1. Volume 23, Issue 4, Pages 725-736 (August 1999)
GFRα3, a Component of the Artemin Receptor, Is Required for Migration and Survival of the Superior Cervical Ganglion 
Jinsuke Nishino, Kyoko Mochida, Yasuhisa Ohfuji, Takuya Shimazaki, Chikara Meno, Sachiko Ohishi, Yoichi Matsuda, Hideta Fujii, Yukio Saijoh, Hiroshi Hamada 
Neuron 
Volume 23, Issue 4, Pages (August 1999)
DOI: /S (01)

2. Figure 1 Generation of Gfrα3 Knockout Mice
(A) Targeting strategy. The structure of the Gfrα3 gene is shown at the top (K indicates KpnI). Homologous recombination resulted in replacement of exons 4–8 of Gfrα3 (closed boxes) by the neomycin resistance gene (neo).
(B) Southern blot analysis of ES cell lines. Genomic DNA was digested with KpnI and subjected to hybridization with the 3′ probe shown in (A). Arrowheads indicate the 25 kb (wild-type) and 10.5 kb (mutant) fragments. The cell line indicated by the closed circle is one of the mutant lines.
(C) Genotype analysis of offspring obtained from intercrossing of Gfrα3+/− heterozygotes. Analysis was performed by PCR with primers shown in (A): 5′ PCR and 3′ PCR1 for the wild-type allele, and 5′ PCR and 3′ PCR2 for the mutant allele. The indicated 780 and 460 bp fragments represent the wild-type and mutant alleles, respectively.
(D and E) In situ hybridization analysis of Gfrα3 expression in wild-type (D) and mutant (E) embryos at embryonic day 14.5 (E14.5). Whereas Gfrα3 mRNA was abundant in PNS ganglia, including the sympathetic chain (SC), DRG, and trigeminal ganglion (TG), of wild-type embryos, it was not detected in mutant embryos. Scale bar, 1 mm.
Neuron  , DOI: ( /S (01) )

3. Figure 2 Defects in the SCG of Adult Gfrα3−/− Mice
(A and B) External appearance of wild-type (A) and Gfrα3−/− (B) adult mice. The Gfrα3−/− mice exhibited ptosis.
(C and D) Whole-mount views of the SCG of wild-type (C) and Gfrα3−/− (D) mice. The SCG of the wild-type mouse is indicated by red arrowheads (C). The SCG was missing (D) or severely reduced in size in the mutant animals. Scale bar, 420 μm.
(E and F) Transverse sections of the SCG dissected from wild-type (E) and Gfrα3−/− (F) mice. The marked reduction in size of the SCG is apparent in the mutant animal. Scale bar, 150 μm.
(G–J) Submandibular glands from wild-type (G and I) and mutant (H and J) mice stained with an antibody against TH (G and H) or an antibody against ChAT (I and J). TH-positive fibers are apparent in the wild-type mice (G) but absent in the mutant (H). ChAT-positive fibers are present both in wild-type (I) and in mutant (J) mice. Scale bar, 70 μm.
(K–N) The superior tarsus muscle from wild-type (K and M) and mutant (L and N) mice stained with an antibody against TH (K and L) or an antibody against ChAT (M and N). TH-positive fibers are apparent in the wild-type mice (K) but absent in the mutant (L). ChAT-positive fibers are present both in wild-type (M) and mutant (N) mice. Scale bar, 50 μm.
(O and P) In situ hybridization anlaysis of Gfrα2 (O) and Gfrα3 (P) transcripts in the parasympathetic submandibular ganglion from wild-type mice on P1. Marked expression of Gfrα2, but not of Gfrα3, was apparent. Scale bar, 75 μm.
(Q and R) Nissl staining of the submandibular ganglion from wild-type (Q) and Gfrα3−/− (R) adult mice. The submandibular ganglion was normal in the mutant animal. Scale bar, 70 μm.
Neuron  , DOI: ( /S (01) )

4. Figure 3
Decrease in the Number of SCG Cells in Gfrα3−/− Mice During Postnatal Development
(A) The number of cells in the SCG of wild-type and Gfrα3−/− mice was counted at various developmental stages. Data are from three animals of each strain.
(B–I) Nissl staining of SCG (B–E) and stellate ganglion (STG) (F–I) neurons from wild-type (B, D, F, and H) and Gfrα3−/− (C, E, G, and I) mice at P1. Higher magnification views of (B), (C), (F), and (G) are shown in (D), (E), (H), and (I), respectively. Many morphologically abnormal neurons were apparent in the SCG, but not in the stellate ganglion, of mutant mice. Arrowheads in (E) indicate pyknotic nuclei.
(J and K) Ret expression in the P1 SCG from wild-type (J) and Gfrα3−/− (K) mice. Ret expression is markedly reduced in the mutant SCG (K).
(L and M) Sections of the P5 SCG from wild-type (L) and Gfrα3−/− (M) mice were processed for the TUNEL detection method of apoptotic nuclei. Increased apoptosis is apparent in the mutant SCG (M).
(N–Q) The innervation of the submandibular gland (N and O) and the superior tarsus muscle (P and Q) were examined at P1 with an anti-TH antibody. Wild-type mice are shown in (N) and (P); Gfrα3−/− mice are shown in (O) and (Q). TH-positive fibers are present in the control (N and P) but absent in the mutant (O and Q).
Scale bar, 100 μm for (B), (C), (F), (G), (J), and (K); 20 μm for (D), (E), (H), (I), (L), and (M); 90 μm for (N) and (O); and 60 μm for (P) and (Q).
Neuron  , DOI: ( /S (01) )

