Organotypic specificity of key RET adaptor-docking sites in the pathogenesis of neurocristopathies and renal malformations in mice (original) (raw)
Parasympathetic, but not enteric or sympathetic, ganglia require RET51 isoform for proper development. To investigate the relative contributions of the 2 major RET isoforms, RET9 and RET51, in PNS development, we studied mice that exclusively express 2 copies of WT human RET9 or RET51. These knockin mice, and each of the homozygous RET mutants studied, were generated previously using homologous recombination of human RET cDNAs into the endogenous Ret locus (knockout-knockin approach) (Figure 1 and Table 1) (29). All analyses performed in this study were on homozygous mice. For PNS analyses, we selected structures that are severely affected in _Ret_- or _GFL_-null mice, including the ENS and the cranial parasympathetic sphenopalatine ganglia (SPG) and superior cervical sympathetic ganglia (SCG) of the ANS (9, 10, 19). Most of the analyses were performed on newborn mice (P0), as these structures are largely developed at birth, and many of these mutant animals die shortly after birth due to severe renal (29) or intestinal abnormalities (see below).
Schematic of the various _RET_-mutant mice used in this study. (A) Simplified diagram of mutant or WT human RET cDNAs homologously recombined into exon 1 (black box) of the mouse Ret locus using knockout-knockin approach (29). (B) Schematic of the different WT RET9, RET51, and their respective mutant knocked in alleles is shown. The different domains and sizes of RET9 and RET51 are indicated. The area of divergence between the 2 RET isoforms is indicated in red or blue. Also indicated are the key docking tyrosine (Y) residues, the major intracellular adapters that dock at these tyrosines, and the downstream signaling cascades. Homozygous mice were generated that harbor Tyr-to-Phe (Y to F) mutations for each of the indicated Tyr except Y1096. In the RET51(Cdel) allele, residues 1063–1072 of RET9 were replaced with residues 1063–1072 of RET51. This results in a receptor that is essentially RET51 with a deletion of residues 1073–1114, including Y1096. In the RET9(51C) allele, residues 1063–1072 of RET51 were replaced with residues 1063–1072 of RET9. This results in a receptor that is essentially similar to RET9 with C terminus of RET51, including Y1096. Note Grb2 can directly bind to RET51 and also indirectly to Y1062 in both RET9 and RET51.
List of RET-mutant mice used
To determine whether the intestinal aganglionosis in _Ret_-null mice could be attributed to a specific Ret isoform, we used acetylcholinesterase (AChE) staining to visualize the ENS in mice expressing solely RET51 (RetRET51/RET51) or RET9 (RetRET9/RET9). In a previous report, Ret9 was found to be critical for intestinal innervation (28); however, we found that either isoform was capable of supporting ENS development. AChE-stained neurons and a normal myenteric neuronal plexus were detected throughout the small and large bowel in these mutant mice (Figure 2A; data not shown).
RET51 is sufficient to support ENS and sympathetic nervous system development and is necessary for the development of cranial parasympathetic ganglia. (A) RET9 and RET51 can both support normal ENS development. Whole-mount AChE staining (to visualize ENS formation) from WT or the indicated RET51 and RET9 mice (at postnatal day zero, P0) show typical dense reticular, honeycomb-like distal colon staining, indicating normal ENS colonization by both RET isoforms. (B) Redundant roles of RET isoforms in development of sympathetic ganglia. Whole-mount TH immunohistochemistry (brown color) shows normally located SCG and normal projections to the eye (white arrowhead) and submandibular gland (SM, black arrowheads). (C) RET9 alone is unable to fully support parasympathetic ganglia development. Nissl-stained sections of head from P0 mice show normal location of SPG in WT and RET51 (arrows) mice, but a subset of RET9 mice show unilateral SPG agenesis (dashed oval) (see E–H for orientation). (D) Reduced harderian gland innervation in RET9 mice (1- to 2-month-old mice) was observed with anti-TuJ1 immunohistochemistry (red fibers). While TuJ1-positive fibers surround almost all acini in WT and RET51 mice, RET9 harderian glands show reduced innervation consistent with incompletely penetrant unilateral agenesis. (E–H) Anatomical and histological landmarks for SPG analysis. Vertical bar in E shows approximate level of histological sections. (F) Coronal section showing SPG location (white arrow); boxed area is shown at higher magnification in G, which also represents images in C and Figure 7A. (H) Closer view of SPG of one side denotes approximate representation of images in Figure 7B. Scale bars: 400 μm (A); 600 μm (B); 200 μm (C); 100 μm (D); 50 μm (inset); F, 1 mm. br, brain; sep, septum; mu, eye muscles; np, nasopharynx; mn, maxillary nerves; oa, orbital artery; ov, orbital vein; spa, sphenopalatine ganglion.
