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Abstract
Our comprehension of the molecular mechanisms underlying embryogenesis has been greatly enhanced by the identification and characterization of associated extracellular matrix macromolecules. Using Xenopus laevis as a model, we investigated the expression and distribution of SPARC (Secreted Protein, Acidic, Rich in Cysteine; also called osteonectin and BM-40) during early embryonic development. SPARC has been found to be enriched in tissues undergoing rapid morphological development, differentiation, and remodeling. In Xenopus, SPARC transcripts are first expressed by primordial cells which give rise to the first embryonic tissues, the notochord and somites. SPARC RNA levels remained high throughout the rapid morphological development and differentiation phase of these tissues, and then rapidly decreased. Of particular interest, SPARC protein began to accumulate within the intersomitic clefts at the onset of trunkmyotome contraction. The intersomitic enrichment of SPARC remained high as long as the myotomes remained electrically coupled, principally by gap junctions. As myotomes became innervated, SPARC expression decreased dramatically within the somites. SPARC was also found to be enriched within other tissues, such as the neural tube and epidermis. In addition, the selective spatial-temporal enrichment of SPARC suggests it makes important calcium-dependent contributions to early morphological development.
Fig. 1. Whole-mount in situ localization of SPARCRNA during early Xenopus development. A 460 bp EcoRI fragment of the 5' region of Xenopus
SPARC was subcloned into pGEM-4Z, and digoxogenin-labeled transcripts were generated using the flanking viral promoters and cognate RNA
polymerases. (A) Shortly after neurulation (stage 18), SPARC RNA was expressed by the notochord (n), and in the segmented (s) and unsegmented (um)
somitic mesoderm (B) At stage 25, SPARC message was abundant in the notochord (n), the neural floor plate (fp), and in the somites (s). (C) By early
tailbud (stage 33). SPARC messages were no longer eVident In the notochord (n), but were abundant m the floor plate (fp) of the neural tube and subnotochordal
rod (sr) of the endoderm. (0) Towards the end of somitogenesis (stage 431 the presence of SPARC message m the dorsal axis was greatly
diminished. Expression was now up-regulated in the anterior region of the embryo where organogenesis was progressing (E) Closer examination of the
distribution of SPARC RNA within maturing somites revealed that SPARC messages at stage 25 were localized principally over the centrally located nuclei
within each myotome (sn). Very little SPARC RNA was found near the anterior and postenor poles of the myotome adjacent to the intersomitic cleft (i).
(F) As somltes matured (stage 33). SPARC RNA was no longer centrally localized above the nuclei (sn), but was now abundant at the anterior and posterior
poles of the somite, adjacent to the intersomitic cleft (i).As controls, Xenopus cyroskeletal actin cRNA (G) and SPARC sense cRNA probes (H) were used.
Fig. 2. Whole-mount immunolocalization of SPARC during early Xenopus embryogenesis. An anti-SPARC monocional anti-body(MAb-ON3), which
cross-reacted with Xenopus SPARC, was used to localize SPARC (A) During early somitogenesis (stage 18).SPARC was detected within the notochord
(n), the unsegmented dorsal mesoderm (urn), segmented somites (51,and eye anlage (ea), (8) By stage 25 the notochord tube (n), eyes (e) and somites
(s) all expressed high levels of SPARC. At higher magnification (E) SPARC was found within the samires. and was becoming detectable within the
intersomitic cleft (i). (C) By stage 33 SPARC levels decreased In the notochord (n), but remain elevated within the neural tube (nt). The protein exhibited
a striated chevron distribution along the somites. At higher magnification (F) this striation could be seen as a result of SPARC accumulation within the
intersomitic clefts (i). (D) The stage-45 embryo showed decreased levels of SPARC in the dorsal a1(;s, bur increased anterior accumulation. As a control.
a neural-specific antibody (G) was used.
Fig. 3. Mid-trunk cross-sectional analysis of SPARC RNA and protein
localization. To more closely examine the distribution of SPARC RNA and
protein within the embryo, mid-trunk cross section were performed. (A) At
stage 18 SPARC message was found to be abundant in the notochord (n),
flanking somites (s). floor plate (fp) of the neural tube (nt), and the
endodermal cells fated to become the sub-notochordal rod (sr). (B) By the
early tailbud stage (33), the highly vacuolated notochord (n) contaIned few
SPARC messages. The somltes (s), neural tube (nt), particularly its floor
plate (fp) and the sub-notochordal rod (sr) all expressed high levels of
SPARC message. SPARC RNA was also detected in the surface ectoderm
(se). To compare rhe dlstribution of SPARC protein to its cognate mRNA.
Immunohistochemistry was performed using ON3. (C) At stage33 SPARC
protein was no longer present in the notochord (n)and sub-notochordal rod
(sr). SPARC staining was still visible in the neural tube (nt), and the sensorial
laver of the surface ectoderm (se)
