condensation markers are aberrantly expressed. We further show that the loxl3b morphant phenotype is not due to P53 mediated cell death but likely to be due to reduced chondrogenic progenitor cell proliferation within the pharyngeal arches. Taken together, these data demonstrate a novel role for loxl3b in the maturation of craniofacial cartilage and can provide new insight into the specific genetic factors important in the pathogenesis of craniofacial birth defects.

Expression of HMA4 cDNAs of the zinc hyperaccumulator Noccaea caerulescens from endogenous NcHMA4 promoters does not complement the zinc-deficiency phenotype of the Arabidopsis thaliana hma2hma4 double mutant

Noccaea caerulescens (Nc) exhibits a very high constitutive expression of the heavy metal transporting ATPase, HMA4, as compared to the non-hyperaccumulator Arabidopsis thaliana (At), due to copy number expansion and altered cis-regulation. We screened a BAC library for HMA4 and found that HMA4 is triplicated in the genome of a N. caerulescens accession from a former Zn mine near La Calamine (LC), Belgium. We amplified multiple HMA4 promoter sequences from three calamine N. caerulescens accessions, and expressed AtHMA4 and different NcHMA4 cDNAs under At and Nc HMA4 promoters in the A. thaliana (Col) hma2hma4 double mutant. Transgenic lines expressing HMA4 under the At promoter were always fully complemented for root-to-shoot Zn translocation and developed normally at a 2-μM Zn supply, whereas the lines expressing HMA4 under Nc promoters usually showed only slightly enhanced root to shoot Zn translocation rates in comparison with the double mutant, probably owing to ectopic expression in the roots, respectively. When expression of the Zn deficiency responsive marker gene ZIP4 was tested, the transgenic lines expressing AtHMA4 under an NcHMA4-1-LC promoter showed on average a 7-fold higher expression in the leaves, in comparison with the double hma2hma4 mutant, showing that this construct aggravated, rather than alleviated the severity of foliar Zn deficiency in the mutant, possible owing to expression in the leaf mesophyll.

Evolutionary Responses to Zinc and Copper Stress in Bladder Campion, Silene Vulgaris (Moench.) Garcke

Terrestrial metal-toxic soils are found all over the world. They originate either from geological anomalies (e.g., ore outcrops, serpentines), or from human activities (e.g., mining, smelting, agricultural use of metal containing fertilizers, pesticides, sludges, etc.). Even extremely metal-toxic soils eventually become vegetated, though often sparsely and without trees. These vegetations are usually poor in species, particularly in parts of the world that have been glaciated during the Pleistocene (Brooks et al., 1985), most probably mainly due to a strong selective effect of metal-toxicity as such (Schat and Verkleij, 1998).

Mechanisms of heavy metal resistance in Silene vulgaris

A few plant species, like Silene vulgaris, have evolved ecotypes, that are capable of growing on metal-enriched soils. These tolerant ecotypes exhibit metal-specific resistance mechanisms, which are not completely understood to date. Resistance to copper, zinc and cadmium seems to be regulated by different major genes and some hypostatic modifiers. Metallothionein (MT2b) expression is involved in the level of copper tolerance, while an enhanced ATP-dependent copper efflux across the root cell plasma membrane may result in a decreased copper-accumulation and contributes to the higher resistance of mine plants compared to the copper-sensitive plants. Given the lack of evidence for the significance of similar strategies for zinc and cadmium (such as reduction of uptake and increased binding to intracellular molecules) vacuolar compartmentation is generally believed to be the basic mechanism of tolerance of zinc and cadmium in higher plant species. An enhanced tonoplast transport of zinc in roots plays a role in zinc tolerance, but the existence of a similar tolerance mechanism for cadmium remains to be demonstrated. In our present research, the use of molecular biological techniques to study the presence and expression of (transporter) genes (like ZAT, ZNT and other families) has been initiated. Introduction