Figure 1.
Strategy for the construction of Pdgfaex4COIN and Pdgfaex4COIN-INV-lacZ alleles.
(a) Outline of the COIN module introduced as an artificial intron in the middle of Pdgfa exon 4. Abbreviations: TM, transmembrane; pA, polyA; SA, splice acceptor. (b) Pdgfaex4COIN allele with the lacZ cassette in anti-sense orientation. The expected splicing that rejoins exon 4a and exon 4b in the Pdgfaex4COIN transcript is indicated. (c) The Pdgfaex4COIN-INV-lacZ allele following Cre-mediated inversion of the lacZ cassette. Splicing from exon 4a now enters into the lacZ cassette and transcription terminates at its polyA site.
Figure 2.
Processing of a PDGF-A-lacZ fusion reporter protein in Pdgfaex4COIN-INV-lacZ expressing cells.
(a) Schematic outline of the Pdgfa exons and their contribution to the normal PDGF-A protein precursor. (b) Predicted transcription, translation and processing of the PDGF-A-lacZ fusion protein. The normal PDGF-A pre-pro and pro-peptide processing sites remain in the fusion protein. After exon1-encoded signal sequence removal, the exons 2-3-encoded pro-peptide is cleaved. The lacZ transmembrane domain anchors the exon 4a-lacZ fusion protein to the plasma membrane with the lacZ catalytic domain facing the cytoplasm. i.c, intracellular; e.c extracellular; UTR, untranslated region; SS, signal sequence; ret, extracellular matrix-binding retention motif.
Table 1.
Genotype distribution from heterozygous crossings of Pdgfaex4COIN-INV-lacZ.
Figure 3.
Comparison of Pdgfa, lacZ and Elastin mRNA expression in selected organs of Pdgfaex4COIN-INV-lacZ/+ mice.
Quantitative PCR analyzes of Pdgfa and lacZ in P5 (a) and adult (b) organs. The relative expression of the two genes follows the same pattern in different organs. Stomach was used as the reference sample (mean+SD) and its values set to 1.0. Wild-type lung mRNA was used as a negative control for lacZ. (c) Expression of Elastin in P5 organs shows a different expression pattern compared to Pdgfa and lacZ, as expected. Fold change on y-axis, Error bars show the standard deviation.
Figure 4.
Embryonic expression of Pdgfaex4COIN-INV-lacZ at E9.5.
(a) X-gal staining of an E9.5 Pdgfaex4COIN-INV-lacZ embryo (right) and a wildtype littermate control (left). (b) X-gal staining is concentrated to 1st branchial arch, the otic vesicles, somites and tail. (c, d) Longitudinal sections of paraffin embedded embryo. (c) Pdgfaex4COIN-INV-lacZ expression in somites. (d) Expression in epithelia of e.g. otic vesicle and 1st branchial arch. b.a., 1st branchial arch; o.v., otic vesicle; s, somites; t, tail. Scale bar 50 µm.
Figure 5.
Embryonic expression of Pdgfaex4COIN-INV-lacZ in E14.5 whole embryo/organs.
Whole mount X-gal staining of an E14.5 (a) Pdgfaex4COIN-INV-lacZ/+ embryo, arrow points at developing hindlimb muscles, arrowheads points at eye brow, whisker hair follicles, back skin hair follicles. (b) Pdgfa+/+ littermate. Note the absence of any X-gal staining. (c–g) Overnight X-gal staining of individual intact organs dissected from E14.5 Pdgfaex4COIN-INV-lacZ/+ (with blue staining) and Pdgfa+/+ (no staining) embryos. (c) jejunum, (d) lung, (e) heart (atria partially detached from the Pdgfa+/+ heart) (f) kidneys flanking the aorta and branches, (g) diaphragm, (h) liver.
Figure 6.
Embryonic expression of Pdgfaex4COIN-INV-lacZ in E17.5 whole organs.
X-gal staining of whole organs from E17.5 Pdgfaex4COIN-INV-lacZ/+ embryos (to the right or below) and wildtype littermate controls (to the left or on top). (a) aorta, (b) brown adipose tissue – note expression in associated vasculature, (c) esophagus, (d) thymus, (e) lung and trachea (arrowhead), (f) heart, (g) diaphragm, (h) stomach, (i) liver, (j), spleen, pancreas and mesenteric vessels (arrow head), (k) intestine, mesenteric vessels (arrow head), (l) adrenal gland and kidney, (m) skin, (n) brain.
Figure 7.
Postnatal expression of Pdgfaex4COIN-INV-lacZ in P5 whole organs.
X-gal staining of whole organs from P5 Pdgfaex4COIN-INV-lacZ/+ pups (to the right or below) and wildtype littermate controls (left or on top). (a) aorta, (b) brown adipose tissue – note expression in associated vasculature, (c) esophagus, (d) thymus, (e) lung, trachea (arrow head), (f) heart, (g) diaphragm, (h) stomach, (i) liver, (j) spleen, (k) pancreas, (l) colon, (m) kidney, (n) skin, (o) brain, (p) urinary bladder, (q) retina, (r) ribs.
Table 2.
Summary of previously published data on Pdgfa expression compared with expression patterns revealed by X-gal staining of Pdgfaex4COIN-INV-lacZ mice.
Figure 8.
Embryonic expression of Pdgfaex4COIN-INV-lacZ in E14.5 tissue sections.
