RBPjkappa-dependent Notch signaling regulates mesenchymal progenitor cell proliferation and differentiation during skeletal development

Abstract

The Notch pathway has recently been implicated in mesenchymal progenitor cell (MPC) differentiation from bone marrow-derived progenitors. However, whether Notch regulates MPC differentiation in an RBPjkappa-dependent manner, specifies a particular MPC cell fate, regulates MPC proliferation and differentiation during early skeletal development or controls specific Notch target genes to regulate these processes remains unclear. To determine the exact role and mode of action for the Notch pathway in MPCs during skeletal development, we analyzed tissue-specific loss-of-function (Prx1Cre; Rbpjk(f/f)), gain-of-function (Prx1Cre; Rosa-NICD(f/+)) and RBPjkappa-independent Notch gain-of-function (Prx1Cre; Rosa-NICD(f/+); Rbpjk(f/f)) mice for defects in MPC proliferation and differentiation. These data demonstrate for the first time that the RBPjkappa-dependent Notch signaling pathway is a crucial regulator of MPC proliferation and differentiation during skeletal development. Our study also implicates the Notch pathway as a general suppressor of MPC differentiation that does not bias lineage allocation. Finally, Hes1 was identified as an RBPjkappa-dependent Notch target gene important for MPC maintenance and the suppression of in vitro chondrogenesis.

Fig. 1.

Notch pathway component expression during MPC differentiation and chondrogenesis both in vitro and in vivo. (A-C) Real-time RT-PCR gene expression analyses of the Notch ligands (A), Notch receptors (B) and the Hes/Hey family of RBPjκ-dependent Notch target genes (C). (Da-j) In situ hybridization for the indicated Notch pathway components at E11.5 (Da-Dh) and for N2 (Di) and Hes1 (Dj) at E12.0. Insets show high magnification of N1- and Dll4-associated endothelial cells surrounding vascular canals on alternative sections. (E) Western blot for cleaved Notch2 protein (NICD2) isolated from limb-bud-derived MPCs (LB-MPCs) cultured in the presence and absence of DAPT or from whole limb-bud (WLB). The y-axis of graphs is the relative gene expression normalized to β-actin represented in arbitrary units. d, days; hr, hours.

Fig. 2.

DAPT-mediated Notch inhibition enhances limb-bud MPC differentiation without biasing lineage determination. (A) Alcian Blue/Orange G (AB/OG) staining of limb-bud MPC micromass cartilage nodules and real-time RT-PCR for Sox9, Col2a1 and Agc1 at 3, 5 and 7 days. (B) Alkaline Phosphatase staining of limb-bud MPC osteogenic cultures and real-time RT-PCR for Col1a1, AP and Oc at 21 days. (C) Oil Red-O staining of limb-bud MPC adipogenic cultures and real-time RT-PCR for Pparg at 21 days. The y-axis of graphs is the relative gene expression normalized to β-actin and to the control. *, P<0.05 versus control. d, days; hr, hours.

Fig. 3.

Loss of RBPjκ-dependent Notch signaling in vivo accelerates chondrogenesis during limb development. (Aa,b) AB/OG staining of wild-type (WT) and Prx1Cre; Rbpjk^f/f (RBPjκ) E12.5 limb-bud sections. (Ac-h) In situ hybridization for Sox9 (Ac,d), Col2a1 (Ae,f) and Agc1 (Ag,h). (B) Real-time RT-PCR analyses from limb-buds of WT and RBPjκ mutant E12.5 hindlimbs. The y-axis of graphs is the relative gene expression normalized to β-actin and to the WT control. *, P<0.05 versus control.

Fig. 4.