Previous analysis of _Ret_-null and _Artemin_-null mice showed that Ret signaling is important for sympathetic neuron precursor migration and axonal projection (10, 30). In these mutants, the SCG fails to migrate to its proper location, resulting in accompanying abnormalities in projections to the submandibular gland and muscles of the eye. The stellate and sympathetic chain ganglia and their axonal projections are also abnormally developed in these mutant mice. We next searched for isoform-specific differences in sympathetic nervous system development. We used tyrosine hydroxylase (TH) whole-mount immunohistochemistry to visualize the sympathetic nervous system in homozygous RET9 and RET51 mice. We found that it developed normally, with properly located SCGs and normal innervation of its targets and normally formed sympathetic chain (Figure 2B and data not shown).
Finally, we investigated to determine whether RET9 and RET51 mice were able to support parasympathetic ganglia development. The SPG, which innervates the harderian/lachrymal glands, fails to develop in _Ret_-null mice due to defective precursor proliferation (9). Histological analysis of P0 head sections using Nissl staining revealed normally localized SPGs in RET51 mice (Figure 2C). However, a subset of RET9 mice had unilateral agenesis of the SPG (25%), which was accompanied by reduced harderian gland innervation compared with WT mice as assessed by TuJ1 immunohistochemistry (WT = 10821 ± 977, n = 3; RET9 = 2040 ± 297, n = 3; mean innervation area pixel2 ± SEM, P < 0.001); innervation in RET51 and WT harderian glands were similar (RET51 = 8862 ± 661, n = 3, mean ± SEM, P = 0.1) (Figure 2D). These results indicate that the WT forms of RET9 and RET51 have redundant roles in morphologically normal ENS and sympathetic nervous system development. However, RET9 alone is insufficient to promote proper SPG development, suggesting that Grb2-mediated signaling through Y1096 is important in this process.
Signaling through RET51-Y1062 is essential for distal colon innervation. Four phosphotyrosines in the RET cytoplasmic domain serve as major docking sites for intracellular adaptors that initiate downstream signaling cascades. We next sought to delineate which of these RET docking sites, Y981 (site for Src interaction), Y1015 (site for Plcγ interaction), Y1062 (interacts with Shc and other intracellular adaptors) and Y1096 (a site only present in RET51 that interacts with Grb2), play critical roles in the ENS in order to gain new mechanistic insights into RET-mediated HSCR.
We used AChE staining to examine the ENS of mice expressing human RET51 in which each of these docking sites was individually mutated (Y to F mutants) or, in the case of Y1096, by using a C-terminal deletion mutant (RET51Cdel) (Figure 1 and Table 1) that lacks this residue (25, 26, 28, 29). We observed remarkable differences in colonic ENS structure in these mutants, which were highly reminiscent of long (aganglionic segment proximal to the rectosigmoid colon) and short segment (aganglionosis confined to rectosigmoid region) HSCR. The enteric plexus and neurons in the distal bowel were absent (i.e., aganglionosis) in mice expressing the RET51(Y1062F) mutant (lacks Shc adaptor site), with the length of aganglionic segment varying from the distal colon only to total colon aganglionosis (Figure 3). Notably, these mice do not have significant renal abnormalities (29), but do not survive to adulthood due to complications of the HSCR-like phenotype (i.e., megacolon, ruptured bowel). Thus, abrogating RET-Shc–mediated signaling has a severe effect on the ENS, but not on urogenital development.
