ABSTRACT During zebrafish fin regeneration, blastema cells lining the epidermis differentiate into osteoblasts and joint cells to reconstruct the segmented bony rays. We show that osteoblasts and joint cells originate from a common cell lineage, but are committed to different cell fates. Pre-osteoblasts expressing runx2a/b commit to the osteoblast lineage upon expressing sp7 , whereas the strong upregulation of hoxa13a correlates with a commitment to a joint cell type. In the distal regenerate, hoxa13a , evx1 and pthlha are sequentially upregulated at regular intervals to define the newly identified presumptive joint cells. Presumptive joint cells mature into joint-forming cells, a distinct cell cluster that maintains the expression of these factors. Analysis of evx1 null mutants reveals that evx1 is acting upstream of pthlha and downstream of or in parallel with hoxa13a . Calcineurin activity, potentially through the inhibition of retinoic acid signaling, regulates evx1 , pthlha and hoxa13a expression during joint formation. Furthermore, retinoic acid treatment induces osteoblast differentiation in mature joint cells, leading to ectopic bone deposition in joint regions. Overall, our data reveal a novel regulatory pathway essential for joint formation in the regenerating fin.

A conserved endothelin 1 signaling pathway patterns the jaw and other pharyngeal skeletal elements in mice, chicks and zebrafish. In zebrafish, endothelin 1 ( edn1 or sucker ) is required for formation of ventral cartilages and joints in the anterior pharyngeal arches of young larvae. Here we present genetic analyses in the zebrafish of two edn1 downstream targets, the bHLH transcription factor Hand2 and the homeobox transcription factor Bapx1, that mediate dorsoventral (DV) patterning in the anterior pharyngeal arches. First we show that edn1 -expressing cells in the first (mandibular)and second (hyoid) pharyngeal arch primordia are located most ventrally and surrounded by hand2 -expressing cells. Next we show that along the DV axis of the early first arch primordia, bapx1 is expressed in an intermediate domain, which later marks the jaw joint, and this expression requires edn1 function. bapx1 function is required for formation of the jaw joint, the joint-associated retroarticular process of Meckel's cartilage, and the retroarticular bone. Jaw joint expression of chd and gdf5 also requires bapx1 function. Similar to edn1, hand2 is required for ventral pharyngeal cartilage formation. However, the early ventral arch edn1 -dependent expression of five genes ( dlx3, EphA3, gsc, msxe and msxb )are all present in hand2 mutants. Further, msxe and msxb are upregulated in hand2 mutant ventral arches. Slightly later, an edn1 -dependent ventral first arch expression domain of gsc is absent in hand2 mutants, providing a common downstream target of edn1 and hand2 . In hand2 mutants, bapx1 expression is present at the joint region, and expanded ventrally. In addition, expression of eng2 , normally restricted to first arch dorsal mesoderm, expands ventrally in hand2 and edn1 mutants. Thus, ventral pharyngeal specification involves repression of dorsal and intermediate (joint region) fates. Together our results reveal two critical edn1 effectors that pattern the vertebrate jaw: hand2 specifies ventral pharyngeal cartilage of the lower jaw and bapx1 specifies the jaw joint.