Sustained activation of Notch signaling suppresses MPC differentiation during skeletal development. (Aa-f) Alcian Blue/Alizarin Red staining of WT and Prx1Cre; Rosa-NICD^f/+ (NICD) mutant E18.5 skeletons (Aa,b), forelimbs (Ac,d) and hindlimbs (Ae,f). Black arrows indicate NICD mutant limbs, green arrow indicates the open sternum of the NICD mutant. (Ba-h) AB/OG staining of WT and NICD hindlimb sections at E12.5 (Ba,b). In situ hybridization for Sox9 (Bc,d), Col2a1 (Be,f) and Agc1 (Bg,h). (Bi,j) Gfp expression indicated NICD expression/activity in WT (Bi) and NICD (Bj) sections. (C) Real-time RT-PCR for Sox9, Col2a1, Agc1, Runx2, Hes1, Hey1 and HeyL from limb-buds. The y-axis of graphs is the relative gene expression normalized to β-actin and to the WT control. *, P<0.05 versus control. d, digits; fe, femur; fi, fibula; h, humerus; il, illium; pu, pubic; r, radius; s, scapula; t, tibia; u, ulna.

Fig. 5.

Sustained activation of Notch signaling in the limb mesenchyme does not significantly affect limb patterning or apoptosis, but increases MPC proliferation during limb development. (Aa-f) In situ hybridization for Fgf8 (Aa,b), Fgf10 (Ac,d), and Ptc1 (Ae,f) on WT (Aa,c,e) and Prx1Cre; Rosa-NICD^f/+ mutant (NICD; Ab,d,f) sections at E11.0. (B) TUNEL staining and statistical analyses of MPC apoptosis for WT and NICD sections at E11.0. (Ca-c) BrdU immunohistochemistry (Ca,b) and statistical analyses of MPC proliferation (Cc) for WT (Ca) and NICD (Cb) sections at E11.5. (Cd) Real-time RT-PCR for the proliferation marker, CyclinD1. *, P<0.05 versus control. AZ, apical zone. Red dashed boxes denote regions analyzed for MPC proliferation.

Fig. 6.

Notch signaling suppresses MPC differentiation in an RBPJκ-dependent manner. (Aa-d) Alcian Blue/Alizarin Red staining of WT and Prx1Cre; Rosa-NICD^f/+ (NICD), Prx1Cre; Rbpjk^f/f (RBPjκ) and Prx1Cre^f; Rosa-NICD^f/+; Rbpjk^f/f (NICD; RBPjκ) mutant E18.5 skeletons. Black arrows indicate NICD mutant limbs. Red arrows mark the length of tibiae. Asterisks identify parietal bones. (Ba-c) AB/OG staining of WT, NICD; RBPjκ^f/+, and NICD; RBPjκ^f/f littermate hindlimb sections at E12.5. (Bd-l) In situ hybridization for Sox9 (Bd-f), Col2a1 (Bg-i), and Agc1 (Bj-l). (Bm-o) Gfp expression assesses NICD activity in WT (Bm), NICD; RBPjκ^f/+ (Bn), and NICD; RBPjκ^f/f (Bo) sections.

Fig. 7.

Hes1 is an important RBPjκ-dependent Notch target gene that suppresses MPC differentiation and chondrogenesis. (Aa-f) Alcian Blue staining of control infected (Aa,c,e) and Hes1 shRNA-infected (shHes1; Ab,d,f) limb-bud MPC micromass cultures. (B) Real-time RT-PCR for Sox9, Col2a1, Agc1 and Hes1. The y-axis of graphs is the relative gene expression normalized to β-actin and to the control at day 3. *, P<0.05 versus control. d, days.

Fig. 8.

Model for Notch regulation of MPC and COP differentiation during cartilage development. Cartilage and bone development begins from the earliest common precursor, the MPC (yellow), which differentiates to become a more lineage-restricted COP (light blue) cell. Further differentiation allows COPs to adopt either an osteoblastic (Ob, green) fate or that of a maturing chondrocyte (MC, dark blue). Arrows indicate induction of proliferation or differentiation; perpendicular lines indicate suppression. Curved arrows indicate cell proliferation/self-renewal. A, RBPjκ-dependent/Hes1 regulation of MPC proliferation and differentiation (Figs 1, 2, 3, 4, 5, 6 and 7); B, Notch regulation of COP differentiation and chondrocyte proliferation (Mead and Yutzey, 2009).