Hemoglobin transition :(2)▶ Organ transformation in metamorphosis occurs through cell

replacement of programed cell death and proliferation▶ Metamorphic event triggered by thyroid hormone (TH), one

example being T3 ▶ After climax of metamorphosis liver morphology changes and

larval hemoglobin (HB) is replaced by new cells, adult HB▶ This happens in all vertebrates, in Xenopus it is TH regulated▶ Change is important in change of oxygen affinity in adults▶ TRα expressed in larval red blood cells (RBC)

Dual function model of thyroid hormone receptors: (1)▶ Two gene loci in vertebrates: TRα and TRβ; TRα

heterodimerizes with RXRα to help with gene regulation▶ In the absence of ligand, TRs represses TH response genes

required for metamorphosis and regulate timing of metamorphosis

▶ No T3 present causes larval growth, with T3 induces TH-response genes repressed by TRα causing metamorphosis

Research Question: What regulates HB transition?

▶ Complex with HB because new adult stem cells are created in order to make new adult HB

▶ Only receptors identified on larval RBC thus far have been TRα thus TRα may be the only receptor that is required in the transition of HB

Hypothesis: TRα is required for HB transition

Introduction

Role of TRα in the hemoglobin transitionduring metamorphosis in Xenopus tropicalis

J. Schoephoerster, D. BuchholzUniversity of Cincinnati, Cincinnati, OH

References Cited

▶ Larval HB decreases as individual approach metamorphosis▶ TRαKO animals HB switch occurs earlier when compared to WT

▶ Adult HB increases, as expected, as individual approaches metamorphosis

Gene expression of adult HB

Figure 2: TRα present at larval stage and onward, delays metamorphosis until T3 present. Low amount of changes during stage 45-54 when low T3. Metamorphosis climax stages 48-66(3)

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*

Figure 4: Columns represent relative expression of A1 in TRαKO and WT animals. Error bars represent SE+/-1 (N=3-8). Significance (p-value<0.05) found at stage 54 and 60/62 between TRαKO and WT.

Figure 1: With the presence of T3 larval RBC go through programed death. T3 causes liver to create new adult RBC

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* Gene expression of larval HB

Figure 3: Columns represent relative expression of L5 in TRαKO and WT animals. Error bars represent SE+/-1 (N=3-8). Significance (p-value<0.05) found at stage 54 and 58 between TRαKO and WT.

Methods and Materials

Figure 5: TRαKO animals created to test hypothesis. Livers harvested from TRαKO and WT to extract RNA and synthesis cDNA to conduct a qPCR of larval HB (L5) and adult HB (A1). ▶ Support for this research was from NSF REU Grant #1262863

Acknowledgements

1. Buchholz, Daniel R., Bindu D. Paul, Liezhen Fu, and Yun-Bo Shi. "Molecular and Developmental Analyses of Thyroid Hormone Receptor Function in Xenopus Laevis, the African Clawed Frog." General and Comparative Endocrinology 145.1 (2006): 1-19.

2. Nishikawa, A., and Hideo Hayashi. "T 3 -hydrocortisone Synergism on Adult-type Erythroblast Proliferation and T 3 -mediated Apoptosis of Larval-type Erythroblasts during Erythropoietic Conversion in Xenopus Laevis." Histochemistry and Cell Biology 111.4 (1999): 325-34.

3. Wen, Luan, and Yun-Bo Shi. "Regulation of Growth Rate and Developmental Timing by Xenopus Thyroid Hormone Receptor α." Development, Growth & Differentiation Develop. Growth Differ. 58.1 (2016): 106-15.

DiscussionHypothesis Reject

Data interpretation

Unexpected results TRα is not required but is somehow involved

Implications 1) TRβ partially compensates for KO of TRα2) TRβ not involved but derepression from KO of TRα is not sufficient for full adult HB switch

New knowledge New adult stem cells are created in order to make new adult HB

Future Studies Look at development of new adult stem cells, may involved TRβ, thus both receptors may be involved in switching of HB rather than just TRα which is known to be expressed in larval RBC

TRαKO have derepression of metamorphic genes, allowing metamorphosis to initiate (larval HB graph), but expression of adult HB is slowed showing lack of TH mediated induction

Conclusions

Results

Figure 5: Timeline of TRαKO and WT HB switching. TRαKO have larval RBC death earlier and take longer for adult RBC to be produced, when compared to WT switching of RBC.