External appearance, palatal, and tracheoesophageal malformations at 15.5 dpc. (a,a′) External appearances of control littermate and mutant (Vcc/Vcc) embryos, respectively. The mutant embryo is smaller, has a prominent exomphalos (Ex) and markedly shortened hindlimbs (HL), and lacks a tail. (b) MRI sagittal section through a control embryo showing a normal palate (P). The tongue (T) and clivus (C) are indicated. (b′) Corresponding section through a littermate mutant Vcc/Vcc embryo showing complete absence of the palate. (c) MRI transverse section through a control embryo showing the normal thymus (Th), esophagus (O), trachea (Tr), and superior caval vein (SCV). (c′) Corresponding section through a littermate mutant Vcc/Vcc embryo showing that the two thymus lobes are smaller and separated, and that the trachea and esophagus are a single unseparated structure (Tr-O). (d) Transverse histological section through a wild-type embryo showing the bronchi (Br) originating from the trachea. The esophagus (O) and aorta (Ao) are indicated. (d′) Corresponding section through a littermate mutant Vcc/Vcc embryo showing the bronchi (Br) originating from the unseparated tracheoesophagus (Tr-O). Axes are anterior (A), posterior (P), right (R), left (L), dorsal (D), and ventral (V). Bar, 0.5 mm.

Cardiac and pulmonary malformations at 15.5 dpc. (a) MRI transverse section through a control embryo (at 15.5 dpc) showing a normal heart. The right and left atria and ventricles (RA, LA, RV, LV), mitral valve (MV), primary atrial septum (PAS), and interventricular septum (IVS) are indicated. (a′) Corresponding section of a mutant littermate Vcc/Vcc embryo showing an ostium primum atrial septal defect (ASD) and common atrioventricular valve (CAVV). (b) Three-dimensional reconstruction of a control heart, left anterior oblique view. The aorta (Ao) arises from the left ventricle (LV), and the main pulmonary artery (PA) rises from the right ventricle (RV). The aortic arch passes to the left of the trachea, over the left main bronchus. (b′) Corresponding reconstruction of a mutant littermate Vcc/Vcc heart showing a ventricular septal defect (VSD); a common arterial trunk (CAT) arising from the right ventricle (RV), giving rise to both cranial and pulmonary arteries; and an aortic vascular ring (AoVR). The trachea is not separated from the esophagus (Tr-O). (c,c′) Three-dimensional reconstruction of lungs from wild-type (wt) and littermate mutant (Vcc/Vcc) embryos. The cranial (Cr), middle (mi), caudal (Ca), and accessory (Ac) lobes of the right lung and the left lung (LL) are indicated. (d) Lung volumes (mean ± SEM, corrected for embryo volume) are shown for control left and right lungs (Con-LL, Con-RL), and littermate mutant Vcc/Vcc left and right lungs (Mut-LL, Mut-RL). The mutant left lung is significantly smaller than the control (t-test, P = 0.003). Axes are anterior (A), posterior (P), right (R), left (L), dorsal (D), and ventral (V). Bars, 0.5 mm.

Visceral malformations. (a) MRI transverse sections through the abdomen of a wild-type embryo (wt) at 15.5 dpc, showing the normal kidneys (LK, left kidney), ureter (LU, left ureter) and adrenals (LAd, left adrenal). (a′) Corresponding sections through a mutant littermate Vcc/Vcc embryo. The kidneys and ureter could not be identified. The pancreas (P) is indicated. (b) MRI sagittal sections through a 15.5-dpc control embryo showing the normal urinary bladder (UB), urethra (U), liver (Li), and vertebral column (VC). The tail (T) is indicated. (b′) Corresponding section through a mutant littermate Vcc/Vcc embryo. A presacral mass (PM) is seen between the vertebral column and the bladder. The liver herniates through the umbilicus forming an exomphalos (Ex). (c) Three-dimensional reconstruction of a control embryo showing the spinal cord (SC), gut (G), urinary bladder (UB), urethra (U), and rectum (R). (c′) Corresponding reconstruction of a mutant Vcc/Vcc embryo showing that the presacral mass (PM) is connected to the spinal cord, and its relation to the bladder and gut. The rectum could not be defined. (d) Sagittal histological section of a 13.5-dpc wild-type embryo showing the vertebral column (VC) and the spinal cord (SC) entering the tail (T). (d′) Corresponding section of a littermate mutant Vcc/Vcc embryo showing the vertebral column and spinal cord entering the presacral mass (PM). Axes are anterior (A), posterior (P), right (R), left (L), dorsal (D), and ventral (V). Bars, 0.5 mm.

