Quantifying the relationship between cell proliferation and morphology during development of the face.

Morphogenesis requires highly coordinated, complex interactions between cellular processes: proliferation, migration, and apoptosis, along with physical tissue interactions. How these cellular and tissue dynamics drive morphogenesis remains elusive. Three dimensional (3D) microscopic imaging poses great promise, and generates elegant images. However, generating even moderate through-put quantified images is challenging for many reasons. As a result, the association between morphogenesis and cellular processes in 3D developing tissues has not been fully explored. To address this critical gap, we have developed an imaging and image analysis pipeline to enable 3D quantification of cellular dynamics along with 3D morphology for the same individual embryo. Specifically, we focus on how 3D distribution of proliferation relates to morphogenesis during mouse facial development. Our method involves imaging with light-sheet microscopy, automated segmentation of cells and tissues using machine learning-based tools, and quantification of external morphology via geometric morphometrics. Applying this framework, we show that changes in proliferation are tightly correlated to changes in morphology over the course of facial morphogenesis. These analyses illustrate the potential of this pipeline to investigate mechanistic relationships between cellular dynamics and morphogenesis during embryonic development.

Transcriptome analysis of FOXO-dependent hypoxia gene expression identifies Hipk as a regulator of low oxygen tolerance in Drosophila.

When exposed to low oxygen or hypoxia, animals must alter their metabolism and physiology to ensure proper cell-, tissue-, and whole-body level adaptations to their hypoxic environment. These alterations often involve changes in gene expression. While extensive work has emphasized the importance of the HIF-1 alpha transcription factor on controlling hypoxia gene expression, less is known about other transcriptional mechanisms. We previously identified the transcription factor FOXO as a regulator of hypoxia tolerance in Drosophila larvae and adults. Here, we use an RNA-sequencing approach to identify FOXO-dependent changes in gene expression that are associated with these tolerance effects. We found that hypoxia altered the expression of over 2,000 genes and that ∼40% of these gene expression changes required FOXO. We discovered that hypoxia exposure led to a FOXO-dependent increase in genes involved in cell signaling, such as kinases, GTPase regulators, and regulators of the Hippo/Yorkie pathway. Among these, we identified homeodomain-interacting protein kinase as being required for hypoxia survival. We also found that hypoxia suppresses the expression of genes involved in ribosome synthesis and egg production, and we showed that hypoxia suppresses tRNA synthesis and mRNA translation and reduces female fecundity. Among the downregulated genes, we discovered that FOXO was required for the suppression of many ribosomal protein genes and genes involved in oxidative phosphorylation, pointing to a role for FOXO in limiting energetically costly processes such as protein synthesis and mitochondrial activity upon hypoxic stress. This work uncovers a widespread role for FOXO in mediating hypoxia changes in gene expression.

Tolerance to Hypoxia Is Promoted by FOXO Regulation of the Innate Immunity Transcription Factor NF-κB/Relish in Drosophila.

Exposure of tissues and organs to low oxygen (hypoxia) occurs in both physiological and pathological conditions in animals. Under these conditions, organisms have to adapt their physiology to ensure proper functioning and survival. Here, we define a role for the transcription factor Forkhead Box-O (FOXO) as a mediator of hypoxia tolerance in Drosophila We find that upon hypoxia exposure, FOXO transcriptional activity is rapidly induced in both larvae and adults. Moreover, we see that foxo mutant animals show misregulated glucose metabolism in low oxygen and subsequently exhibit reduced hypoxia survival. We identify the innate immune transcription factor, NF-κB/Relish, as a key FOXO target in the control of hypoxia tolerance. We find that expression of Relish and its target genes is increased in a FOXO-dependent manner in hypoxia, and that relish mutant animals show reduced survival in hypoxia. Together, these data indicate that FOXO is a hypoxia-inducible factor that mediates tolerance to low oxygen by inducing immune-like responses.

TORC1 modulation in adipose tissue is required for organismal adaptation to hypoxia in Drosophila.

Animals often develop in environments where conditions such as food, oxygen and temperature fluctuate. The ability to adapt their metabolism to these fluctuations is important for normal development and viability. In most animals, low oxygen (hypoxia) is deleterious. However some animals can alter their physiology to tolerate hypoxia. Here we show that TORC1 modulation in adipose tissue is required for organismal adaptation to hypoxia in Drosophila. We find that hypoxia rapidly suppresses TORC1 signaling in Drosophila larvae via TSC-mediated inhibition of Rheb. We show that this hypoxia-mediated inhibition of TORC1 specifically in the larval fat body is essential for viability. Moreover, we find that these effects of TORC1 inhibition on hypoxia tolerance are mediated through remodeling of fat body lipid storage. These studies identify the larval adipose tissue as a key hypoxia-sensing tissue that coordinates whole-body development and survival to changes in environmental oxygen by modulating TORC1 and lipid metabolism.