HPTE-Induced Embryonic Thymocyte Death and Alteration of Differentiation Is Not Rescued by ERα or GPER Inhibition but Is Exacerbated by Concurrent TCR Signaling.

Organochlorine pesticides, such as DDT, methoxychlor, and their metabolites, have been characterized as endocrine disrupting chemicals (EDCs); suggesting that their modes of action involve interaction with or abrogation of endogenous endocrine function. This study examined whether embryonic thymocyte death and alteration of differentiation induced by the primary metabolite of methoxychlor, HPTE, rely upon estrogen receptor binding and concurrent T cell receptor signaling. Estrogen receptor inhibition of ERα or GPER did not rescue embryonic thymocyte death induced by HPTE or the model estrogen diethylstilbestrol (DES). Moreover, adverse effects induced by HPTE or DES were worsened by concurrent TCR and CD2 differentiation signaling, compared with EDC exposure post-signaling. Together, these data suggest that HPTE- and DES-induced adverse effects on embryonic thymocytes do not rely solely on ER alpha or GPER but may require both. These results also provide evidence of a potential collaborative signaling mechanism between TCR and estrogen receptors to mediate adverse effects on embryonic thymocytes, as well as highlight a window of sensitivity that modulates EDC exposure severity.

Methoxychlor metabolite HPTE alters viability and differentiation of embryonic thymocytes from C57BL/6 mice.

Endocrine-disrupting chemicals (EDC) are widespread in the built and natural environments. Heightened public awareness of their potential danger has led to concern about whether EDC and their metabolites have significant negative biological effects. Studies have shown that EDC like DDT and other organochlorine pesticides, such as methoxychlor (MXC), have adverse effects on immune cells, but no studies have addressed the impact of HPTE, the primary metabolite of MXC. To elucidate the presence and significance of HPTE adverse effects, this study explored the impact of HPTE on a critical window and component of immune system development, embryonic T-cell development. Lesions at this phase of development can lead to lifelong immune dysfunction and increased incidence of immune disease, such as autoimmunity. Embry-onic thymocytes (GD 16-18) from C57BL/6 mice were subjected to an in vitro differentiation culture that mimicked early steps in thymocyte development in the presence of 0.005, 0.05, 0.5, 5, or 50 µM HPTE, or a model endocrine disruptor, DES. The results indicated that compared to the vehicle control, HPTE- and DES-induced death of thymocytes. Annexin-V staining and Caspase 8, markers of programed cell death, revealed that the loss of cells was due at least in part to induction of apoptosis. Moreover, HPTE-induced cell death not only resulted in selective loss of double positive thymocytes, but also loss of developing CD4 intermediate cells (post-double positive partially differentiated thymocyte population). Phenotypic analysis of thymocyte maturation (T-cell receptor, TCR) and TCR ligation (CD5) surface markers revealed that surviving embryonic thymocytes expressed low levels of both. Taken together these data demonstrate that immature embryonic thymocytes are sensitive to HPTE exposure and that HPTE exposure targets thymocyte populations undergoing critical differentiation steps. These findings suggest HPTE may play a pivotal role in MXC exposure-induced immune dysfunction.

"Do I need to know this for the exam?" Using popular media, inquiry-based laboratories, and a community of scientific practice to motivate students to learn developmental biology.

One of the greatest challenges instructors face is getting students to connect with the subject in a manner that encourages them to learn. In this essay, we describe the redesign of our Developmental Biology course to foster a deeper connection between students and the field of developmental biology. In our approach, we created a community of scientific practice focused on the investigation of environmental impacts on embryonic development and informed by popular and scientific media, the students' own questions, and the instructor. Our goals were to engage students in meaningful ways with the material, to develop students' science process skills, and to enhance students' understanding of broad principles of developmental biology. Though significant challenges arose during implementation, assessments indicate using this approach to teach undergraduate developmental biology was successful.

Altered development of CD8+ T cell lineages in mice deficient for the Tec kinases Itk and Rlk.

Mutations affecting the Tec kinases Itk and Rlk decrease T cell receptor-induced Ca(2+) mobilization and Erk kinase activation and impair both positive and negative thymic selection. Itk(-/-) and Rlk(-/-)Itk(-/-) mice also have decreased CD4:8 T cell ratios, suggestive of altered CD4:8 lineage commitment. Nonetheless, we find that CD8 single-positive (SP) thymocytes and peripheral CD8(+) T cells in these mice do not resemble conventional CD8(+) T cells. Instead, these cells express memory markers, rapidly produce interferon-gamma, and can be selected on hematopoietically derived cells, similar to MHC class Ib-restricted "innate-type" lymphocytes. Itk deficiency also greatly increases the number of cells selected by MHC class Ib. Expression of a hypersensitive Erk2 mutant partially corrects the CD8(+) T cell phenotypes in Itk(-/-) mice, arguing that altered signaling permits development of this innate-type CD8(+) cell population. Our results suggest that Tec kinases differentially regulate development of conventional versus nonconventional lymphocytes.