Essential role of the RET-Shc multidocking site in distal colon innervation. To determine roles of individual RET adaptor sites in ENS development, AChE whole-mount staining to visualize the ENS was performed on intestines (P0) of the indicated adaptor mutants in the context of RET51 isoform (Y981F, Src adaptor; Y1015F, Plcγ adaptor; Y1062F, Shc adaptor; or null, KO). Neuronal plexus and ganglion formation, depicted by typical reticular, honeycomb like staining pattern at ileocecal (IC) junction and distal colon, occurs in all adaptor mutants except for RET51(Y1062F). The RET51(Y1062F) image shows distal colon with absent enteric ganglia and plexus, but readily visible thick extrinsic nerve fiber bundles similar to those observed in _Ret_-null mice. Varying degrees of colon aganglionosis were seen in RET51(Y1062F) mice. Compared with aganglionosis in terminal ileum (TI) of _Ret_-KO mice, AChE staining of the intestines in RET51(Y1062F) mice detected both a neuronal plexus and ganglia in the TI. The schematic summarizes the extent of bowel colonization by ENS precursors in mutant mice. For RET51(Y1062F), each pentagon represents the location of the most distal enteric ganglion cell in an individual mutant; for other mouse lines, single pentagons represent the entire group, since none of these mice had bowel aganglionosis (refer to graph in Figure 4B for number of mice analyzed for each mutant mouse and associated ENS abnormality). Scale bars: 600 μm (IC junction); 400 μm (distal colon).
Further, we found that RET51(Y1015F) mice (lack RET-stimulated Plcγ signaling) do not have major abnormalities in the ENS in contrast, with the crucial importance of this pathway in supporting normal urogenital development (29). The majority of these mutant mice showed normal distal colon innervation (13/16), with only a small subset manifesting distal colon hypoganglionosis (Figure 3 and Figure 4B). We also found normal intestinal innervation in RET51(Cdel) mice (RET51 C-term deletion, which lack the direct Grb2 binding site Y1096), RET9(51C), or RET51(Y981F) (lack Src binding site) (Figure 3, Figure 4B, and data not shown). The phenotypes of these mutant mice indicate that in the context of RET51, the Src adaptor site, Grb2-binding site, and the C-term tail are largely dispensable for normal ENS (Figure 4B and Table 2) as well as renal development (29). Furthermore, these results demonstrate that the ENS and genitourinary (GU) systems have different thresholds to the loss of RET-Plcγ (results in CAKUT) and RET51-Shc (results in HSCR) mediated signaling for normal development.
Severe ENS defects in RET tyrosine docking mutants lacking the Grb2-binding site. (A) Whole-mount AChE staining was used to visualize the ENS in P0 intestines in RET9 or the indicated RET9 docking tyrosine mutant mice. Regions of the bowel from the distal colon and duodenum are shown. WT RET9 monoisoformic animals have a normal-appearing dense reticular staining pattern in the duodenum and the distal colon, while RET9(Y981F) (Src mutants) have colon aganglionosis but normal duodenum staining. RET9(Y1015F) (Plcγ) mice have neuronal ganglia and plexus in the colon but at reduced density (hypoganglionosis); duodenum innervation is normal. RET9(Y1062F) mice manifest complete intestinal aganglionosis, as no neurons are present in colon or in duodenum. A compound isoformic Y1062F mutant (RET9/51[Y1062F]) has intermediate aganglionosis compared with RET51(Y1062F) (Figure 3) and RET9(Y1062F) mice, indicating isoform dosage influences intestinal innervation. (B) The bar graph summarizes the innervation phenotypes of all the RET9 and RET51 WT and adaptor mutants examined. Numbers in the bars represent the number of mice with that phenotype. Scale bars: 600 μm (duodenum); 400 μm (distal colon).
