Student project by Amy French

Who Flipped the Switch?

Student project by Amy French, amymkfrench@yahoo.com

Purpose: This is an informational student project for Biology 475, a neurobiology course given by Dr. Grant Mastick at the University of Nevada , Reno . Link to course: http://med.unr.edu/homepage/gmastick/BIO475page/index.html.

The goal of this webpage is to explain the primary research paper “Churchill, a Zinc Finger Transcriptional Activator, Regulates the Transition Between Gastrulation and Neurulation.” Sheng, G., Dos Reis, M., and Stern, C.D. 2004. Cell: 115(5):603-613., as it relates to the specific field of neural induction in neurobiology.

Introduction:

One of the most engaging questions to biologists in all fields is how embryos actually make the switch from general tissue formation to neurulation in early development. At this point in development, the higher centers of organization-- namely the brain-- are not present. We are in fact talking about their initial formation, so they clearly cannot be influential during this stage. It is known that the organizer complex secreted proteins which led to neural induction and that entire process is time-sensitive. The initial model was that the neural state was a default state inhibited by the secretion of bone morphogenetic proteins (BMPs) and induced by the secretion of BMP antagonists, but it was merely a skeleton model, missing most of the details which we know must occur in elaborate biological processes. Additionally, one of the practical problems with neural induction is that several of the same proteins, but particularly the FGFs (fibroblast growth factors), are present in the two opposing stages of gastrulation and neurulation. The question became: what dictates the switch?

Researchers Sheng et. al have identified one protein, a zinc finger protein which they named Churchill (ChCh), that could possibly act as a molecular switch in the transition from gastrulation to neurulation in the constant presence of FGFs by carrying out multiple roles within the cell

Figure 1: The pathway from gastrulation to neurulation involving the Churchill crossroad.

Experimental Systems:

many systems used, one system best for visualization of results

cultured frog and chick embryos electroporated and injected with fluorescently tagged molecules, localization of proteins observed through antiflourescein immunostaining, in-situ hybridization, and time lapse photography with a fluorescence-dissecting microscope

genetic and biochemical approaches

transplants, animal cap assays, DNA cloning and PCR reactions, immunoblotting, electroporation and flourescence

>>researchers were able to do very specific and functional tests for the protein, but their systems were complex and required multiple tests

>>additionally, although the researchers attained very strong results for their populations, it may have been useful to increase the population size and see the effects

Figure 2: Main experimental techniques utilized to deduce the model for Churchill function.

Results:

Several key experiments (subsequently outlined) were involved in determining the function of ChCh after its initial discovery within the organizer complex.

Experiment 1.

analysis of structure of ChCh protein to determine that it was a zinc finger protein that had a functionally different domain due to the spacing/arrangement of its amino acids
PCR reactions and database searches revealed homologs of ChCh in several species with distinct domain conserved in all species

Figure 3: A) Homologous domains of ChCh proteins in several species. B) Percent identities between the amino acid sequences of ChCH in different species. Extracted from source: Sheng et. al 2004. Cell 115 (5).

Experiment 2.

in-situ hybridization of normal chick embryos showed that ChCh was present in the right region, where there are cells present which are destined to become/do become the neural plate

Experiment 3.

quail nodes were again grafted into chick embryos, and ChCh assays were performed at certain time intervals to determine when it was its expression was induced
found that it was induced in stages 4-5 (stages are denoted by hours at which events take place)

Experiment 4.

to determine which proteins already present were affecting ChCh induction, heparin coated beads with control complexes and several organizer-secreted proteins including FGFs, Noggin, Chordin, HGF/SF, and Cerberus were injected into chick embryos along with BMP4
ChCh’s expression pattern was observed in conjunction with these other proteins
found that FGFs caused induction even in the presence of high levels of BMP, indicating ChCh was induced by FGF signaling but BMP-signaling independent

Experiment 5.

to determine active site and localization of the protein, the fluorescently tagged ChCh mRNA was injected into frog embryos in the blastula stage of development

-stages 1-8 it was in the cytoplasm, but at stage 8 ½, fully functional and localized to nucleus

-earlier analysis had shown that ChCh had no NLS signal in its amino acid sequence so a transport protein was also required

-ChCh was a fully functional protein at this stage so it was possible for it to be a DNA binding protein and act as transcriptional repressor or activator

Figure 4: Flourescently tagged mRNA in frog embryos localized in cytoplasm (in A), and nucleus (in B) at different stages of development. Extracted from source: Sheng et. al 2004. Cell 115 (5).

