The Interaction of GPC4 and 
FGF in the developing Xenopus forebrain
 

Student project:  Teresa Kegler, kegler@unr.nevada.edu.

Project Purpose: This is intended to summarize the research in the area of developmental biology pertaining specifically to the development of the forebrain 
based on the research article; “Glypican 4 modulates FGF signaling and 
regulates dorsoventral forebrain patterning in Xenopus embryos”. Development 130: 4919-4929.

My project is for the course BIOL 480, Development Biology, Dr. Grant Mastick, Biology Department, University of Nevada, Reno.

Introduction: 

The development of the forebrain derives from a sheet of neuroepithelial cells. Many different signals occur to differentiate the regions

of the forebrain.

A diagram of the developing brain

1. Ectodermal cells must obtain their neural identity. 2. Neural tissue located in the rostral part of the forebrain must obtain their anterior identity. 3. Regional patterning must occur in the rostral neural plate mentioned above.

 
 It has also been shown in experiments that Fgf signaling has a definite role in the patterning and polarization of the telecephalon 
of the forebrain and a partial role in dorso-ventral gene expression. 
 Furthermore, a dorso-ventral patterning must occur in the forebrain and Emx1, Emx2, and Nkx2.1 are important molecules in the 
signaling of the forebrain. These interactions are regulated by cell surface proteins. One type of cell surface proteins are called glypicans. 
They bind to Fgfs, Wnts, and Bmps on their heparin sulphate glycosaminoglycan side chains. The role of glypicans with the signaling of Fgfs, 
Wnts, and Bmps may have a major role in the patterning of the forebrain.

Experimental System: 
1. Make a morpholino oligonucleotide sequence to block the translation of the Gpc4 gene (Gpc4Mo) in Xenopus embryos to see the results 
of Gpc4 depleted embryos by using in situ hybridization. 
 
2. Rescue forebrain patterning by injecting mouse Gpc4 mRNA along with Gpc4Mo in Xenopus embryos. 
 
3. Immunoblotting was performed to find the levels of ERK protein kinases present in Gpc4Mo injected embryos.
 
4. SU5402 was added to non-injected embryos to find the results of blocking Fgf signaling by SU5402 interaction. 
 
Results:
When Gpc4Mo was injected into two cell blastula stage Xenopus embryos it was found that:
 
1. They contained a kinked axis and reduced dorsal fin and head structures, which caused them to be shorter than normal. 
 
 

This is the in situ hybridization results of the control injected embryos.

.	This is the result of Gpc4Mo injected embryos. Notice the overall decrease in length of the embryo.

 
 
 
 
 
 
 
 
 
 
 
 
2. The length of the notochord is reduced.
 
3. Anteroposterior axis elongation was affected during gastrulation.
 
4. Central nervous system structures are affected. Emx2 expression is reduced in the neural tube closure. 
 
5. They contained two to three-fold reduction levels of ERK protein kinases than regularly developing embryos. 
 
When Gpc4Mo injected Xenopus embryos were rescued by injecting mouse Gpc4 mRNA it was found that:
69% of the rescued embryos had normal Emx2 expression compared to 20% normality found in Gpc4Mo injected embryos.
 

Injection	Number of Embryos	Normal
CoMo (control)	14	100%
Gpc4Mo	15	20%
Gpc4Mo+Mouse Gpc4 mRNA	39	69%
CoMo+Mouse Gpc4 mRNA	17	95%

 
This table shows some of the results from the rescue experiment performed in this experiment. 
 
 
When SU5402 was injected into normal embryos it was found that:
  The expression of Emx2 was reduced, and had same phenotypic results as Gpc4Mo injected embryos.

Conclusions:
Injecting Gpc4Mo into two stage embryo:
 Gpc4 is required for the expression of Emx2 during early neural plate stages and onward through the development of the forebrain.
 
Rescuing Gpc4Mo embryos with mouse Gpc4 mRNA:
 Gpc4 function is required to regulate the expression of forebrain patterning genes. Gpc4 is also the enhancer for Fgf signaling, but is not 
essential for Fgf signaling to occur in the developing embryo. 
 
Levels of ERK protein kinases:
  The phosphorylation levels of ERK protein kinases indicate the level of intracellular signaling of Fgfs. When the embryos were injected with 
Gpc4Mo, the levels of ERK phosphorylation were compared to levels found in normal developing embryos. The reduced levels of phosphorylation 
in the embryos when injected with Gpc4Mo indicate that Gpc4 is a key factor in signaling with Fgf molecules to create a normal forebrain. 
 
SU5402 injections in normal embryos:
 The results from this experiment indicate that the blocking the Fgf signaling pathway by SU5402 in the embryos have the same affects as 
blocking the Gpc4 gene. Therefore, both of these components are important for normal forebrain development. And Gpc4 regulates Emx2 and, 
thus, regulates dorsal forebrain patterning by affecting Fgf signaling. 

Future Direction:
 Although the research from this primary article concludes with Gpc4 interacting with Fgf signals to create forebrain differentiation, 
the specific Fgf ligands involved in this process still need to be identified. The interaction of the specific Fgf ligands with Gpc4 will more clearly
explain the process of forebrain development
 
Health Implications:
 Mutations in Gpc4 and Gpc3 genes in human chromosomes are linked to the Simpson-Golabi-Behmel syndrome (SBGS). This is a 
syndrome characterized by pre- and postnatal overgrowth with a tendency for mental retardation. The conclusions from this experiment 
suggest that the characteristics of SBGS may be due to Gpc4 disruptions specifically during the neutralization portion of embryogenesis. 
 
References:
1. Galli, A., A. Roure, R. Zeller, and R. Dono. (2003). Glypican 4 modulates FGF signaling and regulates dorsoventral forebrain patterning in 
Xenopus embryos. Development, 130: 4919-4929.
 Annotation: This was the primary research article that investigates the roles of Gpc4 and Fgf signaling in forebrain development.
2. Rallu, M. JG. Corbin, G. Fishell. (2002). Parsing the prosencephalon. Nature Review Nueroscience 3:943-951.
 Annotation: This paper had the image from the introduction. 
3. Wison, S., and C Houart. (2004). Early steps in the development of the forebrain. Developmental Cell, 6: 167-181.
 Annotation: This paper reviewed the development of the early forebrain determined by the roles of Wnts, Bmps, and Fgfs.

This page was constructed by:  Teresa Kegler, kegler@unr.nevada.edu. 4/12/2004.