Soriano Lab - Research
Overview
Research in this laboratory is centered on the genetic analysis of mouse development, with a particular emphasis on genes implicated in signaling pathways. Our work covers three areas:
Ephrin signaling and boundary formation
Growth factor signaling
Growth factors are involved in controlling cell proliferation and survival, as well as migration along extracellular matrices or guidance by chemotactic or repulsive cues underlying normal development. Among the best understood growth factor regulated pathways are those mediated by receptor tyrosine kinases (RTKs). However, the relative role of individual signaling pathways activated in response to growth factors has not been fully elucidated in vivo. We have studied the receptors for platelet derived growth factors (PDGFs). Loss of function studies in the mouse have indicated a role for these factors in vascular smooth muscle cells, in the kidney, cranial and cardiac neural crest cells, and in somite patterning (Soriano, 1994; Soriano, 1997; Tallquist et al., 2000; Tallquist and Soriano, 2003).
To elucidate the importance of individual signaling pathways in mediating PDGF responses, we have generated allelic series at the PDGFRα and PDGFRβ loci in which docking sites for various effectors (PI3K, PLCγ, RasGAP, SHP-2, and Src) have been mutated. Loss of one or several docking sites leads to hypomorphic mutations of increasing severity (Heuchel et al., 1999; Tallquist et al., 2000; Klinghoffer et al., 2002, Tallquist et al., 2003). These results might help distinguish between various models for signaling modules in mediating growth factor responses. We have also studied phenotypic rescues resulting from kinase domain swaps between the two receptors (Klinghoffer et al., 2001). These results bear on the general question of the origin of receptor specificity. We have also examined the ability of two receptors of increasing divergence, Drosophila torso and the mouse FGF receptor 1, to complement PDGF signaling (Hamilton et al., 2003).
It has been known for many years that activation of growth factor signaling pathways leads to the expression of multiple Immediate Early Genes (IEGs), yet their role in specifying particular growth factor responses remained controversial as similar IEGs are often engaged following induction of multiple signaling pathways. We have used gene trap-coupled microarray analysis to identify targets of PDGF signaling, and their function in PDGF regulated processes (Chen et al., 2004). Mutations in these genes lead to a high frequency of phenotypes that affect the same cell types and processes as those controlled by PDGF signaling (Schmahl et al., 2007). These results suggest that these genes form a network that control specific processes downstream of PDGF signaling.
We are also dissecting the signaling pathway of fibroblast growth factor receptors 1 and 2 (Fgfr1/2), which are thought to signal primarily through the multidocking proteins FRS2/3. In contrast to FGFR1 null mutants, which have been shown to exhibit defects in gastrulation and somitogenesis, embryos in which the Frs2/3 binding site on Fgfr1 is deleted die during late embryogenesis and exhibit defects in neural tube closure and the development of the tail bud and pharyngeal arches (Hoch and Soriano, 2006). We are extending these studies by creating a more extensive allelic series of mutations at both receptor loci. We are also testing how these signaling pathways affect stem cell formation in the early embryo.
During development, cells are simultaneously exposed to multiple signals. We have studied how Smads, which are traditionally viewed as mediators of the TGFβ superfamily signaling pathway, act as convergence points for TGFβ and RTK signaling. Our efforts have focused on creating small mutations in the gene encoding Smad1, a mediator of BMP signaling (Aubin et al., 2004).
Ephrin signaling and boundary formation
Ephrin-B1 and ephrin-B2 are transmembrane ephrins that can activate both forward signaling to Eph receptors as well as reverse signaling through their cytoplasmic domain. We have investigated the function of both ephrins during mouse development. Complete ablation of the X-linked ephrin-B1 resulted in peri-natal lethality associated with a range of phenotypes, including defects in neural crest cells (NCC)-derived tissues, incomplete body wall closure and abnormal skeletal patterning. Conditional deletion of ephrin-B1 demonstrated that ephrin-B1 acts autonomously in NCCs, and controls their migration. These results demonstrate that ephrin-B1 acts both as a ligand and as a receptor in a tissue-specific manner during embryogenesis. (Davy et al., 2004). Current efforts are underway to identify critical effectors of ephrin-B1 forward and reverse signaling by making point mutations in the cytoplasmic domain.
We noticed that mutations in X-linked ephrin-B1 lead to similar phenotypes as in humans, causing Craniofrontonasal Syndrome (CFNS), a disease which affects female patients more severely than males. We have found that ephrin-B1+/- mutants exhibit calvarial defects of NCC origin that correlates with sorting of ephrin-B1 positive and negative cells following X-inactivation. We have traced the causes of calvarial defects to impaired differentiation of osteogenic precursors. Moreover, we showed that gap junction communication (GJC) is inhibited at ectopic ephrin boundaries and that ephrin-B1 interacts directly with Connexin43 and regulates its distribution. In turn, regulation of GJC influences cell sorting. These results uncover a novel role for Eph/ephrins in regulating GJC in vivo and suggest that the pleiotropic defects seen in CFNS patients are due to improper regulation of GJC in affected tissues (Davy et al., 2006).
Genetic studies have previously implicated ephrin-B2 in blood vessel formation, cardiac development and remodeling of the lymphatic vasculature. We have found that loss of ephrin-B2 also leads to defective somite development and to defects in populations of cranial and trunk NCCs. In addition, Efnb1/Efnb2 double heterozygous embryos exhibit phenotypes in various NCC derivatives. Expression of one copy of a mutant version of Efnb2 that lacks tyrosine phosphorylation sites was sufficient however to rescue the embryonic phenotypes associated with loss of Efnb2. These results uncover an important role for ephrin-B2 in somites and NCCs during embryogenesis and suggest that ephrin-B2 may exert many of its embryonic function via activation of forward signaling (Davy and Soriano, 2007).
