Hapln1a Is Required for Connexin43-Dependent Growth and Patterning in the Regenerating Fin Skeleton

<div><p>Cell–cell communication, facilitating the exchange of small metabolites, ions and second messengers, takes place via aqueous proteinaceous channels called gap junctions. Connexins (cx) are the subunits of a gap junction channel. Mutations in zebrafish <i>cx43</i> produces the <i>short fin</i> (<i>sof ^b123</i>) phenotype and is characterized by short fins due to reduced segment length of the bony fin rays and reduced cell proliferation. Previously established results from our lab demonstrate that Cx43 plays a dual role regulating both cell proliferation (growth) and joint formation (patterning) during the process of skeletal morphogenesis. In this study, we show that Hapln1a (<u>H</u>y<u>a</u>luronan and <u>P</u>roteoglycan <u>L</u>i<u>n</u>k Protein 1a) functions downstream of <i>cx43</i>. Hapln1a belongs to the family of link proteins that play an important role in stabilizing the ECM by linking the aggregates of hyaluronan and proteoglycans. We validated that <i>hapln1a</i> is expressed downstream of <i>cx43</i> by in situ hybridization and quantitative RT-PCR methods. Moreover, in situ hybridization at different time points revealed that <i>hapln1a</i> expression peaks at 3 days post amputation. Expression of <i>hapln1a</i> is located in the medial mesenchyme and the in the lateral skeletal precursor cells. Furthermore, morpholino mediated knock-down of <i>hapln1a</i> resulted in reduced fin regenerate length, reduced bony segment length and reduced cell proliferation, recapitulating all the phenotypes of <i>cx43</i> knock-down. Moreover, Hyaluronic Acid (HA) levels are dramatically reduced in <i>hapln1a</i> knock-down fins, attesting the importance of Hapln1a in stabilizing the ECM. Attempts to place <i>hapln1a</i> in our previously defined <i>cx43</i>–<i>sema3d</i> pathway suggest that <i>hapln1a</i> functions in a parallel genetic pathway. Collectively, our data suggest that Cx43 mediates independent Sema3d and Hapln1a pathways in order to coordinate skeletal growth and patterning.</p>