Functional analyses of type IIA procollagen in embryo development

Abstract

Type II collagen is the major extracellular matrix (ECM) protein present in

cartilage and is detected in non-chondrogenic tissues such as the heart and the

neural tube during developmental stages involving rapid tissue morphogenesis

indicating an active role played by the collagen in embryogenesis. Type II

collagen is synthesized as a procollagen precursor which has amino- and

carboxyl-terminal globular extensions (N- and C-propeptides) flanking a central

triple helical domain. Two isoforms of type II procollagen are generated by

alternative mRNA splicing of the exon 2: IIA and IIB. Sequence present in the

N-propeptide of IIA, translated from the spliced-in exon 2, encodes a von

Willebrand factor-like C cysteine rich (CR) domain. This domain is homologous

to those present in regulators of the bone morphogenetic protein (BMP) signaling

such as chordin (Chd), twisted gastrulation (Tsg) and crossveinless (Cv).

Previous in vitro binding assays and overexpression studies in frog embryo

suggest that the CR domain of IIA antagonized BMP signaling. In order to give

a better understanding of the function of IIA in embryonic development and

cellular signaling, several approaches including expression pattern analyses,

phenotypic analyses of null mutant and gain of function studies are employed in

this study. Expression studies of IIA mRNA in early postimplantation mouse

embryos find that it is present in the axial mesendoderm (including the anterior

definitive endoderm [ADE] and the prechordal plate) which is a critical head

organizer at neural plate (E7.5) and head process (E8.0) stages. Characterization

of the IIA deficient mice (IIA-/-), constructed by removing exon 2 from type II

collagen (Col2a1) gene by homologous recombination, indeed reveals that the

anterior-most neural tissue is deficient at early somitogenesis denoted by

reduction/loss of the forebrain/optic cup markers. Marker studies indicate that

the ADE may already be affected at the neural plate stage in IIA-/-. The neural

phenotype of IIA-/- displays significant similarities with mutants deficient in BMP

pathway components such as Chd-/-;Nog+/-, Tsg-/- and Tsg-/-;BMP4+/- suggesting

that IIA plays a role in maintaining the specification and/or regulating the

signaling properties of the anterior midline tissue which involves regulation of

BMP signaling. Results of ectopic expression of IIA in Xenopus laevis embryos

suggest that IIA regulate BMP and the related Nodal signaling pathways in a

context dependent manner which has significant implications in normal anterior

neural plate development. Based on the work described in this thesis and the

body of existing evidence, a model is presented which suggests that IIA

promote/maintain anterior neural plate development by regulating the range and

extent of BMP signaling in the anterior neural plate. This study sheds light on

the role of an ECM component in regulating tissue patterning and cellular

signaling during early mouse development and also provides putative function for

the CR domain of other fibrillar procollagens including type I, III and V which is

poorly understood currently. This work will provide the framework for the

design of subsequent studies in re-examining the role of these fibrillar

procollagens in embryogenesis.