Skeletal malformations at 15.5 dpc. (a) Wild-type (wt) embryo showing normal tail (T), hindlimbs (HL), and forelimbs (FL). Mutant littermate embryo (Vcc/Vcc) showing absence of tail, and severely hypoplastic hindlimbs. The forelimbs are relatively spared. (b, top) Left hindlimb from the wild-type embryo (left), and the mutant embryo (right). (Bottom) The ilium (I), femur (F), tibia (Ti), and fibula (Fi) are indicated. Wild-type (left) and mutant (right) forelimb. Ulna (U), radius (R), humerus (H), and scapula (S) are indicated. (c) Axial skeletons from wild-type and littermate mutant Vcc/Vcc embryos, respectively. Vertebrae are numbered by craniocaudal position (left), and by identity (right). The wild-type embryo has seven cervical (C1–C7), 13 thoracic (T1–T13, identified by the presence of ribs), six lumbar (L1–L6), and four sacral (S1–S4) vertebrae. The first caudal vertebra (Ca1) is also indicated. There are 26 presacral vertebrae. The mutant embryo has 18 thoracic vertebrae, and lacks sacral and caudal vertebrae, and has a total of 31 presacral vertebrae. (d) Rib cage from the wild-type embryo (top), and the mutant embryo (bottom). In the wild-type embryo, the clavicle (C) and xiphisternum (X) are indicated, as are the true ribs (T1–T7, joining the sternum directly). The mutant embryo lacks the xiphisternum, has 11 true ribs on the right, and nine true ribs on the left. Axes are anterior (A), posterior (P), right (R), eft (L), dorsal (D), and ventral (V). Bars, 0.5 mm.

Functional analysis of PCSK5A and PCSK5A-C470R. (a) Western blot of Cos-1 cell extracts and conditioned medium following transfection with C-terminally Flag-tagged PCSK5A (915-amino-acid isoform, wild type) and the corresponding C470R mutant. The blot was probed with anti-Flag monoclonal antibody. (b, top panel) Autoradiogram of anti-PCSK5 immunoprecipitates from HEK293 cells transfected with the indicated plasmids and radiolabeled with [³⁵S]Met + Cys. (Bottom panel) Autoradiogram of anti-PCSK5 immunoprecipitates from the conditioned media of the transfected cells. (*) PCSK5A; (**) PCSK5A cleavage product that deletes the C terminus (De Bie et al. 1996). (c,c′,d,d′) Immunofluorescence confocal microscopy of Cos-1 cells transfected with Flag-tagged PCSK5A and PCSK5A-C470R. Cells were stained with DAPI (blue) to localize nuclei, anti-Flag monoclonal antibody (green), anti-TGN46 polyclonal antibody (red, in c,c′) to localize the trans-Golgi network, and anti-calnexin polyclonal antibody (red, in d,d′) to localize the endoplasmic reticulum. The merged images are shown, in which colocalizing signals appear yellow. (e) Western blot of Cos-1 cell conditioned medium following cotransfection with a mouse BMP2-mycBMP4 hybrid plasmid and either PCSK5A (wild type, 0.8 or 0.4 μg, lanes 5,6, respectively) or the corresponding C470R mutant (0.8 or 0.4 μg, lanes 3,4, respectively). BMP2-mycBMP4 plasmid expresses a protein containing the mouse BMP2 prodomain fused to the BMP4 mature peptide in which the BMP4 cleavage site (TRRRAKR↓SP) is followed by an internal myc tag. The blot was probed with anti-myc monoclonal antibody. (f) Western blot of Cos-1 cell conditioned medium following cotransfection with LEFTY1 and either PCSK5A (wild type, lanes 3) or the corresponding C470R mutant (lane 4). The blot was probed with anti-LEFTY1 antibody.

