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In Development this week (Vol. 143, Issue 23)

Posted by , on 29 November 2016

Here are the highlights from the current issue of Development:


Syndecan 4 lets lymphatic endothelial cells go with the flow

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Fluid flow is known to play a role in the development and remodelling of both blood and lymphatic vessels. But how is fluid flow sensed and transduced into a response? Here, Michael Simons and colleagues identify a role for syndecan 4 (SDC4) in regulating flow-induced remodelling of the lymphatic vasculature in mice (p. 4441). They first show that Sdc4/ mice exhibit lymphatic vessel remodelling defects during the late stages of embryonic development. Notably, the alignment of valve-forming lymphatic endothelial cells (LECs), and hence valve formation, is perturbed in these mutants. The authors note that these defects are similar to those seen in mice mutant for Pecam1, which encodes a known flow-sensing molecule, but that Sdc4/; Pecam1/ double knockouts exhibit a more severe phenotype, suggesting that SDC4 and PECAM1 act via distinct pathways. Following on from this, the researchers demonstrate that SDC4 acts by regulating the planar cell polarity protein VANGL2; SDC4knockdown LECs express increased levels of VANGL2 in response to flow and fail to align under flow, whereas the reduction of VANGL2 levels in these cells restores flow-induced alignment. Together, these findings uncover new regulators of flow-mediated remodelling in the lymphatic vasculature.


Optimized inducible gene knockdown and knockout in hPSCs

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Human pluripotent stem cells (hPSCs) are emerging as an attractive model for studying human development and disease. However, functional studies of these cells are limited due to a lack of efficient methods for manipulating their gene expression. Here, Alessandro Bertero and co-workers devise platforms that allow for the inducible knockdown or knockout of specific genes in hPSCs and their derivatives (p. 4405). They first validate the ROSA26 and AAVS1 loci as genomic safe harbours that can be engineered in hPSCs to support stable transgene expression in a large panel of mature cells obtained from hPSCs. The authors then develop single-step optimized inducible knockdown (sOPTiKD) – an inducible shRNA-mediated approach for gene knockdown. This method allows for strong inducible expression of shRNAs, resulting in efficient gene knockdown even following hPSC differentiation. It also uses an optimized tetracycline-responsive repressor protein that eliminates leaky shRNA expression. Importantly, the authors show that this method can be used to knock down individual and multiple genes to study developmental mechanisms. They also develop a conditional knockout system based on CRISPR/Cas9 technology, named single-step optimized inducible knockout (sOPTiKO), and show that gene knockout using this system is possible in hPSCs and mature cell types. Given their robustness, high efficiency and scalability, these platforms promise to be valuable tools for the field.


Transposing from ligament to bone

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Phenotypic variation among mutant animals is common, with some mutants displaying dramatic phenotypes while their genetically similar siblings seem less affected. Why is this? Here, on p. 4430, Charles Kimmel and colleagues reveal that phenotypic variation, in the case of zebrafish mef2cab1086mutants, can be caused by a fate-switching event during development. Zebrafish mef2cab1086 mutants express a truncated form of Mef2c – a protein involved in skeletal development – and are known to develop variable ectopic bones in their heads. The researchers now reveal that these bones arise due to a fate-switching event during development, such that cells that are normally destined to be ligament variably turn into bone. Selective breeding demonstrates that the penetrance of the bone phenotype is heritable. The authors further show that the mef2cab1086 transcript is differentially expressed in low and high penetrance strains. Finally, they report that a transposon that resides upstream of the mef2calocus exhibits differential levels of DNA methylation; in high penetrance strains, which express high levels of the mef2cab1086 transcript, DNA methylation of the transposon is significantly reduced. These findings lead the authors to propose that variable epigenetic silencing of transposons underlies the variable mef2cab1086 phenotypes and could explain other cases of phenotypic variability.


Bringing in fresh blood: SOX7, RUNX1, AP-1 and TEAD4

Despite being the focus of intense research in recent years, the precise mechanisms that regulate the development of haematopoietic stem and progenitor cells (HSPCs), which give rise to all differentiated blood cells, remain unclear. In particular, it is not clear how various transcription factors function together to drive the emergence of HSPCs from haemogenic endothelium (HE) during development. In this issue, two papers attempt to tackle this problem.