Experiment 6.

misexpressed ChCh in stages 2-4, determined that it could have been influencing FGF signaling (seen in two opposing stages of gastrulation and neurulation) because results were similar to those when FGF signaling was directly perturbed

-FGF signaling known to be responsible for mesoderm formation by activating XBra, a mesodermal marker

-FGF signaling also known to influence early neural induction by activating ERN1 and SOX1, pre-neural genes

Experiment 7.

to observe effects of ChCh on XBra (Brachyury), mesodermal marker, ChCh was fused to activator domain, VP16 activator, and repressor domain, engrailed repressor EnR
animal cap assays with these constructs were examined, found that repressor complex had no effect, but activator complex did
led to hypothesis of ChCh as a primary messenger activating a secondary messenger which caused direct repression of XBra

Experiment 8.

Sip1 (Smad-interacting protein-1) previously identified as a direct repressor of XBra so Sip1 thought to be possible target of ChCh
DNA cloning techniques found optimal binding site on Sip1 codons for ChCh

Experiment 9.

when binding site on ChCh for Sip1 expression was blocked with fluorescently labeled morpholino oligonucleotides (MO) in stage 4 embryos or when ChCh was misexpressed (in stage 3+) cells, gave three results

in stage 4

>Sip1 was not expressed

>neurulation did not occur or was defective

in stage 3+

>cells failed to ingress to form mesoderm

researchers were able to conclude that Sip1 was indeed a target of ChCh, and that Sip1 was directly repressing XBra, which functions in regulating formation of the mesoderm

Experiment 10.

injection of Sip1 rescued neurulation—showed interactive nature of ChCh and Sip1 proteins because Sip1 is not present in-vivo without expression of ChCh (figures from paper?)

Conclusions:

ChCh has diverse functions in the embryo

-ChCh is expressed regardless of BMP signaling, allowing it to activate Sip1

-the activation of Sip1 by ChCh acts to inhibit further ingression of cells into the mesoderm by inhibiting XBra, essentially leaving these cells available for formation of the neural tube

-Sip1 has also been identified in an interaction with Smad1 when Smad1 is phosphorylated, and because Smad1 is responsible for the activation of BMP, it is possible that Sip1 could be a sensor for BMP levels within the embryo, allowing it to de-sensitize itself to BMP signaling and therefore leading more directly to neural induction

Figure 5. The function of Churchill in gastrulation and neurulation. Modified from source: Sheng et. al 2004. Cell 115 (5).

Significance:

The researchers identified at least one step in the very complex pathway of neurulation, that of the switch from gastrulation to neurulation. Their discovery sheds light on the possibility the embryo can effectively recycle proteins, that it can undergo a very complex process but maintain some sort of simplicity and continuity by having different functions for multiple copies of a single molecule, most notably the FGFs, which are present in gastrulation and neurulation. Churchill was shown to directly influence gastrulation and probably indirectly influences neurulation by providing a sensor for BMP levels. In the larger context, the researchers have identified that the neural state is indeed induced, it is not the default state. The formation of mesoderm must be closely regulated or there are no cells available to form neural tissue. It is amazing to look at the body’s most complex system in its most rudimentary form and witness how the protein signaling pathways converge in the right time and place to form what will become the control center of the body.

Future Directions:

Researchers will probably look for a co-factor that acts together with Churchill, because without a NLS signal it cannot get to the nucleus-- its site of action-- by itself.
When researchers are examining very early problems with neural development, they now have a starting point for investigation because a critical point in development has been identified.

References:

Primary research article: "Churchill, a Zinc Finger Transciptional Activator, Regulates the Transition between Gastrulation and Neurulation>" Sheng, G, Dos Reis, M., Stern, C., 2003. Cell 115: 603-613. This article was focused on determining which protein is responsible for the transition from gastrulation to neurulation when many of the same proteins are present at each stage. S0092-8674(03)00927-9