Phenotypic effects of Gdf11 deletion and its activation by PCSK5. (A,B) MRI sagittal section and 3D reconstruction of a 15.5-dpc control wild-type (wt) embryo showing a normal urinary bladder (UB), urethra (U), gut loops (G), and vertebral column (VC). The tail (T) and rectum (R) are indicated. (a′,b′) Corresponding section and 3D reconstruction of a mutant littermate Gdf11^−/− embryo. A presacral mass (PM) arising from the spinal cord is seen entering the bladder. The rectum could not be defined. (c,c′) MRI transverse sections of wild-type and mutant littermate Gdf11^−/− embryo, respectively, at 15.5 dpc. The liver (L) and gut loops (G) are present in the exomphalos (Ex) of the Gdf11^−/− embryo. (d) Luciferase reporter assay for GDF11 activation. Flag-tagged PCSK5A-Flag wild-type and C470R mutant-expressing plasmids were cotransfected in 293T cells together with plasmids expressing GDF11, and FOXH1 expression plasmids to monitor induction of the reporter construct AR3-lux. Results are presented in relative luciferase units (mean ± SEM of three independent transfections). Values for GDF11 alone, reflecting baseline activity, were set at 1. In this assay, PCSK5A does not activate luciferase in the absence of GDF11 (data not shown). (e) Chromatographic profiles of a 12-mer mouse GDF11-derived peptide (NTKRSRRNLGLD) containing a consensus proprotein convertase cleavage site, incubated overnight with conditioned media from vector control, PCSK5A wild-type, and PCSK5A-C470R transfected HEK293 cells. Axes are anterior (A), posterior (P), right (R), left (L), dorsal (D), and ventral (V). Bars, 0.5 mm.

Gene expression analysis using in situ hybridization. Wild-type (wt) control and littermate mutant Pcsk5^Vcc/Vcc (Vcc/Vcc) embryos, respectively, are shown at the time points indicated. (a,a′) Pcsk5 is expressed in the caudal-most somites (S), and the hindlimb and forelimb buds (in the apical ectodermal ridge) in both wild-type and mutant embryos. (b,b′) Gdf11 is expressed strongly in the tailbud (TB), hindlimb bud (HLB), and forelimb bud (FLB) in both wild type and mutant. (c,c′) Hoxa10 is expressed in the otic vesicle (OV), forelimb bud (FLB), hindlimb bud (HLB), tailbud region (TB), and caudal end of the wild-type embryo, but is reduced in the mutant. (d,d′) Hoxa11 is expressed strongly in otic vesicle (OV), hindlimb bud (HLB), and forelimb bud (FLB) of the wild type, but is reduced in forelimb bud (FLB), and absent in hindlimb bud (HLB) of the mutant. (e,e′) Hoxa11os is expressed in otic vesicle (OV), forelimb bud (FLB), and tailbud (TB) of the wild-type embryo, but is reduced in the mutant. (f,f′) Hoxc10 is expressed strongly at the caudal end of the wild-type embryo, and the tailbud region (TB), but only weakly in the mutant embryo. (g,g′) Hoxd10 is expressed in otic vesicle (OV), forelimb bud (FLB), hindlimb bud (HLB), tailbud (TB), and caudal end of the wild-type embryo, but is reduced in the mutant. (h,h′) Hoxd11 is expressed in otic vesicle (OV), forelimb bud (FLB), hindlimb bud (HLB), tailbud (TB), and caudal end of the wild-type embryo, but is reduced in the mutant. (i,i′) Hoxd12 is expressed in otic vesicle (OV), forelimb bud (FLB), hindlimb bud (HLB), tailbud (TB), and caudal end of the wild-type embryo, but is reduced in the mutant. (j,j′) Mnx1 (Hlxb9, Hb9) in wild-type embryos is expressed in the motor neurons of the spinal cord (MN) and in the dorsal aspect of tailbud (arrowhead). In the mutant embryo, Mnx1 is expressed on the ventral aspect of the tailbud, extending apparently into the gut (G) (arrowhead). (k,k′) T is expressed in the tailbud (TB) of both wild-type and mutant embryos. (l,l′) Tbx6 is expressed in the tailbud (TB) and caudal end of both wild-type and mutant embryos. Bars, 0.5 mm.