Paraffin sections of developing organs at E14.5, counterstained with nuclear fast red. (a) Close-up of the head region from the whole mount stained Pdgfaex4COIN-INV-lacZ/+ embryo in Fig. 3a. Note expression in hair follicles and in surface ectoderm. Strong staining occurs in the ectoderm covering developing eyelids and outer ear. (b–l) Sections from different regions/organs of a Pdgfaex4COIN-INV-lacZ/+ embryo. (b) Eye and surrounding tissue; expression is seen in cornea (arrowhead), eyelid ectoderm, eyebrow follicles and lacrimal gland. (c) Expression in whisker hair follicles, epidermis, and developing facial muscles. (d) Expression in the tail and genital tubercle ectoderm (m, skeletal muscle; vb, cartilage primordium of vertebral body; drg, dorsal root ganglia). (e) Expression in the inner ear (ep, epithelium of otic vesicles; mes, mesenchyme). (f) Expression in bronchial epithelium of lung. (g) Expression in testis seminipherous ducts and mesonephric duct in epididymis (epi.). (h) Expression in developing renal epithelium and surrounding structures (med, medulla; ao. aorta). (i) Developing jejunum. Expression in pseudostratified epithelium (ep.) and developing muscular layers (m, arrow heads; mes., mesenchyme). (j) In the stomach, expression is mainly seen in the epithelium and developing muscle layers (arrowheads). (k) Expression in the heart, particularly in the outflow tract (oft). Arrowheads in k and h point at aortic VSMC. (l) Sagittal section of the developing brain showing expression in the dorsal horn of the lateral ventricle, and in amygdaloid and hippocampal epithelium. Scale bars 50 µm.
Table 3.
PDGFA/Pdgfa expression based on publicly available expressed sequence tag (EST) data.
Figure 9.
Expression of Pdgfaex4COIN-INV-lacZ in adult whole organs.
Whole mount X-gal staining of postnatal whole organs or organ slices. Organs were cut or sliced before staining. (c–i) Tissues from Pdgfaex4COIN-INV-lacZ/+ and Pdgfa+/+ littermates. Pdgfa+/+ negative controls appear in (c–i) and (k) to the right or below of the Pdgfaex4COIN-INV-lacZ/+ sample. (a) Coronal slice of a P12 cerebrum shows specific staining in neuronal layers. (b) Sagittal section of a P12 cerebellum. Arrowheads indicate Purkinje cell layer. (c) Sagittal section of a P15 brain. Inserted picture shows the same brain together with negative control. (d) P15 heart. (e) P15 uterus. Note staining of uterine blood vessel VSMC. (f) P60 retina. (g) P60 adrenal glands. (h) Slice of P60 liver lobe. (i) Dorsal view of P60 spinal cord. (j) P60 Pdgfaex4COIN-INV-lacZ/+ kidney slice, note the strong staining in the medullary papilla. (k) P60 Pdgfa+/+ kidney slice, note background staining in the cortex.
Figure 10.
Cell type-specific expression of Pdgfaex4COIN-INV-lacZ in adult organs.
Expression of Pdgfaex4COIN-INV-lacZ in organs and organ sections from a 4-month-old male. (a) Expression in hyaline cartilage and respiratory epithelium around the trachea. (b) Expression in lung, arrowhead points at bronchi, note the strong epithelial staining. (c) Lung alveolar region, arrowhead indicate positive cell with the morphological appearance of a type II pneumocyte. (d) Stomach. Note staining in the mucosa and in the underlying muscular layer. (e) Stomach epithelium, staining at the base of the gastric pits. (f) Colon. Note staining in surface enterocytes. (g) Hair follicle epithelium. (h) Kidney medulla with strong expression in the renal papilla. (i) Expression in Henley’s loop cells in the renal papilla. (j) Expression in tubular epithelium and the juxtaglomerular apparatus in the kidney cortex. (k) Close view of expression in the juxtaglomerular apparatus (white asterisk). (l) Dissected pituitary, whole mount stained, note expression in Rathke’s cleft cysts (RCC). (m) Ciliated epithelium of RCC. (n, o) Cerebellum. Expression restricted to Purkinje cells. (p) Expression in forebrain neurons. (q) Expression in diaphragm muscle fibers. (r) Expression in mesenteric arteries. (s) Expression in mesenteric arterial VSMC. (t) Expression in aortic VSMC. Scalebars 50 µm (except figures b, d, n where scale bars are 25 µm).
Figure 11.
Expression of Pdgfaex4COIN-INV-lacZ in neuromuscular junctions.
(a) Whole mount X-gal staining of musculus quadriceps femoris from P20 Pdgfaex4COIN-INV-lacZ/+ and Pdgfa+/+ littermates. (b) Higher magnification shows a band staining coinciding with the location of neuromuscular junctions. (c) Paraffin section of X-gal stained P60 Pdgfaex4COIN-INV-lacZ/+ muscle, counterstained with hematoxylin-eosin. (d) Paraffin section of X-gal stained P60 muscle, weakly counterstained with nuclear fast red. (e) Confocal imaging of free-floating sections of P60 muscle, stained with X-gal (seen as black shadows) and alpha-bungarotoxin (red). (f) Confocal z-stack of X-gal stained P60 muscle with superimposed alpha-bungarotoxin staining (green). Scale bar 50 µm in (e) and 25 µm in (f).
Figure 12.
Cell-type specific expression in P5 Pdgfaex4COIN-INV-lacZ mice.
(a–c) Immunofluorescence labeling of Surfactant protein-C (SPC) and beta-galactosidase in lung. Note co-expression in SPC positive type II pneumocytes, whereas bronchial epithelium is beta-galactosidase positive only (arrowheads in a and b). (d–h) Co-expression of alpha-smooth muscle actin (ASMA) and beta-galactosidase in vascular smooth muscle cells of blood vessels in brown adipose tissue. (d–f) Immunofluorescent stainings of (d) ASMA and (e) beta-galactosidase. (g) X-gal staining visualized with transmitted light overlap with ASMA and b-gal (h). (i–l) X-gal staining (blue) in megakaryocytes in liver, counterstained with hematoxylin/eosin. Scale bar 25 µm.