Gene trap mutagenesis
To identify growth factor regulated genes, we are using gene traps in embryonic stem (ES) cells. In this approach, a promoterless reporter gene (for instance encoding βgalactosidase, or βgeo) is introduced in ES cells. Selection for expression of the gene requires transcription from a cellular promoter, and consequently a mutation in a cellular gene, and the activity of the tagged gene can be followed by staining for βgalactosidase activity. Detailed description of methods used for gene trap mutagenesis may be found in Friedrich, G., and Soriano, P. (1993) and Chen, W.V., and Soriano, P(2003). Large scale sequencing of ES cell clones was conducted and sequence tags have been deposited to NCBI's dbGSS. Sequence searches for genes that we have trapped can be performed by running a BLAST search of the dbGSS database. A list of the genes we have trapped can be found in our gene trap database.
Vector design
The basic gene trap vectors we have used include a reporter gene downstream of a splice acceptor sequence (Figure 1). They are therefore designed to function when inserted in an intron. The gene trap cassette is inserted in reverse orientation in a retroviral vector, and vectors and mutant lines derived from our screens are referred to as ROSA (Reverse-Orientation-Splice-Acceptor). Retroviruses insert as a single copy per locus, with no rearrangement of flanking sequences. They have a preference for insertions at the 5' end of genes, often upstream of the initiator ATG, and the splice acceptor sequence we use does not appear to be bypassed. As a result, the majority of the mutations generated using our gene trap vectors are predicted to lead to null alleles. This has in fact been verified in all of the insertions we have analyzed to date at a molecular level.
Figure 1: Gene Trap Design
Phenotypic analysis
Among 70 mutations that have been transmitted through the germ line (all called ROSA), about one third result in a recessive phenotype, either affecting embryonic development at different stages, or the adult.
Lines
SA βgeo 1-5
ROSA 1-29
ROSA 30-39
ROSA 40-45
ROSA 46-52
ROSA 53-55
ROSA 56-61
ROSA 62-65
ROSA 66-70
ROSA 71
ROSA 72-83
Vector
FUSA βgeo
Screen
Random
Random
Random
Induction(EB)
Induction
(TGFβ)
Random
Sequence
Induction (RA)
Induction (serum)
Gene Trap Array
Gene Trap Array
Phenotype
2/ 5 Emb. lethal
7/29 Emb. lethal
1/29 Male sterile
7/10 Emb. lethal
1/ 7 Emb. lethal
1/ 7 Male sterile
1/ 7 Emb. lethal
1/ 3 Emb. lethal
1/ 3 Growth retarded
1/ 3 Emb. lethal
1/ 4 Neuropathy
1/ 4 Male sterile
1/ 5 Growth retarded
Emb. lethal
Neonatal lethal
postnatal phenotypes
Gene
Sec8 ("Spock")
TEF1
Transmembrane
Nuclear RNA
Bcl-W
CD98
Shroom
HMG Box
CTBP2
βIV-spectrin
E-MAP 115
EFII
AA109197
Strap
Zfand5, BC058969, Myo1e
Arid5b, BC055757, Schip1
Sgpl1, Tiparp, Axud1,
Mzf6d, Plekha1, r-Txnip
Induction trapping
To identify and mutate growth factor or retinoid regulated genes, we initially performed induction trapping by monitoring differential expression of the reporter gene, in ES cells or their differentiated derivatives. In some cases, such as ROSA49, we have replica plated ES cells infected with ROSAβgal and tested for differential lacZ activity upon exposure to exogenous factors. In other instances, such as ROSA62 or ROSA63, we have used a modified version of βgeo for the same purpose. βgeo encodes a βgalactosidase-neomycin phosphotransferase fusion protein and carries a mutation in the neo moiety that reduces its activity. This mutation has been corrected in βgeo*. As a result, induction trapping using ROSAβgeo* is more efficient as all neo resistant colonies are gene trap events yet only a fraction (~60%) exhibit βgalactosidase activity. We have also designed a novel vector system to isolate inducible gene traps by flow sorting (Medico et al., 2001).
It has been known for many years that activation of growth factor signaling pathways leads to the expression of multiple Immediate Early Genes (IEGs), yet their role in specifying particular growth factor responses remained controversial as similar IEGs are often engaged following induction of multiple signaling pathways. We have used gene trap-coupled microarray analysis, using a modified gene trap vector system in which 3' RACE products from mutated genes are spotted on arrays (Chen et al., 2004), to identify targets of PDGF signaling, and their function in PDGF regulated processes. Mutations in these genes lead to a high frequency of phenotypes that affect the same cell types and processes as those controlled by PDGF signaling (Schmahl et al., 2007). These results suggest that these genes form a network that control specific processes downstream of PDGF signaling. We are also using gene traps to perform gain of function or suppressor genetics on sensitized backgrounds.
Results from large scale sequencing of ES cell clones and sequence tags have been deposited to NCBI's dbGSS. Sequences searches can be performed by running a BLAST search of the dbGSS database. A list of the genes we have trapped can be found in our gene trap database. A number of laboratories performing gene trap mutagenesis, including ours, have formed a gene trap consortium. Through the gene trap consortium, cell lines are made available to investigators at non-profit institutions for a modest fee (to recover costs). These cell lines may be ordered from the MMRRC. You can also search for our clones in the MMRRC catalog.
© 2008 Mount Sinai Medical Center
