Crkl deficiency disrupts Fgf8 signaling in a mouse model of 22q11 deletion syndromes

Abstract

Deletions on chromosome 22q11.21 disrupt pharyngeal and cardiac development and cause DiGeorge and related human syndromes. CRKL (CRK-Like) lies within 22q11.21, and Crkl-/- mice have phenotypic features of 22q11 deletion (del22q11) syndromes. While human FGF8 does not localize to 22q11, deficiency of Fgf8 also generates many features of del22q11 syndrome in mice. Since Fgf8 signals via receptor-type tyrosine kinases, and Crk family adaptor proteins transduce intracellular signals downstream of tyrosine kinases, we investigated whether Crkl mediates Fgf8 signaling. In addition to discovering genetic interactions between Crkl and Fgf8 during morphogenesis of structures affected in del22q11 syndrome, we found that Fgf8 induces tyrosine phosphorylation of FgfRs 1 and 2 and their binding to Crkl. Crkl is required for normal cellular responses to Fgf8, including survival and migration, Erk activation, and target gene expression. These findings provide mechanistic insight into disrupted intercellular interactions in the pathogenesis of malformations seen in del22q11 syndrome.

Figure 1

Crkl and Fgf8 Genetically Interact during Cardiovascular, Pharyngeal, and Skeletal Development (A-D) Pharyngeal arch artery (PAA) formation assessed by intracardiac ink injections at embryonic day (E) 10.5. Normal PAA formation and patterning in (A) Fgf8^+/-;Crkl^+/+and (B) Fgf8^+/+;Crkl^-/- embryos. PAA4 or PAA3-PAA6 vasculogenesis fails (red arrowheads) in (C) Fgf8^+/-;Crkl^+/- and (D) Fgf8^+/-;Crkl^-/- mutants associated with a dilated aortic sac (AS, [D]). PAAs are numbered. (E-M) (E, G, I, K, and M) Transverse sections through the neck and thorax of E15.5 Fgf8^+/-; Crkl^+/+ control embryos reveal a (E) normal left aortic arch, (G) right ventricular OFT, (I) continuity of aortic and mitral valves, (I and K) intact atrial and ventricular septae, and (K) atrioventricular valves. Thymus (thy, [E]), thyroid (tr, [E]), and parathyroids (arrowheads, [M]) are normally located and sized. (F, H, J, and L) Fgf8^+/-;Crkl^-/- mutants display: ectopic, single-lobed thymi (thy, [F]) or bilateral thymic aplasia; right aortic arch (arrow, [F]) and aberrant subclavian artery (arrowhead, [F]); (H and J) outflow tract defects, including a Double Outlet Right Ventricle with a dysplastic common aortic/pulmonary valve (arrow, [H]) or an aortic valve communicating with the right ventricle (arrow, [J]); endocardial cushion defects, including a primum atrial septal defect (red arrow, [L]) and mitral and tricuspid valve dysplasia (black arrow, [L]). (N) Fgf8^+/-;Crkl^+/- mutant with unilateral parathyroid aplasia (arrowhead) and ectopy (arrow). (O-Q) Skeleton preparations at E15.5 reveal markedly short femurs (red double arrows) in Fgf8;Crkl mutants, even in mutants with longer crown-rump lengths than (P) controls (the black, vertical double arrow is the same length in all specimens). (R-U) Palate and jaw defects. Sections through the palate reveal complete bony fusion of the palatal shelves (ps) in (R) control versus unfused shelves causing cleft in (S) mutants (arrow). (U) 30% of Fgf8^+/+;Crkl^+/-, 50% of Fgf8^+/-;Crkl^+/-, and 70% of Fgf8^+/-; Crkl^-/- mutants have short, narrow mandibles (arrowhead). Note the severe edema in the Crkl^-/- mutant (arrows, [U]). fl, forelimbs.

