Genetic analysis of SMOC1 and SMOC2 in eye and limb development

Abstract

Secreted modular calcium-binding proteins (SMOCs) are extracellular glycoproteins of the secreted protein, acidic, and rich in cysteine-related modular calcium-binding protein (SPARC) family and include two paralogues in humans: SMOC1 and SMOC2. They have been characterised and studied in several vertebrates showing a crucial function in eye and limb development. To date, no study of SMOC genes has been undertaken in chicken, despite its utility as a highly tractable model organism.

Loss-of-function mutations in SMOC1 cause severe eye and post-axial limb defects in vertebrates. Similarly, there is evidence for an essential requirement for SMOC2 in normal craniofacial development in multiple species, including canines. Currently, the exact function of SMOC family genes and their interactions with other genes during vertebrate development remain largely unknown. This study aimed to use the chicken embryo as a new model for elucidating SMOC gene expression and, by focusing on their role during eye and limb development, determine how SMOC proteins influence embryonic development, RT-PCR and whole mount in situ hybridisation confirmed the presence of SMOC gene expression in chicken embryos during key stages of both eye and limb development. Comparing their expression patterns with mouse orthologues, similar expression patterns were observed in chick embryos. These data suggest that SMOCs perform similar developmental roles across divergent vertebrates. Further analysis with qRT-PCR confirmed high expression of SMOC1 in the developing eye and limbs at early stages (HHSt.20-25), whilst SMOC2 was mostly expressed in the limbs at later stages (HH.St.27-33).

The optic fissure in the ventral eye must fuse for normal eye development to occur. Previously published data suggested SMOC1 expression was enriched in the ventrally developing eye. RNA-seq data from whole eyes and optic fissure regions of humans, mice, chick and zebrafish were compared to assess SMOC gene expression before, during, and after fissure closure. Focusing first on chick data produced by the Rainger lab, I observed that SMOC1 expression was enriched in the fissure tissue. However, contrary to the hypothesis of SMOC1 being a fusion-specific gene during optic fissure closure, I observed increasing SMOC1 expression during fusion that that was also maintained at high levels afterwards. By performing cross-species transcriptome comparison, I confirmed that SMOC1 expression is also upregulated in the ventral eye in mouse, zebrafish and humans. This evolutionarily conserved gene expression underscores SMOC1’s importance in eye development and reduces its candidacy as a direct mediator of fusion.

SMOC2 did not show up-regulation in eye tissues but was enriched during later stages of development and in non-fissure regions of the eye. This expression profile appeared to complement that of SMOC1 in the eye. In chicken, human and mouse, SMOC2 showed up-regulation at pre-fusion stages in the fissure, vi while it appeared not differentially expressed once the tissue was fused. Expression levels of SMOC2 were higher in the dorsal eye than in the fissure. For zebra fish, however, smoc2 appeared up-regulated in pre-fusion stages, while no significant changes were observed once the fissure has completed fusion.

Finally, the SMOC1 locus in chicken was extensively studied from the perspective of isoform expression. Three distinct SMOC1 isoforms were found and one of them implied the loss of a complete functional domain of the SMOC1 protein. The relevance of these isoforms in eye and limb development was tested by measuring their expression levels by qRT-PCR in both embryo and adult tissues.

Taken together, these findings confirm the utility of the chicken embryo for revealing developmental genetic information at the single gene and whole transcriptome levels and confirm that SMOC1 is likely to have the same functional requirement in chickens as it has in other vertebrates. This work also provides a framework to follow these genetic studies up at the protein function level, including understanding the roles of SMOC proteins and individual isoforms, as well as their molecular interactions and importance for normal development and disease.