Role of Oct4 in The Early Embryo Development Assignment

Assignment Task

Genetics of Development

We have learned that Oct4 regulates multiple developmental events during early embryogenesis. (A) What key developmental decisions require Oct4, and which fail in its absence? There is also experimental evidence that Oct4 function is required for definitive endoderm development. (B) What is the definitive endoderm, and what is its final fate in adult animals? (C) To study formation of definitive endoderm, which factor(s) (proteins) would you choose to distinguish definitive endoderm from other cell lineages, and why is your choice sufficient?

Oct4 is required for maintenance of the naive and primed stem cell states and for definitive endoderm development. (D) Please provide a mechanism supported by experimental evidence in the literature that explains how Oct4 can perform these different functions within a short developmental window. An important factor to consider is Oct4’s expression pattern throughout embryogenesis.

Interestingly, the experimental evidence regarding the role of Oct4 in definitive endoderm development is equivocal. Loss of Oct4 was shown to result in the upregulation of definitive endoderm markers during early embryogenesis, whereas other studies showed that Oct4 is required for definitive endoderm gene expression. Again, consider Oct4’s expression pattern throughout embryogenesis and how a factor that is no longer present can influence gene expression at a later time. (E) Please propose a model system and design an experiment to resolve this discrepancy. This will require you to interpret the outcome of your experiment in the context of these seemingly contradictory conclusions.

Reproductive System Development

A. Outline three experimental approaches (one must include an in vivo study) that you consider important in studying the differentiation of the human bi-potential gonad into an ovary or a testis and explain your rationale for each one.

B. State a hypothesis regarding the factors controlling gonadal differentiation, and design an experiment to test this hypothesis. Explain the methods you would use and the controls for your experiment. What results do you expect? Please include an explanation of why your experimental approach has the rigor to test the hypothesis.

Epigenetics

We recently discussed a paper by Velker et al. in which methylation of CpG islands in the promoter of the imprinted mouse gene Mest was reduced by superovulation when followed by embryo culture. Please answer the following questions within the page limit for each section:

(A) Please describe the cycle of DNA methylation of CpG islands during gametogenesis, early and late embryonic development, and specification of primordial germ cells. Specifically, when are methyl groups added to or removed from cytosines in most of the CpG sequences in gene promoters? Does the cycle differ for genes in maternal and paternal chromosomes, and if so, how?

(B) What is imprinting? Define a HDMI. How does the DNA methylation of an imprinted gene differ from that of other genes during the cycle you described in part (A)? Are CpG islands in the promoters of imprinted genes ever demethylated and remethylated? If so, when?

(C) In Velker et al., the authors showed that methylation of the Mest gene promoter on mouse maternal chromosome 7 was reduced by superovulation when followed by embryo culture. How do these results relate to concerns about individuals conceived by IVF, and how would a child or adult be affected by loss of maternal imprinting at the Mest gene? How do you think superovulation and embryo culture could lead to demethylation and expression of the maternal Mest gene?

Neural Tube Defects

A. Folic acid (FA) supplementation of pregnant mothers reduces the incidence of neural tube defects (NTDs) in their offspring by ~30%. NTDs can be modeled using mice with genetic mutations that cause defects. In the paper on neural tube defects caused by a Pax3 gene mutation (Sudiwala et al., 2019), we learned that the cranial neural tube fails to close in mice that are homozygous for the Pax3Sp2H mutation, causing exencephaly. In the mutant mice, neurons in the midbrain dorsal folds differentiated prematurely and cell division was reduced. However, folic acid (FA) promoted cell cycle progression in the dorsal folds, resulting in neural tube closure and rescuing the NTD phenotype.

B. Based on the known mechanisms by which folic acid is thought to regulate gene expression and other papers you should find), propose a specific hypothesis to explain how FA supplementation causes changes in gene transcription that promote cell cycle progression and growth in neural precursor cells of the midbrain dorsal folds in embryos of the Pax3 mutant mouse embryos. This will require some research. Please cite appropriate references, including some that were not assigned in Week.

Describe an experimental strategy to test your hypothesis and to identify the genes of which mRNA expression is upregulated or downregulated by folic acid in cells of the dorsal midbrain folds in the mutant mouse embryos. Include controls and identify the methods you would use, but don’t explain the methods in detail. 

C. Suggest one signaling pathway that you would expect to be upregulated or downregulated by folic acid (FA ) and propose a mechanism by which it would promote cell division and/or retard differentiation in neural precursor cells in the midbrain dorsal folds when the embryo is treated with FA. How would you determine the extent of upregulation of this pathway and identify the cells in which it is upregulated in the neural tube?

Teratology/Cranial neural crest cells and ethanol

This week, we read a review article by Kiecker on chick embryos as a model system for studying mechanisms of prenatal ethanol exposure effects on craniofacial development. Most of the following statements were made in this review, and are followed by the references in the review that he uses to back them up. If you need to check these references, you may, but it’s not necessary. You may accept the following conclusions as true when answering the first part of this question, which is an exercise in logic rather than evaluating the science of these references.

To fit these statements together to make a model (Part A), it may be helpful to make a drawing with arrows to indicate signaling interactions and changes in cell behavior or gene expression. This part should not be difficult!

1. (Sonic Hedgehog) is required for cranial neural crest cell (CNCC) survival. Without Shh, these cells undergo apoptosis (Ahlgren et al., 2002).

2. Chemotactic signal for CNCC cells (Tolosa et al., Eur. J. Cell Biol. 95:136-152, 2016). CNCC cells will migrate toward a source of Shh. If you cannot find this article and want it, I can send it to you. 

3. Anterior endoderm (AE, in the head) is required for the survival of CNCC cells in the chick embryo. If the AE is surgically removed, CNCC cells die by apoptosis and the lower jaw does not form (David et al., 2002).

4. Shh rescues NCC cells from apoptosis and restores normal patterning of cartilage to form the lower jaw in embryos that have had the AE removed (Couly et al., 2002).

5. Ethanol downregulates Shh, Ptc, and Gli genes in the head (Ahlgren et al., 2002).

6. Propose a model (hypothesis involving several steps) to explain how Shh from anterior endoderm regulates NCC cells and how ethanol interferes with this signaling. A drawing would help you to create this model and may be submitted with your written answer.

7. Propose two novel experiments to test your model that have not been done and reported in the literature. Within these experiments, you should propose both manipulations of embryonic tissues (e.g., ablation, transplantation, or cell culture), and assays involving molecular biology techniques, although these techniques could be used in the same or different experiments.

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