Conclusion
Looking to the future, elucidating the cellular and molecular changes
that occur between reproductively active and inactive neuroendocrine
tissues of resident and migrant birds respectively will require
additional approaches, such as localization of proteins or
ultrastructure imaging of the hypothalamus and pituitary. The observed
differentially expressed proteins involved in calcium ion channel
activity, synapse, neuron differentiation, dendrite formation, axon
guidance all raise a possibility of seasonal changes in the morphology
of nerve terminals in the hypothalamus or in endocrine secretory cells
in the pituitary. Cellular localization of specific proteins that play a
role in GnRH synthesis, stability, and release in residents, or that
play a role in migratory processes such as increased food intake or
synaptogenesis in migrants are needed and will provide added insight
into the seasonal regulation of key life history stages in birds.
Seasonal neurogenesis in the hypothalamus and other brain areas has been
reported in diverse species ranging from zebrafish to hamsters, sheep,
and birds (Sousa-Ferreira, de Almeida, & Cavadas 2014; Pozner,
Vistoropsky, Moaraf, Heiblum, & Barnea, 2018). By considering the
potential role of proteins in neurogenesis, synapse formation, and cell
proliferation, we found a few key molecules such as PURA, MAP2K1,
DPYSLL, PAFAH1B1, STONIN 2, and Hypoxanthine-guanine
phosporibosyltransferase that are either directly or indirectly involved
in neuron, dendrite morphology and synapse formation. The proteomics
analysis of the brain regions controlling the neuroendocrine system
could be helpful in introducing neuro-proteomics as a new area of study,
enabling the improved understanding of the molecular mechanisms and
correlates associated with the underlying differential brain response
depending on the time of the day, duration of the day length or other
environmental factors.
Declaration: Authors have no conflict of interest.
Data Accessibility: The proteomics data are available to the
Proteome Xchange Consortium via the MassIVE partner repository with the
dataset identifier ftp://MSV000085662@massive.ucsd.edu and
password: Junco. Data are available in the Dryad Digital Repository:
 https://doi.org/10.5061/dryad.f1vhhmgv4
Authors’ contribution : DS and EDK conceived the idea. DS and
AMF collected field samples. DS carried out the data collection and
analysis. TS performed the hydrogen stable isotope analyses and
geographic assignments. DS wrote the manuscript with the help of all
authors. All authors approved the final draft.
Funding. The funds were provided by Indiana University Grand
Challenge Initiative, Prepared for Environmental Change to EDK and TMS,
National Science Foundation (IOS- 1856423) and, IU Faculty Research
Support Program, (FRSP, account number # 2224640) to AMF and EDK.
Acknowledgements: We thank Douglas B. Rusch and Ram Podicheti
from the Center for Genomics and Bioinformatics (CGB), IU Bloomington
for providing junco protein database generated from recently annotated
junco genome. We thank Jonathan C. Trinidad for performing proteome
sequencing and access to the Biological Mass Spectrometry Laboratory, IU
Bloomington. Mountain Lake Biological Station provided access to field
sites. We are grateful to Kathryn Evans for help with sample preparation
and Peter Sauer at the Indiana University Stable Isotope Research
Facility for assistance with isotopic analysis.
Figure 1. Schematic showing geographical distribution of
resident (J. h. carolinesis ; red circle, year-round) and migrant
(J. h. hyemalis ; orange as breeding area, pale blue as wintering
area; based on IUCN, 2019) dark-eyed juncos on the North America map
(a). The middle left panel showing the difference in the early breeding
resident juncos gonad development than migrants in early spring (b). The
middle right panel showing schematic for the isobaric tandem mass tag
(TMT) labeling of individual hypothalamic protein samples pooled to run
Liquid Chromatography with tandem mass spectrometry (LCMS/MS) (c). The
flow-chart outline steps of protein sequencing, differentially expressed
protein (DEPs) analysis, enriched gene ontology (GO) terms, and
significant kyoto encyclopedia of genes and genomes (KEGG) pathway
analysis (d).