5. Figure 4
Expression of Gfrα1, Gfrα2, Gfrα3, and Ret in Developing and Mature SCG
The SCG from wild-type mice at the indicated ages was sectioned and examined by in situ hybridization for the expression of the indicated genes. Transient drop of Ret expression at E14.5 (H) and of Gfrα2 expression at E18.5 (O) was confirmed with four embryos. Scale bar, 50 μm for (A) through (P) and 100 μm for (Q) through (T).
Neuron  , DOI: ( /S (01) )

6. Figure 5
Defect in the Rostral Migration of SCG Precursors in Gfrα3−/− Embryos
(A–C) Transverse sections prepared from wild-type and Gfrα3−/− embryos at E11.5 (A), E12.5 (B), and E14.5 (C) were analyzed for the number of cells in sympathetic ganglia. Cell number was then plotted along the anteroposterior axis. Three embryos were analyzed for each stage. The positions of the cervical vertebrae (C1–C8) served as indicators of the level of sections along the anteroposterior axis. STG, stellate ganglion.
(D–I) Representative sections from wild-type (D, F, and H) and Gfrα3−/− (E, G, and I) E14.5 embryos that were stained with hematoxylin and eosin. Planes for transverse sections are indicated by arrowheads in (C). SCG and STG precursors are indicated by closed and open arrowheads, respectively. In the E14.5 wild-type embryo, the future SCG is apparent in (D) but not in (F). However, in the Gfrα3−/− mutant, SCG precursors are apparent in (G) but not in (E), indicating their caudal shift. The position of the stellate ganglion is not affected in the mutant mice (H and I). Abbreviations; ao, aorta; cca, common carotid artery; ica, internal carotid artery; ng, nodose ganglion; nX, vagus nerve; svc, superior vena cava; and thy, thyroid primordium. Scale bar, 100 μm for (D) through (I).
Neuron  , DOI: ( /S (01) )

7. Figure 6
Expression of Gfrα3, artemin, and Ret in or near Migrating SCG Precursors
(A–F) Sagittal sections from wild-type (A–C) and Gfrα3−/− (D–F) embryos at E11.5 (A and D), E12.5 (B and E), or E14.5 (C and F) were subjected to in situ hybridization. The developing SCG were detected with a Ret probe (A and D), a mixture of TH and dopamine β-hydroxylase (DBH) probes (B and E), or a mixture of TH and Ret probes (C and F). At E11.5, the expression pattern of Ret was similar in the wild-type and mutant embryos. The caudal shift of the mutant SCG is apparent at E12.5 and E14.5 (E and F).
(G–K) Expression of Gfrα3 (G and H) and artemin (I–K) in wild-type embryos was examined by whole-mount in situ hybridization at E11.5 (G and I) and E12.5 (H and J). (K) shows a transverse section of the embryos in (J). The region corresponding to the SCG is marked in (K). At E11.5, both SCG and stellate ganglion precursors form a uniform column and express Gfrα3 (G). At E12.5, SCG and stellate ganglion precursors are separated and continue to express Gfrα3 (H). artemin is not expressed at E11.5 (I) but is expressed within SCG and in a region surrounding the SCG precursors at E12.5 (J and K). artemin expression at E12.5 was confirmed with three embryos.
Scale bar, 200 μm for (A), (D), (G), and (I); 300 μm for (B), (E), (H), and (J); 500 μm for (C) and (F); and 75 μm for (K).
Neuron  , DOI: ( /S (01) )

8. Figure 7 Characterization of Other PNS Ganglia in Gfrα3−/− Mice
(A–F) DRG from wild-type (A), (C), and (E) and Gfrα3−/− (B), (D), and (F) adult mice were stained with antibodies to CGRP (A and B), to neurofilaments (NFH) (C and D), or to Ret (E and F).
(G–J) The enteric nervous system from wild-type (G and I) and Gfrα3−/− (H and J) adult mice was stained with antibodies to neuron-specific enolase (NSE) (G and H) or to β-tubulin (βT) (I and J).
(K–P) Trigeminal ganglia from wild-type (K, M, and O) and Gfrα3−/− (L, N, and P) mice were examined at P7 for expression of Gfrα1 (K and L), Gfrα2 (M and N), and Ret (O and P) by in situ hybridization.
Scale bar, 50 μm for (A) through (F); 30 μm for (G) through (J); and 100 μm for (K) through (P).
(Q) Total cell numbers and various subpopulations of DRG and TG are summarized. There is no significant difference between wild-type and Gfrα3−/− mice.
Neuron  , DOI: ( /S (01) )