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DVDY_21982_sm_SupFigS1.tif402KSupplemental Figure 1. The imputed MfngΔ1 sequence does not produce stable protein. To address the possibility that a functional fringe protein might be produced from the MfngΔ1 mRNA we examined protein production in cell lines. We utilized results from the 5'RACE analysis to construct Mfng expression vectors that mimic the MfngΔ1 allele. The first construct contains the longest 5' sequence we identified in our RACE procedures, linked to exons 2-8 of MFNG. While this construct most closely reflects the deduced MfngΔ1 mRNA (MfngΔ1long), this mRNA has a long 5'UTR with multiple stop codons upstream of the methionine, which might interfere with translation initiation. Therefore, we also constructed an Mfng cDNA that begins at methionine 122 to assess whether any stable protein could be produced if M122 is used as the initiator methionine (MfngΔ1short). NIH3T3 cells were co-transfected with AP-fringe plasmid and pSVβgal (Promega) using Lipofectamine 2000 (Invitrogen). After 24 hours, media was collected and cells were lysed with Passive Lysis Buffer (Promega). 50μl of the cellular extracts or 50μl of heat inactivated culture were mixed with 50μl of AP Assay Reagent A (GenHunter) to determine AP activity following manufacturers instructions, and was normalized to β-gal activity levels as a control for transfection efficiency. Steady-state AP activity was measured in the cell extracts and media of cells transfected with vectors encoding AP-tagged fringe proteins. MFNG is observed robustly both in intracellular and extracellular fractions, but neither vectors encoding MFNGΔ1short or MFNGΔ1long drive measurable levels of protein. supporting our contention that the MfngΔ1 allele will not produce a stable, active protein. Results are shown as relative AP activity units normalized to βgal used as a control for transfection efficiency. Values are the mean ± SD of three independent experiments.
DVDY_21982_sm_SupFigS2.tif7029KSupplemental Figure 2. Rfng is widely expressed during mouse embryogenesis. A,B) βgal staining at early embryonic stages in RfnglacZ/lacZ embryos. At 7.5 d.p.c., staining is observed throughout embryonic and extraembryonic tissues. C,D) At 8.5 d.p.c., Rfng is expressed in the midbrain, hindbrain, optic vesicle, and branchial arch, similar to results observed with in situ hybridization (Fig. 1C,D and Johnston et al., 1997). In addition, we observe βgal expression in the spinal cord and epithelial somites. Views are lateral (B,D), dorsal (C) and headfolds (A). E) At 9.5 d.p.c., Rfng is expressed throughout the spinal cord, branchial arches, at the midbrain/hindbrain boundary (large arrowhead), somites, limb buds, and the trigeminal and facial nerves (*). F) At 10.5 d.p.c. Rfng expression is observed in the forebrain, midbrain, midbrain/hindbrain boundary (arrowhead), branchial arch, nasal pits, and the trigeminal, facial, glossopharyngeal, and vagus nerves (*), as well as in the heart, dorsal root ganglia, spinal cord, spinal nerves and limb buds. G) At 12.5 d.p.c. Rfng expression is similar to that observed at 10.5 d.p.c. Expression is also observed in the mammary gland (arrows). H-J) Expression in transverse sections of the neural tube demonstrates Rfng expression in the ventricular zone and notochord (arrowhead) at 9.5 d.p.c. (H). At 10.5 d.p.c. (I), expression is found in the notochord (arrowhead) and floorplate, along with weak expression in the ventricular zone and strong expression in the mantle zone, dorsal root ganglia and spinal neurons. At 12.5 d.p.c. (J) expression is observed in the floor plate and mantle layer. Dynamic expression is observed in the ventral neural tube, apparently correlating with the differentiation of motor neurons and/or ventral interneurons. K) Rfng expression is also observed in the embryonic ectoderm at 11.5 d.p.c. after prolonged staining. L, M) At 13.5 d.p.c. Rfng is expressed in the lens of the eye (L) and weakly in developing vibrissae (arrowhead), limb ectoderm, AER, and mammary glands (arrow) (M). N) In saggital sections of 13.5. d.p.c. embryos Rfng expression is expressed in the ribs and remaining cartilagenous skeleton. O, P) At 17.5 d.p.c. Rfng expression is observed in the vibrissae in the snout (O), and developing hair follicles in the rump (P). Q) Expression in hair follicles is maintained in neonatal skin.
DVDY_21982_sm_SupFigS3.tif2796KSupplemental Figure 3. Rfng is robustly expressed in the adult mouse brain. Expression is observed in the hippocampus (hi), thalamus (t), cortex (cx), basal ganglia (bg), globus pallidus (gp), striatum (stm), anterior olfactory nucleus (oa), superior colliculus (sc), inferior colliculus (ic), dentate gryrus (dg), olfactory bulb (ob), pons (p), and the internal granule layer (il) and Purkinje cell layer (pc) of the cerebellum. All images are βgal staining of 200 um sections of adult brains. In the hippocampus, Rfng expression in the CA1 region and dentate gyrus are more intense than expression in the CA3 region (F). In the diencephalon, Rfng expression is observed in the thalamus and hypothalamus, and expression is detected in the superior and inferior colliculus, the anterior olfactory nucleus, the olfactory bulb and the pons (G). In the cerebellum, Rfng expression is limited to the purkinje and inner granule cell layers (H). A-D) Coronal sections of adult brains. (A) is more posterior than (C), (B) is a magnified view of the cerebral cortex in (A), while (D) is a magnified view of the basal ganglia in (C). G-H) Saggital sections of adult brain. (E) is more lateral than (G). (F) is a magnified view of the hippocampus in (E), and (H) is a magnified view of the posterior cerebellum in (G).

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