Figure 2

Decreased Neural Crest Population of the Outflow Tract and Caudal Pharyngeal Arches of Crkl^-/- Embryos Is Associated with Abnormal Apoptosis Genotypes are listed above the columns, and assay type is listed at the left; pharyngeal arches are numbered. (A-E) Whole-mount E10.5 allelelic series assayed for apoptosis with antiactivated caspase3. Right, lateral views at 803. The yellow arrowheads indicate domains of abnormal apoptosis in Fgf8;Crkl and Crkl^-/- mutants, including streams of neural crest (NC) originating in the hindbrain. (F-J) Whole-mount preparations of E9.5 allelic series assayed for the NC/ectoderm marker AP2α (green) and antiactivated caspase3 (red); right, lateral views at 3203. Yellow colabeled cells are apoptotic NC and ectoderm. (K-M) E9.5 transverse cryosections through the second pharyngeal arch assayed for AP2α (green) and apoptosis (TUNEL, red); nuclei are stained blue with Hoechst. Note the multiple cell types undergoing aberrant apoptosis including: NC (double-labeled, yellow), pharyngeal ectoderm, endoderm, and splanchnic mesoderm. nt, neural tube. (K′-M′) As above, sections through the third pharyngeal arch. fg, foregut. (N and O) Wnt1Cre and Rosa26-Cre reporter (R26R) strains were used to label NC derivatives in E10.5 Crkl^-/- and wild-type embryos. Cells stained red outside the neural tube are NC derivatives. The outflow tract of the (O) mutant is minimally stained compared to (N) wild-type. NC cells extend from dorsal to ventral in the PAs of wild-type embryos, but these extensions are not continuous or as extensive in the caudal PAs of the Crkl^-/- mutants (arrowheads). Arrows indicate the OFT/aortic sac.

Figure 3

Crkl Dosage Regulates Expression of Fgf8 Target Genes and Alters MAPK Activation In Vivo Genotypes are listed above the columns; all views are tight. Lateral and pharyngeal arches are numbered. (A-E) Right, lateral views of pharyngeal arches of E10.5 embryos after whole-mount in situ hybridization with an Erm antisense riboprobe. Note the decreasing levels of expression in the pharyngeal arches (red arrowheads) of (C) Fgf8^+/-;Crkl^+/-,(D) Crkl^-/-, and (E) Fgf8^+/-;Crkl^-/- mutants relative to (A and B) controls. Expression in the lung bud (black arrowheads) is intact. (F-J) As above, after whole-mount in situ hybridization with a Pea3 antisense riboprobe. (K-O) As above, after whole-mount in situ hybridization with a Barx1 antisense riboprobe. (P-T) Anti-phospho-Erk1/2 staining of E10.5 embryos. Optical confocal sections were obtained at 2 μm intervals; representative, anatomically matched section images are shown through the casudal PAs. The levels of these activated MAP kinases are decreased (red arrowheads) in the pharyngeal epithelia and mesenchyme in response to decreased Crkl and/or Fgf8 gene dosage. HT, heart.

Figure 4

Fgf Receptors 1 and 2 Are Activated, and Interact with Crkl, in Response to Fgf8 (A and B) Fgf8 stimulates tyrosine phosphorylation of FgfR1 and FgfR2. Anti-receptor antibodies were used to immunoprecipitate Fgf8b-treated MEF lysates (IP), and the products were separated by electrophoresis. Immunoblotting (WB) with anti-phosphotyrosine antibody (pY) reveals that phosphorylation of both receptors occurs in response to 10 ng/ml Fgf8b. (C) Activated FgfRs interact with Crkl via its SH2 domain. MEFs were treated with 10 ng/ml Fgf8b, and proteins from lysates that bind to the SH2 domain of Crkl were pulled down with GST-Crkl SH2 coupled to glutathione-Sepharose beads. Total MEF lysates (total lysate) or proteins pulled down (CRKL SH2 pull-down) were blotted and probed with anti-FgfR1 or anti-FgfR2 antibodies (WB). (D) Endogenous FgfRs associate with Crkl, preferentially with Crk. Proteins that interact with endogenous FgfR1 or FgfR2 were precipitated from control, and Fgf8b-treated MEF lysates were precipitated with anti-FgfR antibody-coupled protein A agarose beads (IP). Immunoprecipitates were separated by electrophoresis and probed with anti-Crkl or -Crk antibody (WB). The relative level of Crkl or Crk in 10 mg total lysate was determined in parallel compared to that associated with FgfR (total lysate).

