2025 Theses Doctoral

Regulation of hlh-2 in the C. elegans proximal somatic gonad

Kim, Jee Hun

Cell fate specification is a crucial mechanism for generating different cell types in complex multicellular organisms. In the larval somatic gonad of C. elegans hermaphrodites, the anchor cell (AC) is a terminally differentiated cell that coordinates uterine and vulval development.

Four cells in the proximal lineage of Z1 and Z4 precursor cells, Z1.ppa, Z1.ppp, Z4.aaa, and Z4.aap, are born with the competence for AC fate. The two distal cells, the β cells (Z1.ppa and Z4.aap), invariably assume the ventral uterine precursor (VU) fate. The two proximal cells, the α cells (Z1.ppp and Z4.aaa), undergo the AC/VU decision mechanism to determine which one becomes the AC and the other the third VU. The E protein HLH-2 plays key and sequential roles in this process. First, HLH-2 endows the α and β cells with the potential for AC fate.

Second, it orchestrates the decision mechanism that determines which of the α cells become the AC and the third VU. HLH-2 is required for the expression of the Notch gene lin-12 and its ligand lag-2/DSL, and the interaction of LIN-12 and LAG-2 amplifies lin-12 in one α cell that becomes the VU whereas the other α cell becomes the AC. In the VUs, HLH-2 is degraded post-translationally by a ubiquitination-dependent mechanism, whereas it is maintained in the AC and required for AC functions.

I first tested the hypothesis that lin-12 regulates degradation of HLH-2. I found that in a lin-12(0) background, in which the AC/VU decision does not occur and both α cells become ACs, HLH-2 expression is greater in the ACs than in the wild type AC, and greater in lin-12(0) β cells compared to wild type β cells. My results suggest that lin-12 contributes to degradation of HLH-2 in all four α and β cells.

I then focused on hlh-2 transcription in the proximal gonad. Previous work had established that HLH-2 begins to be expressed in the parents of α and β cells, Z1.pp and Z4.aa, and that which parent cell visibly expresses HLH-2 first is predictive of the outcome of the AC/VU decision; the α cell daughter of the first parent cell to express HLH-2 is biased towards VU fate. Therefore, the initial activation of hlh-2 transcription early on in the parents of α and β cells has crucial downstream implications for the AC/VU outcome.

I identified early activation elements (EAEs) required for initial transcription of hlh-2 and mutated them in the endogenous gene, and showed that it disrupts initial HLH-2 expression in α and β cells and their parents and induces phenotypes consistent with defective AC function. I also showed that similar sequence elements previously identified in another gene, nhr-67, which is also expressed in α and β cells and plays a role in the AC/VU decision, is required for transcription of nhr-67 in α and β cells and that mutation of those sequences in endogenous contexts disrupts its initial expression in α and β cells and their parents. I also performed an RNAi screen and identified several candidate transcription factors that may regulate the initial transcription of both hlh-2 and nhr-67 in the proximal gonad.

Hlh-2 transcription, which occurs in α and β cells initially, is diminished in β cells and maintained in the α cells over time. I tested the hypothesis from earlier studies that the difference between α and β cells in part reflects activity of the Wnt/β-catenin asymmetry pathway. I found that endogenous POP-1 is present at asymmetric levels in α and β cell nuclei and that depletion of its regulator LIT-1, which abolishes the wild type asymmetric pattern of POP-1 accumulation, results in maintenance of hlh-2 transcription and elevated HLH-2 expression in the β cells while not transforming them to AC fate. Finally, I identified regions in hlh-2prox that appear to regulate the continued maintenance of hlh-2 transcription after initial expression and lsy-12 to likely be involved in that role. Thus, my experiments provide greater context into the regulation of hlh-2 transcription in the proximal somatic gonad from initial onset in the parents of α and β cells to later patterning in α and β cells.