Comparative summary of RET isoforms/docking site mutants and associated main developmental abnormalities
Docking site mutations in RET9 cause severe ENS defects. The RET9 isoform differs from RET51 in that it lacks the C-terminal tail that contains the Grb2 docking site (Y1096), important for activation of the AKT/MAPK pathways. This site provides redundancy to the Src and multidocking Shc sites for normal kidney development, as its absence in several of the RET9-Tyr mutants leads to increased severity of kidney abnormalities (29). Therefore, we explored whether this site was also important in ENS formation. AChE staining of postnatal gastrointestinal tracts from mice expressing RET9 mutations (Y981F, Y1015F or Y1062F) revealed a spectrum of phenotypes that were more severe than those of mice expressing the corresponding RET51 mutations. For example, while the ENS of RET51(Y981F) mutant mice appeared normal, RET9(Y981F) mice manifested partially penetrant distal intestinal aganglionosis (9/18) in which the extent of aganglionosis ranged from the distal third of the colon only to the entire colon and terminal ileum (Figure 4). Interestingly, the RET9(Y981F) mutant mice previously showed the pattern of incomplete penetrance and variable expressivity in CAKUT (29) (Table 2). Thus, abrogated Src adaptor site binding in the face of reduced Grb2-mediated signals (i.e., RET9 context) increases the propensity to develop both HSCR and CAKUT phenotypes.
In contrast with the abnormalities in the RET9(Y981F) mutants, RET9(Y1015F) mutants (lack Plcγ binding) have normal-appearing small intestinal innervation and show only a partially penetrant distal colon hypoganglionosis (15/17) (Figure 4, A and B). While of moderate severity, these colon ENS abnormalities are significantly worse than those observed in RET51(Y1015F) mice. We noted that the differences in penetrance and severity of ENS deficits between _RET9_-Plcγ and _RET51_-Plcγ mutants (Figure 4) were not observed in the genitourinary system in which both mutants manifested severe genitourinary defects (29) (Table 2). We also examined the ENS of RET9(Y1062F) mice (lack binding to Shc and other adaptors). We found that these mice have complete intestinal aganglionosis with 100% penetrance, a phenotype that is similar to _Ret_-null and mouse-human-chimeric-Ret9(Y1062F) mutant mice (19, 31). These deficits were more severe than in RET51(Y1062F) mutant mice analyzed above in which aganglionosis is limited to the colon and further support the idea that Grb2 signaling via the RET51-specific Y1096 docking site is important for proper development of the ENS. Finally, compound isoformic RET(Y1062F) mutant mice (RetRET9(Y1062F)/RET51(Y1062F)), which harbor single mutant RET9(Y1062F) and RET51(Y1062F) alleles, had an intermediate phenotype, with aganglionosis affecting colon and terminal ileum that is reminiscent of long-segment HSCR (32) (Figure 4A). We did not observe any overt differences between males and females for aganglionosis or hypoganglionosis phenotypes in the mutant mice analyzed above. Taken together, these results suggest that activation of AKT and MAPK signaling through Y1062 that serves as a docking site for Shc and other adaptor proteins is necessary for complete colonization of the colon.