Figure 5

Crkl Is Required for Fgf8-Induced Chemotaxis and Activation of Downstream Biochemical Signaling Mediators WB, western blot; IP, immunoprecipitation. (A and B) Modified Boyden chamber assay of Crkl^-/- MEFs migrating toward Fgf8b ([A], 10 ng/ml), fetal bovine serum ([A], 5%), PDGF ([B], 50 ng/ml), or EGF ([B], 20 ng/ml) compared to wild-type MEFs, or Crkl^-/- MEFs expressing CRKL as a transgene at a level comparable to wild-type cells. Control groups (CTR) had buffer with no growth factor or serum in the chambers. Error bars indicate standard deviation of sample data (n = 4). (C and D) Maximal MAPK activation in response to Fgf8b is Crkl dependent. (C) Cell lysates were prepared after incubation with 25 ng/ml Fgf8b and phosphorylated Erk1 and Erk2 were detected by Western blot. Average phospho-Erk levels (n = 2) are graphically represented as fold activation relative to that at 0 min in wild-type cells. Reblotting with anti-Erk antibody indicated equal levels of total Erk1/2 proteins (not shown). (D) MEFs exposed to varying concentrations of Fgf8b were tested for activation of Erk1 (p44) and Erk2 (p42). Cell lysates were prepared after 10 min of incubation with Fgf8b at the concentrations listed. The average phospho-Erk levels (Erk1 + Erk2) in two experiments are graphically represented below as fold activation relative to unstimulated levels. The levels of phospho-Erk were normalized relative to the corresponding total Erk levels (Erk1 + Erk2). (E-H) (E) Crkl regulates the level of Fgf receptor substrate 2 (Frs2) phosphorylation in response to Fgf8b. Anti-phosphotyrosine (pY) blot of total cell lysates prepared at the time indicated after incubation with 25 ng/ml Fgf8b. Black arrowheads indicate a 90 kDa protein phosphorylated in response to Fgf8b; note the decreased intensity of this band in Crkl^-/- MEFs at all time points. (F) Anti-Frs2 antibody reprobe of blot shown in (E); the black line denotes Fgf8-stimulated phosphorylation of Frs2 (corresponds to the black arrowhead on the pY blot), This species of Frs2 is relatively hypophosphorylated in Crkl^-/- MEFs. (G) Anti-phosphotyrosine blot of Frs2 immunoprecipitated from Fgf8b-stimulated MEFs. “+Crkl” samples are two different Crkl^-/- cell lines infected with Crkl-producing retrovirus (see [H]); Fgf8b-stimulated phosphorylation of Frs2 occurs at 1.3× and 1.0× wild-type levels in these cell lines, while that in Crkl^-/- MEFs is only 0.5×. (H) Quantitation of Crkl levels: the +Crkl cell line 1 produces Crkl at 2× compared to wild-type levels, and the +Crkl cell line 2 produces Crkl protein at 1.2× compared to wild-type levels.

Figure 6

A Model for Interaction between Crkl-Modulated Fgf Signaling and Tbx1 Function in the Developing Pharyngeal Arches Secreted Fgf8 signals to target cells via paracrine (1) or autocrine (2) pathways. Ligandstimulated FgfR activation triggers receptor autophosphorylation and interaction with signaling mediators, including Crkl. Erk-dependent and -independent pathways affect gene expression, cell survival, and other functions. Phosphorylated MAPK/ERKs downstream of Crkl activate existing ETS proteins (e.g., Erm) and also stimulate their transcription. Tbx1 is expressed in FgfR-expressing pharyngeal mesoderm, ectoderm, and endoderm; in these cells, Tbx1 and Fgf signaling may regulate expression of common transcriptional targets such as Gbx2. Furthermore, both Fgf8 and Tbx1 have been shown to have non-cell autonomous effects on neural crest survival and function.