**9. Aggrecan–GAG Interactions Are of Importance in Heart Development**

During early embryonic development, ectodermal NC cells migrate to form the neural tube and notochord under the direction of HNK-1 substituted aggrecan. This HNK-1 substituted form localises to the peri-notochordal space where its repulsive cell interactive properties guide the NC cells to form the notochord in a co-ordinated pattern-dependant manner (Figure 9). Equally impressive is the direction of assembly of tissue structures through HNK-1 aggrecan with NC cells migrating outwards along specific guidance tracts to form the cardiac neural crest region and the cardiac septae, outflow tracts and aortic arches [171] (Figure 9). Development of the heart valves and cardiac muscle with electroconductive properties from the endocardial cushions is also regulated by distinct spatiotemporal distributions of aggrecan and versican [16,172]. Heart tissues have remarkable mechanical properties of elasticity, compressibility, stiffness, strength and durability achieved through careful guidance of cell-mediated ECM assembly of collagen fibril and versican- and aggrecan-rich tissue regions to provide these tissues with highly specialised functional properties. Cardiac tissues are electroconductive and the charge transfer properties of GAG side chains of heart PGs may contribute to tissue properties in a similar manner to how electrosensory properties are conveyed to neural tissues. Similar developmental processes are also evident in the formation of cartilage, tendon and bone using the same ECM components but in a different manner to effect specialised tissue function [11]. The development of co-ordinated electroconductive cardiomyocyte networks with synchronised pulsatile properties is a particularly impressive achievement [173,174]. The properties of the heart valves and heart strings are equally important to heart function and these have material properties more similar to cartilage and tendon. It is not surprising therefore that aggrecan and transcription factors such as Sox 2, Sox 9 and growth factors/morphogens such as FGFs/BMPs play such prominent roles in the development of cardiac tissues [11].

**Figure 8.** Demonstration of: Sox 10 (**a**,**e**); Seraf (**b**); and HNK-1 epitope (**c**,**d**) expression in migratory

neural crest cells (**a**,**c**) and in whole mount chick embryos (**b**,**d**,**e**). (a,c,d) In-situ hybridisation images. (**b**,**e**) Immunolocalisations with specific antibodies. Seraf (Schwann cell-specific EGF-like repeat autocrine factor) is a unique protein expressed by avian embryo Schwann cell precursor cells [175]. Images reproduced from [176] under the auspices of attribution-non-commercial-no derivatives 4.0 international licence (CC BY-NC-ND 4.0).

**Figure 9.** Fluorescent immunolocalisation of HNK-1 and aggrecan S103L epitope in 2–5-day-old chick trunk sections associated with the neural tube (NT) and notochord (N) development (**a**,**b**) and in Western blots (**c**) of purified chick notochordal and cartilage aggrecan. Keratan sulphate was also immunolocalised on blots using MAb 5-D-4. Notochordal aggrecan was S103L and HNK-1 positive but did not contain KS and was of a smaller molecular weight; the 400-kDa cartilage aggrecan species was not detected. Cartilage aggrecan did not stain with the HNK-1 antibody. The S103L antibody identifies the sequence <sup>585</sup>XXX Glu Ileu Ser Gly Phe Leu Ser Gly Asp Arg<sup>615</sup> in the CS attachment domain of aggrecan. Images reproduced from [177–179].

The calcineurin/nuclear factor of activated T cells (NFatc1), which regulates osteoclast differentiation [180], is also required for valve formation [181,182]. Myocyte-specific enhancer factor 2C (Mef2c), a master transcription factor which regulates hypertrophy and osteogenic differentiation of chondrocytes [183], is also essential for normal cardiovascular development, and loss of function mutations in Mef2c contribute to congenital heart defects [184]. Moreover, activated BMP signalling has been shown to increase expression of cartilage and bone-type collagens, and increased expression of the osteogenic marker Runt-related transcription factor 2 (Runx2)/core-binding factor subunit alpha-1 (CBF α-1) is observed in adult aortic valve disease [185]. Thus, there is considerable overlap in cartilage synonymous transcriptional factors in the integrated development of functional heart tissues. The cell directive properties of HNK-1 aggrecan not only makes a particularly important contribution to the sculpting of embryonic cardiac tissues but it is also a functional component of these tissues.