To further determine the importance of Grb2-mediated signaling in ENS development, we isolated and cultured ENS precursors from E14.5 WT rat bowel using P75 immunoselection (see Methods). We infected these with lentiviruses expressing siRNAs directed against either Grb2, Shc, or _Plc_γ and performed analysis using TuJ1 and Tau1 immunocytochemistry (33) (see Methods). Grb2 knockdown resulted in a marked reduction in enteric neuron number compared with control or Shc siRNA–expressing cells, further confirming the essential role of Grb2 in ENS development; both Tau1 and TuJ1 immunocytochemistry revealed similar reductions in neuronal number (Figure 5 and data not shown). The fact that Shc knockdown did not have a major effect on enteric neuron number suggests redundancy/compensation through other adaptors and is consistent with milder ENS phenotype observed in RET51(Y1062F) mutant mice, which harbor the additional Grb2-binding site. Interestingly, _Plc_γ knockdown also caused a dramatic reduction in neuronal numbers despite the minor ENS deficits in RET(Y1015F) mice, suggesting that Plcγ activation via signals other than RET may be important in ENS development.
Grb2 and Plcγ are essential for maintaining normal enteric neuron number. Roles of Grb2, Plcγ, and Shc adaptor proteins in regulating enteric neuron number were examined in vitro. ENS precursors were harvested from E14.5 rat intestines using p75NTR immunoselection and propagated in culture for 7 days. Indicated siRNAs or control (scrambled siRNA) were delivered by lentivirus infection 1 day after plating the cells. Tau1 immunofluorescence was used to detect neurons and their neurites (arrows). Shc siRNA did not affect cell number compared with control. _Plc_γ and Grb2 siRNA caused a severe reduction in neuron numbers. The y axis represents neurons remaining relative to the beginning of the culture. The graph at the bottom shows quantification results from 3 independent experiments (mean ± SD, *P < 0.05 versus control). Scale bar: 25 μm.
Sympathetic nervous system development is relatively resistant to mutation of individual RET docking sites. Artemin-stimulated RET signaling is important for sympathetic neuron precursor migration and subsequent axonal projection. To decipher RET-dependent pathways that govern sympathetic ganglia formation, we used whole-mount TH immunohistochemistry to visualize the sympathetic nervous system in these _RET_-mutant mice. We found that individual docking sites for Plcγ, Src, Shc, and Grb2 in RET51 isoform are all dispensable for sympathetic nervous system development (Figure 6, Table 2, and data not shown). When we examined mice expressing RET9 with these mutations, we again found few deficits in the sympathetic nervous system. These included moderate, unilateral abnormalities in SCG migration, sympathetic chain development, and neuronal projection defects in a subset of RET9(Y1062F) mutant mice (5/8) (Figure 6). These results are in stark contrast to the deficits observed in the ENS and in kidney development in these mice (Table 2), suggesting that sympathetic nervous system development has a different threshold to aberrant Ret-Tyr signaling than other systems or depends on a high degree of Ret-mediated signaling redundancy.
Sympathetic nervous system defects in RET mutants lacking Shc and Grb2 binding sites. Whole-mount TH immunohistochemistry was performed on P0 pups to assess sympathetic nervous system development in RET9 and RET51 signaling mutants. (A–C) Normal sympathetic nervous system develops in (A) RET9(Y981F) (Src mutant) and (B) RET9(Y1015F) (Plcγ mutant) mice, as demonstrated by normal SCG location and normal innervation to submandibular gland (SM, black arrowheads) and the eye (blue arrowhead). (C) _Ret_-null mice show abnormal caudal location of the SCG, next to the stellate ganglion (STG) instead of the expected normal location (dashed oval); innervations to the SM or the eye are absent. (D–H) RET9(Y1062F) adaptor mutants (lack Shc- and Grb2-binding sites) show a spectrum of sympathetic nervous system defects albeit milder than _Ret_-null animals. These include normal SCG location and projections to eye (blue arrowhead) and submandibular gland (black arrowhead in D), failure of SCG to migrate normally (E and F; dashed circle depicts expected location), mislocalization of the SCG near the stellate ganglion (STG) in G, and fusion of rostral sympathetic chain ganglia (black arrowhead) with the STG, resulting in a gap in the sympathetic chain (double arrowhead) in H. (I) Sympathetic chain (sc) ganglia in RET9(Y1062F) mice are small (black arrows), or absent (dashed oval) and have diminished to absent axonal outgrowths compared with WT mice (blue arrows) in J. (K–M) Docking site mutations in the context of RET51 do not disrupt sympathetic nervous system development. Representative pictures are shown for the indicated adaptor mutants in RET51 context, highlighting normal sympathetic ganglia development. Scale bars: 600 μm (A–H and K–M); 400 μm (I, J).
