A new Adamts9 conditional mouse allele identifies its non‐redundant role in interdigital web regression

Abstract

ADAMTS9 is the most conserved member of a large family of secreted metalloproteases having diverse functions. Adamts9 null mice die before gastrulation, precluding investigations of its roles later in embryogenesis, in adult mice or disease models. We therefore generated a floxed Adamts9 allele to bypass embryonic lethality. In this mutant, unidirectional loxP sites flank exons 5 through 8, which encode the catalytic domain, including the protease active site. Mice homozygous for the floxed allele were viable, lacked an overt phenotype, and were fertile. Conversely, mice homozygous for a germ-line deletion produced from the floxed allele by Cre-lox recombination did not survive past gastrulation. Hemizygosity of the deleted Adamts9 in combination with mutant Adamts20 led to cleft palate and severe white spotting as previously described. Previously, Adamts9 haploinsufficiency combined with either Adamts20 or Adamts5 nullizygosity suggested a cooperative role in interdigital web regression, but the outcome of deletion of Adamts9 alone remained unknown. Here, Adamts9 was conditionally deleted in limb mesoderm using Prx1-Cre mice. Unlike other ADAMTS single knockouts, limb-specific Adamts9 deletion resulted in soft-tissue syndactyly (STS) with 100% penetrance and concurrent deletion of Adamts5 increased the severity of STS. Thus, Adamts9 has both non-redundant and cooperative roles in ensuring interdigital web regression. This new allele will be useful for investigating other biological functions of ADAMTS9.

The evolutionary conservation of the A Disintegrin-like and Metalloproteinase domain with Thrombospondin-1 motif metzincins across vertebrate species and their expression in teleost zebrafish

“…During limb development, reduced interdigital tissue resorption leading to soft tissue syndactyly (webbed feet) occurred in combinatorial ADAMTS5, ADAMTS9 and ADAMTS20 knockout mice and mesodermal Prx-1 Cre conditionally deleted ADAMTS9 (which is non-redundant in this developmental context) [28,29]. In this current study, adamts1 , adamts5 and adamts15 were abundant in adult zebrafish skeletal muscle and we previously reported a role for ADAMTS5 and ADAMTS15 during myogenesis and muscle regeneration [30].…”

ADAMTS9-Mediated Extracellular Matrix Dynamics Regulates Umbilical Cord Vascular Smooth Muscle Differentiation and Rotation

“…S3A). Like Adamts9 LacZ/+ ; Adamts20 Bt/Bt or Adamts9 Del/+ ; Adamts20 Bt/Bt mice, these mice died at birth with cleft palate (Dubail et al, 2014; Enomoto, 2010), and lacked skin melanoblasts (Silver, 2008) (Fig. S3A-F).…”

“…Adamts9 LacZ/+ (Kern et al, 2010), and the Adamts20 mutant Adamts20 bt-Bei1 (referred to as Adamts20 Bt ) (Rao et al, 2003; Silver, 2008) were described previously. Germline deletion of Adamts9 fl (RRID:JAX_026103) provided the Adamts9 Del allele (Dubail et al, 2014). Adamts fl mice were crossed extensively with ROSA mT/mG reporter mice (Muzumdar et al, 2007) to achieve linkage of the two transgenes on chr 6.…”

“…ADAMTS9 is highly conserved; its C.elegans ortholog, Gon-1 is required for gonadal morphogenesis (Blelloch et al, 1999) and the D. melanogaster ortholog AdamTS-A is essential for salivary gland morphogenesis and germ cell migration (Ismat et al, 2013). Adamts9 null mice die at the onset of gastrulation (Enomoto, 2010), but haploinsufficient and conditionally targeted mice identified later developmental roles (Dubail et al, 2014; Enomoto, 2010; Kern et al, 2010). Here, an Adamts9 gene-trap mutant, where ADAMTS9 is restricted to the cell surface, revealed distinct pools of ADAMTS9 activity, cell-proximal and distal, and requirement of the distal pool for umbilical cord vascular development in mice.…”

Genetic and biochemical evidence that gastrulation defects in Pofut2 mutants result from defects in ADAMTS9 secretion

“…To generate the Pofut2 tm2.2bch knockout allele ( Pofut2-LoxP ), Pofut2-Floxed heterozygotes were mated to PGK-Cre-recombinase expressing mice obtained from Ozgene (C57BL/6 background) (Taibi et al, 2013) and were then maintained by backcrossing to C57BL/6J for 6 generations. The Adamts9 tm1.2Apte knockout ( Adamts9-del) and Floxed ( Adamts9-Floxed ) alleles were previously described (Dubail et al, 2014). Adamts9-del and Adamts9-Floxed were subsequently maintained by backcrossing to C57BL/6J .…”