Bonacci G , Fletcher J , Devani M , Dwivedi H , Keller R , Chang C .

R01-GM083029 NIGMS NIH HHS , R01 GM083029-01A2 NIGMS NIH HHS , R01 GM083029-02 NIGMS NIH HHS , R01 GM083029 NIGMS NIH HHS

Xla Wt + abl2 (fig.2.b)
Xla Wt + abl2 (fig.2.b, c)
Xla Wt + abl2 (fig.4.b)
Xla Wt + abl2 (fig.4.c)
Xla Wt + abl2 (fig.5.a)
Xla Wt + abl2 (fig.5.a)
Xla Wt + abl2 (fig.9.a)
Xla Wt + abl2 (fig.9.a, b)
Xla Wt + abl2 (fig.S5.c)
Xla Wt + abl2 MO (fig.3.c)
Xla Wt + abl2 MO (fig.3.c)
Xla Wt + abl2 MO (fig.3.d)
Xla Wt + abl2 MO (fig.3.d)
Xla Wt + abl2 MO (fig.4.b)
Xla Wt + abl2 MO (fig.4.c)
Xla Wt + abl2 MO (fig.5.a)
Xla Wt + abl2 MO (fig.5.a)
Xla Wt + abl2 MO (fig.9.c)
Xla Wt + abl2 MO (fig.S5.c)
Xla Wt + abl2{del}{1} (fig.9.a, b)
Xla Wt + abl2{del}{2} (fig.9.a, b)
Xla Wt + {dn}abl2{K306R} (fig.S5.c)

