XB-ART-35957EMBO J June 20, 2007; 26 (12): 2955-65.
We present a loss-of-function study using antisense morpholino (MO) reagents for the organizer-specific gene Goosecoid (Gsc) and the ventral genes Vent1 and Vent2. Unlike in the mouse Gsc is required in Xenopus for mesodermal patterning during gastrulation, causing phenotypes ranging from reduction of head structures-including cyclopia and holoprosencephaly-to expansion of ventral tissues in MO-injected embryos. The overexpression effects of Gsc mRNA require the expression of the BMP antagonist Chordin, a downstream target of Gsc. Combined Vent1 and Vent2 MOs strongly dorsalized the embryo. Unexpectedly, simultaneous depletion of all three genes led to a rescue of almost normal development in a variety of embryological assays. Thus, the phenotypic effects of depleting Gsc or Vent1/2 are caused by the transcriptional upregulation of their opposing counterparts. A principal function of Gsc and Vent1/2 homeobox genes might be to mediate a self-adjusting mechanism that restores the basic body plan when deviations from the norm occur, rather than generating individual cell types. The results may shed light on the molecular mechanisms of genetic redundancy.
PubMed ID: 17525737
PMC ID: PMC1894760
Article link: EMBO J
Genes referenced: admp bmp4 chrd.1 egr2 en2 gsc hoxb9 myod1 nodal nodal1 otx2 rax six3 smad1 smad2 sox2 szl tal1 tbx2 ventx1.2 ventx2.2
Morpholinos: chrd MO1 chrd MO2 gsc MO1 ventx1.1 MO1 ventx2.1 MO2
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|Figure 1. Gsc knockdown in Xenopus embryos causes loss of head structures and affects patterning of the AP and DV axes. (A) Gsc marks Spemann organizer endomesoderm at early gastrula. (B) Gsc MO targets both pseudoalleles of the X. laevis Gsc gene. (C–I) Gsc MO injection (136 ng total) causes loss of head structures, marked by Otx2 (forebrain), Six3 and Rx2a (forebrain and eyes), and En2 (midbrain/hindbrain border) (n=106; Supplementary Table I). Expression of the ventral marker Szl is reduced anteriorly and expanded posteriorly in the ventral blood island. (E) Co-injection of mGsc mRNA (200 pg total, radial injection) rescues the Gsc MO phenotype (n=78). (H, I) Knockdown of Gsc reduces head size and affects patterning of the posterior somites, including loss of MyoD expression at the tip of the tail (arrows). (J, K) Moderately affected embryos survive until tadpole stage and have cyclopic eyes (indicating holoprosencephaly) and no mouth opening.|
|Figure 2. The dorsalizing effects of mGsc mRNA injection require Chd. (A–E) Gsc MO reduces Chd expression at gastrula 2.5-fold, whereas overexpression of mGsc mRNA greatly expands Chd expression. (F–H) Injection of 50 pg mGsc mRNA into one ventral blastomere at the four-cell stage leads to a range of dorsalized phenotypes, of which 50% develop secondary axes (38% partial; 12% complete with eyes, notochords, and cement glands). (I, J) Co-injection of Chd MO (34 ng) prevents second axis induction and dorsalization by mGsc mRNA in 97% of the embryos. (K, L) mGsc mRNA microinjection (200 pg total) induces Chd expression in UV-ventralized embryos at gastrula. (M–O) The rescue of head (Otx2) and pan-neural marker (Sox2) in UV embryos by mGsc overexpression (n=54) has a complete requirement for Chd (co-injection of 136 ng Chd MO; n=59).|
|Figure 3. Gsc is required for secondary neural induction and mesoderm patterning in Activin-treated animal cap explants. (A) Experimental design (n=15 or more per experimental set) (B, C) Untreated animal caps develop into atypical epidermis, whereas Activin treatment leads to elongation and brain formation, visualized by Otx2 at the anterior pole (arrows). In addition, Otx2 expression in anterior endoderm can be seen in one of the explants (arrowhead). (D, E) Gsc-depleted caps elongate after treatment with Activin, but lack Otx2 neural staining. (F, G) Chd-depleted caps treated with Activin are unable to elongate, confirming the requirement of Chd for dorsal mesoderm and neural induction by Activin (Oelgeschläger et al, 2003). Insets show whole sibling embryos. (H) Quantitative RT–PCRs showing genes affected by depletion of Gsc include markers of anterior CNS, organizer, somites, and ventral mesoderm. Note that Vent1 expression is increased more than 20-fold by Gsc knockdown.|
