A most formidable arsenal: genetic technologies for building a better mouse.

The mouse is one of the most widely used model organisms for genetic study. The tools available to alter the mouse genome have developed over the preceding decades from forward screens to gene targeting in stem cells to the recent influx of CRISPR approaches. In this review, we first consider the history of mice in genetic study, the development of classic approaches to genome modification, and how such approaches have been used and improved in recent years. We then turn to the recent surge of nuclease-mediated techniques and how they are changing the field of mouse genetics. Finally, we survey common classes of alleles used in mice and discuss how they might be engineered using different methods.

FGF signaling regulates development by processes beyond canonical pathways.

FGFs are key developmental regulators that engage a signal transduction cascade through receptor tyrosine kinases, prominently engaging ERK1/2 but also other pathways. However, it remains unknown whether all FGF activities depend on this canonical signal transduction cascade. To address this question, we generated allelic series of knock-in Fgfr1 and Fgfr2 mouse strains, carrying point mutations that disrupt binding of signaling effectors, and a kinase dead allele of Fgfr2 that broadly phenocopies the null mutant. When interrogated in cranial neural crest cells, we identified discrete functions for signaling pathways in specific craniofacial contexts, but point mutations, even when combined, failed to recapitulate the single or double null mutant phenotypes. Furthermore, the signaling mutations abrogated established FGF-induced signal transduction pathways, yet FGF functions such as cell-matrix and cell-cell adhesion remained unaffected, though these activities did require FGFR kinase activity. Our studies establish combinatorial roles of Fgfr1 and Fgfr2 in development and uncouple novel FGFR kinase-dependent cell adhesion properties from canonical intracellular signaling.

Conditional fluorescent mouse translocation reporters for ERK1/2 and AKT signaling.

Understanding how cells activate intracellular signaling pathways in response to external signals, such as growth factors, is a longstanding goal of cell and developmental biology. Recently, live-cell signaling reporters have greatly expanded our understanding of signaling dynamics in response to wide-ranging stimuli and chemical or genetic perturbation, both ex vivo (cell lines) and in vivo (whole embryos or animals). Among the many varieties of reporter systems, translocation reporters that change sub-cellular localization in response to pathway activation have received considerable attention for their ease of use compared to FRET systems and favorable response times compared to transcriptional reporters. We reasoned that mouse reporter lines expressed in a conditional fashion would be a useful addition to the arsenal of mouse genetic tools, as such lines remain undeveloped despite widespread use of these sensors. We therefore created and validated two novel mouse reporter lines at the ROSA26 locus. One expresses an ERK1/2 pathway reporter and a nuclear marker from a single transcript, while the second additionally expresses an AKT reporter in order to simultaneously interrogate both pathways.

The Wnt1-Cre2 transgene is active in the male germline.

The Wnt1-Cre transgenic mouse line is widely used to express the CRE recombinase in neural crest lineages, but it overexpresses WNT1 itself, which can cause undesired phenotypes. To address this, we and others previously developed a Wnt1-Cre2 line based on the same regulatory elements as Wnt1-Cre but without ectopic Wnt1 expression. However, while Wnt1-Cre2 exhibits normal activity when transmitted from female mice, it exhibits unexpected activity in the male germline. The Wnt1-Cre2 transgene was previously mapped to the E2f1 locus. Several genes in this genomic region exhibit significant expression in spermatogonia or spermatocytes, suggesting that local regulatory elements may be driving ectopic transgene expression. The Wnt1-Cre2 line can therefore be used both as a neural crest specific and a general deleter, and care should be taken when setting up genetic crosses.

MAPK and PI3K signaling: At the crossroads of neural crest development.

Receptor tyrosine kinase-mediated growth factor signaling is essential for proper formation and development of the neural crest. The many ligands and receptors implicated in these processes signal through relatively few downstream pathways, frequently converging on the MAPK and PI3K pathways. Despite decades of study, there is still considerable uncertainty about where and when these signaling pathways are required and how they elicit particular responses. This review summarizes our current understanding of growth factor-induced MAPK and PI3K signaling in the neural crest.

Differential regulation of cranial and cardiac neural crest by serum response factor and its cofactors.

Serum response factor (SRF) is an essential transcription factor that influences many cellular processes including cell proliferation, migration, and differentiation. SRF directly regulates and is required for immediate early gene (IEG) and actin cytoskeleton-related gene expression. SRF coordinates these competing transcription programs through discrete sets of cofactors, the ternary complex factors (TCFs) and myocardin-related transcription factors (MRTFs). The relative contribution of these two programs to in vivo SRF activity and mutant phenotypes is not fully understood. To study how SRF utilizes its cofactors during development, we generated a knock-in SrfaI allele in mice harboring point mutations that disrupt SRF-MRTF-DNA complex formation but leave SRF-TCF activity unaffected. Homozygous SrfaI/aI mutants die at E10.5 with notable cardiovascular phenotypes, and neural crest conditional mutants succumb at birth to defects of the cardiac outflow tract but display none of the craniofacial phenotypes associated with complete loss of SRF in that lineage. Our studies further support an important role for MRTF mediating SRF function in cardiac neural crest and suggest new mechanisms by which SRF regulates transcription during development.

A Fateful Decision: Tgif1 and Cardiac Neural Crest Identity.

Neural crest cells have different developmental potencies at different levels along the body axis of the embryo. In this issue of Developmental Cell, Gandhi et al. identify transcription factors that define one subtype of neural crest, the cardiac crest, and demonstrate their ability to reprogram trunk into cardiac crest.

Function of ammonium transporter A in the initiation of culmination of development in Dictyostelium discoideum.

The histidine kinase DhkC controls a phosphorelay involved in regulating the slug versus culmination choice during the multicellular developmental program of Dictyostelium discoideum. When the relay is active, slug migration is favored due to the activation of a cyclic AMP (cAMP) phosphodiesterase and the resultant lowering of the intracellular and extracellular levels of cAMP. Ammonia signaling represents one input into the DhkC phosphorelay, and previous studies indicated that the ammonium transporter C inhibits the relay in response to low ammonia levels. Evidence is presented that another member of the family of ammonium transporters, AmtA, also regulates the slug/culmination choice. Under standard conditions of development, the wild-type strain requires a transitional period of 2 to 3 h to go from fingers to culminants, with some slugs forming and migrating briefly prior to culmination. In contrast, amtA null cells, like cells that lack DhkC, possessed a transitional period of only 1 to 2 h and rarely formed slugs. Disruption of amtA in an amtC null strain overcame the slugger phenotype of that strain and restored its ability to culminate. Strains lacking AmtA were insensitive to the ability of ammonia to promote and prolong slug migration. These findings lead to the proposal that AmtA functions in ammonia sensing as an activator of the DhkC phosphorelay in response to perceived high ammonia levels.