	Fig. 1. Expression of Arg during early Xenopus development. A) Schematic representation of sequence alignment between human and Xenopus Arg proteins. The SH2, SH3 and tyrosine kinase domains are over 92% identical, while the actin binding domains (BDs) are 74–76% identical between the two proteins. B) Temporal expression of Arg assayed by RT-PCR (25 cycles) showed that Arg was expressed maternally and its transcripts persisted till at least tailbud stages. C) Spatial expression of Arg assayed by in situ hybridization of gastrula (panels a and b), neurula (panel c), tailbud (panels d and e) and early tadpole (panel f) embryos. The orientations of the embryos are: panel a, side view with dorsal to the right; panel b, vegetal view with dorsal to the right; panels c and e, dorsal view with anterior to the left; panels d and f, side view with anterior to the left.
	Fig. 2. Overexpression of Arg induced gastrulation defects in early Xenopus embryos. Ectopic expression of Arg led to failure of blastopore closure, reduced head structure and shortened body axis at neurula (A, dorsal view) or tadpole (B, side view) stages. The effects were dose-dependent, so that more severe defects (open back) were observed in a larger portion of the embryos when the doses of Arg RNA were increased (C). The expression level of Arg was shown in panel D.
	Fig. 3. Knockdown of Arg protein production led to gastrulation defects. A) Antisense Arg-MO specifically blocked translation of Arg from the RNA that contained its target sequence, but had no effect on protein translation from a RNA containing a modified 5′-UTR [Arg(UTR*)]. 1 ng RNA and 20 ng Arg-MO were used. B) Arg-MO (40 ng) reduced endogenous Arg protein to about 35% of its original level. C) Expression of Arg-MO (40 ng) in early frog embryos resulted in delayed blastopore closure and neurulation, severe reduction of head structures, shortened and bent body axis, and open back phenotypes. D, E) The defects induced by Arg-MO (40 ng) were partially rescued by co-expression with the modified Arg(UTR*) RNA (5–10 pg).
	Fig. 4. Arg regulated gastrulation without affecting mesodermal cell fate. A) Alteration of Arg levels did not affect expression of the mesodermal markers XBra, Chordin, Gsc and Wnt11 at gastrula stages. B) Arg modulated migration of Gsc-expressing anterior mesendoderm cells during neurulation. C) Arg regulated the shape of the expression domains of the mesodermal markers Chordin and MyoD as well as the neural marker Sox2. D) RT-PCR studies revealed that Arg did not influence mesodermal cell fate determination.
	Fig. 5. Arg modulated migration of anterior mesendoderm. A) Alteration of Arg levels interfered with spreading of Gsc-expressing cells anteriorly. The position of the dorsal blastopore is marked by the red arrowhead. B) Arg modulated elevation of dorsal mesendoderm (yellow arrowhead) over the endodermal mantle of the blastocoel floor. Dorsal side is on the right. C) Arg influenced migratory behaviors of anterior mesendoderm on fibronectin, and the continuous area covered by spreading mesendoderm was significantly reduced when Arg levels were changed. D) The defects in anterior mesendoderm migration in Arg morphants were partially rescued by the modified Arg(UTR*).
	Fig. 6. Arg regulated cell spreading and dynamic changes in protrusive activity. A, B) Arg reduced, while Arg-MO enhanced, significantly the spreading of anterior mesendodermal cells on fibronectin. C) Arg modulated cell morphology. D) Distribution of cells with different numbers of lamellipodia or blebs only suggested that overexpression of Arg promoted formation of blebs, whereas reduction of Arg favored formation of multiple lamellipodia. E) Arg regulated dynamic changes of protrusive activities. Changes in lamellipodia or blebs were counted from recorded time lapse videos and the average changes per cell per minute were calculated and plotted here. Reduction of Arg significantly increased lamellipodia dynamics, whereas elevation of Arg levels resulted in augmented changes in membrane blebs. F) Changes in lamellipodia and blebs in selected cells from time lapse movies. Equal time intervals between each frame were used for all samples. The arrowheads pointed to the following changes: white: blebs; red: split lamellipodia; blue: withdrawal of existing lamellipodia; purple, expansion of lamellipodia; yellow, formation of a new lamellipodium.Fig. 7. Arg controlled convergent extension cell movements. A) DMZ explants taken from embryos with altered levels of Arg did not extend as much as those from control embryos. B) Ectopic expression or reduction of Arg led to statistically significant decrease in length over width ratio of the DMZ explants. C) Ectopic expression of Arg reduced mediolateral cell intercalation, but Arg-MO did not prevent, and seemed to enhance, cell mixing in the trunk mesoderm. 0.5 ng Arg RNA and 40 ng Arg-MO were used in these experiments.
	Fig. 7. Arg controlled convergent extension cell movements. A) DMZ explants taken from embryos with altered levels of Arg did not extend as much as those from control embryos. B) Ectopic expression or reduction of Arg led to statistically significant decrease in length over width ratio of the DMZ explants. C) Ectopic expression of Arg reduced mediolateral cell intercalation, but Arg-MO did not prevent, and seemed to enhance, cell mixing in the trunk mesoderm. 0.5 ng Arg RNA and 40 ng Arg-MO were used in these experiments.
	Fig. 8. Arg controlled actin organization. A) Anterior mesendodermal explants stained with phalloidin revealed that Arg controlled F-actin distribution at the cell–cell contact and in membrane protrusions. B) Arg modulated F-actin distribution and cell polarity in trunk mesoderm. C) Localization of F-actin in dissociated anterior mesendodermal cells revealed by phalloidin staining. D) Dynamic changes in F-actin remodeling in individual cells. Selected frames with equal time intervals for all samples were taken from time lapse video (Green = Utropin-GFP-labeled F-actin, red = membrane-tethered Cherry fluorescent protein).
	Fig. 9. The tyrosine kinase domain was required for efficient induction of gastrulation defects by Arg. A, B) The mutant Arg-N induced gastrulation defects at similar doses in similar percentages of embryos as Arg, but Arg-C was ineffective. C) Arg-N, but not Arg-C, partially rescued gastrulation defects in Arg morphant embryos.
	Fig. 10. Arg regulated phosphorylation of CrkII and paxillin. A) Arg enhanced, and Arg-MO reduced, phosphorylation of endogenous CrkII and paxillin, but neither significantly affected phosphorylation of Erk. B) Arg was required for activation of CrkII phosphorylation by RTKs. Protein phosphorylation was normalized with the total amount of that protein and the level in the control extracts was considered as 1.
	Supplemental Figure S2. Arg-MO2, which was ineffective in reducing endogenous Arg protein level, did not induce gastrulation defects. A) Unlike Arg-MO, Arg-MO2 did not effectively block Arg protein production in vivo. B) Arg-MO2 did not interfere with anterior mesendoderm migration on fibronectin. C) Arg-MO2 did not inhibit convergent extension in DMZ explants. D) Embryos injected with Arg-MO2 did not show gastrulation defects. 40 ng Arg-MO2 was used in all the experiments.
	Supplemental Figure S3. Arg did not directly regulate neural patterning. Animal caps from noggin-injected embryos expressed the pan-neural marker Sox2 and the anterior neural marker Otx2. Wnt8 both caudalizes and dorsalizes the neural tissues, so that the neural crest marker Twist, the hindbrain marker Krox20, and the spinal cord marker HoxB9 were all expressed. Overexpression or knockdown of Arg did not change the expression of any of the markers. The doses of RNAs used were: 10 pg Noggin, 20 pg Wnt8, and 0.5 ng Arg; and 40 ng Arg-MO was used.
	Supplemental Figure S4. Arg did not directly regulate cadherin-mediated cell adhesion. Dorsal mesodermal cells taken from early gastrula embryos were dissociated in calcium (Ca2 +)nd magnesium (Mg2 +)ree buffer. The cells were then transferred into the buffer containing Ca2 + and Mg2 + in tissue culture dishes coated with agarose, swirled to the center of the dishes, and either left alone without further disturbance (left column) or placed on the shaker with continuous slow swirling (right column) for 2 to 4 h. Reaggregation of the cells resulted in formation of cell clusters. Formation of cell clusters was similar in samples with elevated or reduced Arg expression, suggesting that cadherin-mediated cell adhesion was not affected by the changed levels of Arg.
	Supplemental Figure S5. The kinase-dead mutant Arg acted as a weak dominant-negative molecule and induced minor defects in body axes only at high doses. A) Arg(KR) contained a point mutation of a conserved lysine at the amino acid position 306 to arginine. B) Arg(KR) acted as a weak dominant negative molecule. At low doses (0.2.5 ng RNA), Arg(KR) did not block phosphorylation of CrkII by Arg (not shown); while at high doses (2 ng), Arg(KR) reduced CrkII phosphorylation by Arg (0.25 ng) and decreased endogenous CrkII phosphorylation. Arg-MO was used at the 40 ng dose. The amount of Arg or Arg(KR) proteins was shown by Western blot using an anti-Arg antibody from Santa Cruz Biotechnology. C) While Arg induced gastrulation defects at 0.25 ng (shown here), Arg(KR) did not alter embryonic development at the same dose (not shown). At the 2 ng RNA dose, Arg(KR) induced minor axial defects in Xenopus embryos (bottom panel). 40 ng Arg-MO was used.
	abl2 (ABL proto-oncogene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, dorsal right, animal hemisphere up.
	abl2 (ABL proto-oncogene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, vegetal view, dorsal right.
	abl2 (ABL proto-oncogene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, dorsal view, anterior left.
	abl2 (ABL proto-oncogene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.
	abl2 (ABL proto-oncogene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior left.
	abl2 (ABL proto-oncogene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, lateral view, anterior left, dorsal up.

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