|Figure 4. Double depletion of Vent1 and Vent2 causes severe dorsalization of the embryo. (A–D) Injection of either Vent1 or Vent2 MO expands the neural plate at neurula stage (insets), but only the combination of both MOs strongly dorsalizes tailbud stage embryos, with shortened body axes and large heads and cement glands (n=122; Supplementary Table I). (E, F) Vent1/2 depletion leads to transcriptional upregulation of Gsc (hemisections at stage 10; n=15) and Chd (insets in panels E and F; whole embryos, vegetal view; n=18). (G, H) Loss of Gsc increases Vent1 and Vent2 expression (hemisections at stage 10; n=21 and 15) (I) Quantitative RT–PCR analyses showing 3- to 4-fold upregulation of the organizer genes Gsc, Chd, and Admp in animal caps at gastrula stage after Vent1/2 depletion.|
|Figure 5. Gsc is required for the dorsalization caused by Vent1/2 knockdown. (A–I) Co-injection of Gsc MO restores normal pattern in Vent1/2-depleted whole embryos (n=53; Supplementary Table I). At the neurula stage, knockdown of Vent1/2/Gsc reduces the neural plate (Sox2) back to normal size (insets in panels A–C). In addition, the expansion of the cement gland and midbrain in Vent1/2 morphants is rescued in triple knockdown embyros (insets in panels G–I). Note that blood formation (Scl) is not rescued in the triple depletions (I). All MOs were injected at the same dose (45 ng each). (J) The upregulation of Chd expression by Vent1/2 MO is restored to control levels in Vent1/2/Gsc-depleted animal caps at gastrula stage. (K) Expression of Szl is downregulated by Vent1/2 MO, but restored to normal levels when Gsc is also depleted.|
|Figure 6. Knockdown of Gsc and Vent1/2 restores normal development of dorsal and ventral half-embryos (n=52 or more per experimental set). (A) Embryos were bisected into dorsal and ventral halves at blastula stage. (B) Control sibling at the same magnification as the other panels. (C, D) Bisectioned control embryos form smaller but well-proportioned dorsal half-embryos, whereas ventral halves differentiate into belly-pieces that express HoxB9 in the ventral mesoderm (Wright et al, 1990) but are devoid of neural tissue, as indicated by the lack of Sox2 expression (inset). (E, F) Gsc depletion (136 ng MO) causes a reduction of the head in dorsal halves, whereas ventral halves are not affected. (G, H) Dorsal halves of Vent1- and Vent2-depleted embryos (45 ng each) are dorsalized, but retain overall DV patterning. The corresponding ventral halves are strongly dorsalized, including expression of spinal cord (HoxB9), brain (Krox20, Six3), and pan-neural Sox2 marker (inset). (I, J) Remarkably, both halves of triple knockdown embryos (45 ng each) develop as the uninjected control half-embryos.|
|Figure 7. Model of regulatory mechanisms for pattern formation at gastrula. In the dorsal center, Activin/Nodal signals phosphorylate Smad2/3 to activate Gsc expression. The expansion of Chd and ADMP can also be achieved by Gsc-independent pathways. In the ventral center, BMP4/7 signals phosphorylate Smad1/5/8 and lead to the expression of Vent1/2. BMP4 is also able to activate ventral center secreted proteins by Vent-independent mechanisms. The function of Gsc and Vent is to regulate each other, providing an intracellular compensatory mechanism that works in concert with the extracellular networks of growth factors and their antagonists.|