Regulation of TGF-β signalling has become a major focus in understanding the complex mechanisms of vertebrate and invertebrate development. This thesis characterises and investigates the function of a novel gene, termed Crim1, a possible modulator of TGF-β signalling in development. The Crim1 gene has been conserved throughout vertebrate development and a putative orthologue exists in the nematode worm, C.elegans. Crim1 encodes a large cysteine rich protein with a signal peptide and a transmembrane domain. The Crim1 protein also contains multiple cysteine rich repeat motifs found in several TGF-β regulatory proteins including the secreted BMP antagonists Chordin and Drosophila Short gastrulation.
By transient transfection assays, Crim1 protein was localised on the plasma membrane and trafficking pathway. In addition Crim1 was also cleaved and secreted into the extracellular matrix as an ectodomain. This secreted 'ectodomain' was not able to bind to mature BMP4 or BMP7 in the extracellular matrix. However, Crim1 was able to bind to the precursor of BMP4 and BMP? when co-expressed. This interaction was sufficient to tether BMP? to the cell surface suggesting Crim1 may act to mediate BMP processing, secretion or extracellular diffusion.
In mouse and chicken, Crim1 was strongly expressed in multiple tissues during post-gastrulation development, including the neural tube, somites and lens of the eye. In the developing spinal cord, Crim1 was expressed in the floor plate and motor neurons consistent with a role in patterning the dorsal-ventral axis. Overexpression of Crim1 by electroporation into the neural tube did not result in changes in the dorsal-ventral restriction of progenitor or post-mitotic cell markers. This suggests that Crim1 does not regulate early BMP and Shh-dependant dorsal-ventral patterning of the neural tube. However, overexpression of a constitutively secreted ectodomain was able to reduce the numbers of specific ventral postmitotic neuronal cell types. This established a putative role for Crim1 in later patterning of the neural tube, possibly mediated through ventrally expressed BMPs.
To determine if Crim1 could inhibit or activate BMP signalling in vivo, the zebrafish overexpression assay was employed. Crim1 was able to dorsalise zebrafish embryos consistent with inhibition of BMP signalling. The normal function of zebrafish Crim1 in development was then investigated. Zebrafish Crim1 was expressed ubiquitously during pregastrulation development. At somitogenesis stages, Crim1 expression was initially restricted to the caudal notochord, but later found in the eye, ear and urogenital system. Morpholino knockdown of zebrafish Crim1 resulted in a proportion of abnormal post-gastrulation stage embryos. This is consistent with weak Chordino mutants, suggesting a role for Crim1 in BMP signalling during gastrulation. Embryos that were normal during gastrulation stages generally had caudal defects affecting the somites, circulatory system and the shape of the tail. These defects suggest a role for Crim1 in mediating signals from the caudal notochord at segmentation stages.
To determine the function of Crim1 in mouse, a 'gene trapped' allele of Crim1 (Crim1KST264) was analysed. In a preliminary analysis, mice homozygous for Crim1KST264 displayed multiple and variant phenotypes during mid-late gestation affecting the eye, kidney, head and limbs. Analysis of the hindlimb of these mice revealed fully penetrant soft-tissue syndactyly of digits 3 and 4. These defects are consistent with expression of Crim1 and possible roles in mediation of BMP signalling.
In summary, Crim1 is an evolutionary conserved gene that is part of a family of BMP regulators. Crim1 can interact with members of the TGF-β superfamily and may act to mediate their action in vivo. Finally, Crim1 is expressed at multiple tissues in vertebrate embryos and is required for normal development.