Severe parasympathetic nervous system defects in RET9 docking tyrosine mutant mice. In all the RET-dependent organs examined (ENS, sympathetic nervous system, kidneys), RET9 docking site mutants (lack Grb2 binding at Y1096) manifest more severe defects than mice expressing their RET51 counterparts. To extend this analysis to the parasympathetic nervous system, we examined SPG development in these mutant mice. The SPG failed to develop in a large number of _RET9_-Tyr mutants, and these were generally more severe than mice expressing WT RET9 (Figure 2C and Figure 7). For example, RET9(Y981F) (lack Src binding) and RET9(Y1015F) (no Plcγ binding) mutants showed partially penetrant unilateral and bilateral SPG agenesis, with accompanying reduced innervation to the harderian gland in the postnatal period (Figure 7, A–C and E). Importantly, RET9(Y1062F) mutants (no Shc or Grb2 binding) have completely penetrant bilateral SPG agenesis, as observed in _Ret_-null mice (Figure 7A). We counted the number of Nissl-stained neurons in these mutant mice using serial sections of the entire SPG and found that when the SPG is present (i.e., no agenesis), the ganglia contained a normal number of neurons (Figure 7D). Notably, histological analysis of P0 head sections using Nissl staining revealed normal localization of SPGs and normal neuronal numbers in all mutants in the RET51 context (Figure 7 and data not shown). More severe SPG defects in RET9 mutants again indicate that Grb2 signaling from RET51 C terminus is important for normal development of the parasympathetic nervous system.
Severe parasympathetic nervous system defects in RET9 docking tyrosine mutant mice. Cranial parasympathetic SPG and their innervation of the harderian gland using Nissl staining (P0 pups) and TuJ1 immunohistochemistry (1- to 2-month-old mice), respectively. Refer to Figure 2, E–H, for anatomical landmarks for SPG and key for annotations. (A and B) RET9(Y1062F) mice exhibit (n = 14) bilateral SPG agenesis, a phenotype similar to that of _Ret_-null mice (dashed oval, expected normal SPG site); RET51(Y1062F) SPG were normally located (arrows in A and B). RET9(Y981F) and RET9(Y1015F) mice showed incomplete penetrance of SPG agenesis, including normal SPG location bilaterally (arrows in A), unilateral, or bilateral SPG agenesis (SPG agenesis indicated by dashed ovals in B and summarized in E). (C) Differences in innervations (TuJ1 staining, red fibers surrounding each acinus) of the 2 harderian glands from the same mutant mouse. Note that gland 1 of each mutant had TuJ1-positive fibers surrounding all acini, but gland 2 innervation was markedly decreased or absent, consistent with SPG unilateral agenesis (closer view shown in the inset). (D) Summary of SPG neuronal numbers in RET mutants, in which SPG are formed. Quantification of neuron numbers in SPG show no significant differences in mutant and WT mice except RET9(Y1062F) mice, which had SPG agenesis. The graph depicts number of neurons in each completely sectioned SPG (mean ± SEM; numbers at the bottom denote SPG used per genotype). (E) Spectrum of SPG abnormalities observed in _RET_-mutant mice. Results of SPG location from both RET51 and RET9 mutant mice are summarized in the bar graph. Numbers in each colored bar represent the number of SPG with the corresponding defect. Scale bars: 200 μm (A); 50 μm (B); 100 μm (C); 50 μm (inset).