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In the first paper (p. 4341), Valerie Kouskoff and co-workers examine how SOX7 and RUNX1 regulate haemogenic fate in the yolk sac of mouse embryos. These two factors are thought to play opposing roles: RUNX1 acts as a master regulator of endothelial-to-haemogenic transition (EHT) while SOX7 downregulation is needed for this event. Now, the authors report that, when overexpressed in ESC-derived HE, SOX7 inhibits the expression of RUNX1 target genes but has no effect on the expression of RUNX1 itself. They further reveal that SOX7 and RUNX1 are co-expressed in the yolk sac and dorsal aorta HE of mouse embryos and, importantly, can physically interact with each other via their respective HMG and RUNT domains. This interaction, the authors report, inhibits the transcriptional activity of RUNX1; the binding of SOX7 to RUNX1 prevents RUNX1 from interacting with its co-factor CBFβ and with its target DNA sites. Together, these findings highlight how direct protein-protein interactions between endothelial and haematopoietic transcription factors can regulate cell differentiation programmes during development.


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In a second paper (p. 4324), Nadine Obier, Constanze Bonifer and colleagues investigate how AP-1 transcription factors regulate cell fate during the differentiation of mouse embryonic stem cells (ESCs) into haematopoietic cells. They demonstrate that the global inhibition of AP-1 factors (using inducible overexpression of a dominant-negative FOS peptide) affects various stages of ESC differentiation, as cells transition from haemangioblasts (HB) into haemogenic endothelium (HE) and haematopoietic cells. In particular, inhibition at the HB stage enhances cell proliferation and affects the balance between smooth muscle and blood cells, shifting cells towards a blood cell fate. Finally, the authors reveal that AP-1 factors bind to target genes involved in vasculogenesis; these target sites colocalize with binding motifs for TEAD transcription factors, and the authors further show that AP-1 factors are required for the de novo binding of TEAD4 to these genes. In summary, these results suggest that cis-regulatory elements that bind both AP-1 and TEAD4 act as ‘hubs’ that integrate multiple signals to regulate specific gene expression programmes during haematopoiesis.


CHK2 mediates DNA damage in adult stem cells

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Adult stem cells are often exposed to genotoxic stress, whether directly from the environment or from within their own stem cell niche. DNA damage accumulates in the stem cells of aged tissues and has been proposed to accelerate both cellular aging and cancer formation, yet the mechanism through which this occurs is not well understood. Now, on p. 4312, Ting Xie and colleagues investigate this issue and demonstrate that DNA damage disrupts germline stem cell (GSC) self-renewal and lineage differentiation in a checkpoint kinase 2 (CHK2)-dependent manner. The authors use an inducible system to generate widespread double-stranded breaks (DSBs) in the GSCs of the Drosophila ovary. These DSBs resolve over time but leave the tissue with significantly fewer GSCs. By contrast, the number of GSC daughter cells initially increases then remains constant, suggesting that differentiation is blocked. The authors go on to identify a role for CHK2, showing how the induction of DSBs in flies lacking CHK2 is sufficient to prevent damage-induced GSC loss. Finally, the authors provide some evidence to suggest that the loss of GSCs may be partly due to reduced BMP signalling and cell adhesion. This study offers insight into how DNA damage might affect stem cell-based tissue regeneration and provides a mechanistic target – CHK2 – for further investigation.




An interview with David McClay

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David McClay is the Arthur S. Pearse Professor of Biology at Trinity College of Arts and Sciences, Duke University, North Carolina. His lab works on the transcriptional control of morphogenesis in the sea urchin embryo. We caught up with David at the 2016 Society for Developmental Biology – International Society of Differentiation joint meeting in Boston, where he received the Lifetime Achievement Award. Read the Spotlight article on p. 4289.


A common framework for EMT and collective cell migration

Fig. 2.It has long been considered that epithelial cells either migrate collectively as epithelial cells, or undergo an epithelial-to-mesenchymal transition and migrate as individual mesenchymal cells. Here, Kyra Campbell and Jordi Casanova hypothesise that such migratory behaviours do not fit into alternative and mutually exclusive categories. Rather, they propose that these categories can be viewed as the most extreme cases of a general continuum of morphological variety. See the Hypothesis article on p. 4291.


Cycling through developmental decisions: how cell cycle dynamics control pluripotency, differentiation and reprogramming

Fig. 1.A strong connection exists between the cell cycle and cell fate decisions in a wide-range of developmental contexts. Terminal differentiation is often associated with cell cycle exit, whereas cell fate switches are frequently linked to cell cycle transitions in dividing cells. In recent years, progress to address the connection between cell fate and the cell cycle has been made in pluripotent stem cells, in which the transition through mitosis and G1 phase is crucial for establishing a window of opportunity for pluripotency exit and the initiation of differentiation. Here, Abdenour Soufi and Stephen Dalton summarize recent findings in this area and place them in a broader context that has implications for a wide range of developmental scenarios. See the Review on p. 4301.



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