Bilogan CK and Horb ME (2012), Xenopus staufen2 is required for anterior endod...

XB-ART-44557

Genesis March 1, 2012; 50 (3): 251-9.

Xenopus staufen2 is required for anterior endodermal organ formation.

Bilogan CK , Horb ME .

Abstract
Defining the regulatory molecular networks involved in patterning the developing anterior endoderm is essential to understand how the pancreas, liver, stomach, and duodenum are discretely specified from each other. In this study, we analyzed the expression and function of the double-stranded RNA-binding protein Staufen2 in Xenopus laevis endoderm. We found that staufen2 was broadly expressed within the developing endoderm beginning at gastrulation becoming localized to the anterior endoderm at later stages. Through morpholino-mediated knockdown, we demonstrate that Staufen2 function is required for proper formation of the stomach, liver, and pancreas. We define that its function is required during gastrulation for proper patterning of the dorsal-ventral axis and that it acts to regulate expression of BMP signaling components.

PubMed ID: 22162130
PMC ID: PMC3294188
Article link: Genesis
Grant support: [+]
Genes referenced: cer1 chrd.1 gata4 gcg hhex ilf2 ins onecut1 pdia2 pdx1 ptf1a sfrp5 sox17b.2 stau1 stau2 tspan8 ventx2.2
Morpholinos: stau2 MO1

Article Images: [+] show captions

	Figure 1. Staufen2 expression in the endoderm and isolated gut tissue. a: Transverse section through embryo at NF10 showing punctate expression of staufen2 throughout the mesendodermal precursor cells (arrowheads) as well as in the dorsal marginal zone (DMZ). b: Transverse section through embryo at NF15 showing Staufen2 expression within the archenteron roof (Ac) endoderm (arrowheads). Expression is also detected in the mesoderm (Mes). c: NF35 embryos showing Staufen2 expression throughout the head, in the spinal cord and in the dorsal (dp) and ventral (vp) pancreatic buds. d: Sagittal section through Staufen2 expressing region of NF35 embryo showing broad expression in the endoderm (en) and in the spinal cord (sc). e,f: Whole guts from NF40 and NF44 tadpole, expression was found throughout the pancreas, liver and stomach, becoming slightly stronger at later stages. St, stomach; L, liver; dp, dorsal pancreas; vp, ventral pancreas. Reproduced with permission of the Publisher, John Wiley & Sons.
	Figure 2. Staufen2 is required for anterior endodermal organ formation. a,b:Insulin expression at NF44 is almost completely abolished (75%, n = 16). c,d:Glucagon expression at NF44 is almost entirely lost in the stomach and pancreas (67%, n = 6). e,f: Expression of PDIp is almost completely lost in Staufen2 knockdown animals at NF44 (75%, n = 12). g,h:tm4sf3 gene expression is lost at NF44 (78%, n = 9). i,j: Expression of hex is lost at NF44 (100%, n = 11). k,l: Real-time PCR analysis of endocrine and exocrine markers in Staufen2 knockdown embryos for amylase in control and Staufen2 knockdown whole guts at NF44 and insulin expression in whole embryos at NF35. Each bar is an average of eight individual whole tadpoles. Amylase expression is almost lost, and insulin expression is decreased by more than twofold. m:Flag-Staufen2 mRNA rescues the morpholino knockdown phenotype. m: Control NF45 whole gut stained for ptf1a mRNA showing expression in the pancreas. n: Whole gut from embryo injected with 40 ng of Staufen2 morpholino. Ptf1a expression is almost completely lost in injected embryos (n = 20). o: Isolated whole gut stained for ptf1a expression from embryo injected with 40 ng of Staufen2 morpholino and 2,000 pg of flag-Staufen2 mRNA. Ptf1a expression was restored in 31/45 embryos. p: In vitro transcription translation of staufen2 mRNA with either the morpholino (MO) or the mismatch morpholino (MM). Reproduced with permission of the Publisher, John Wiley & Sons.
	Figure 3. Staufen2 is required for the patterning of the developing anterior endoderm. a,b:Pdx1 expression by whole mount in situ hybridization in control and Staufen2-MO-injected embryos at NF35, expression of pdx1 is lost in the developing stomach and pancreatic anlagen (83%, n = 12). c,d: NF35 embryo, expression of ptf1a is almost completely lost in the developing dorsal and ventral pancreatic bud (80%, n = 10).
	Figure 4. Patterning of anterior endoderm is perturbed in Staufen2 morpholino injected embryos. Sagittal sections through the anterior endoderm of NF35 control and Staufen2 morpholino injected embryos. a,b:Insulin expression is almost completely abolished (93%, n = 14). Arrowhead in B points to the small remaining insulin expression in the dorsal pancreas. c,d: Expression of PDIp is lost in the future dorsal and ventral anlagen (100%, n = 6). e,f: Expression of frp5 is almost completely lost (100%, n = 7). g,h: Expression of hnf6 is lost (63%, n = 8). dp, dorsal pancreas; vp, ventral pancreas; st/du, stomach/duodenum; li, liver. Reproduced with permission of the Publisher, John Wiley & Sons.
	Figure 5. Staufen2 is required for the earliest stages of endoderm regionalization. a: Transverse section through the embryo at NF18. a,b:Sox17β expression by whole mount in situ hybridization in control and Staufen2-MO-injected embryos, expression of sox17β remains unchanged throughout the endoderm (80%, n = 20). c,d: Expression of gata4 is lost in the anterior endoderm (82%, n = 17). e,f: Expression of hnf6 is lost in the anterior endoderm (67%, n = 9). g,h: Expression of hex is almost completely lost (72%, n = 18). Brackets identify anterior endoderm. Reproduced with permission of the Publisher, John Wiley & Sons.
	Figure 6. Staufen2 regulates early endoderm patterning through the BMP pathway. a,b:Chd expression by whole mount in situ hybridization in control and Staufen2-MO-injected embryos at NF10 transverse section, expression of chd is lost in the dorsal half of the embryo (80%, n = 10). Arrows indicate dorsal lip. c,d:Vent2 expression is significantly up-regulated in the anterior half of the embryo at NF18, transverse section (75%, n = 12). Anterior is to the left and dorsal is up. e,f: Expression of cerberus is unaffected in NF10 embryos (100%, n = 10). Arrows indicate dorsal lip. Reproduced with permission of the Publisher, John Wiley & Sons.
	stau2 (staufen double-stranded RNA binding protein 2) gene expression in bissected Xenopus laevis embryo, NF stage 10, dorsal right. Key: endomesoderm, arrowheads; dorsal marginal zone =DMZ.
	stau2 (staufen double-stranded RNA binding protein 2) gene expression in transverse section of a Xenopus laevis embryo, NF stage 15, anterior left, dorsal up. Key: Ac= archenteron roof ; endoderm=arrowheads); Mes= mesoderm.
	stau2 (staufen double-stranded RNA binding protein 2) gene expression in Xenopus laevis embryo, NF stage 35 & 36, head region, lateral view, anterior left, dorsal up. Key: dp =dorsal pancreatic bud; vp= ventral pancreatic bud.
	tspan8 (tetraspanin 8) gene expression in an isolated gut tube of Xenopus laevis embryo, NF stage 44, with expression in stomach and bile duct (darker staining, towards bottom)
	hhex (hematopoietically expressed homeobox) gene expression in an isolated gut tube of Xenopus laevis embryo, NF stage 44, with expression in the liver ( upper out pocketing of foregut) and gall bladder (darker staining, below liver)

References [+] :

Allison, Two distinct Staufen isoforms in Xenopus are vegetally localized during oogenesis. 2004, Pubmed, Xenbase