Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse

Tbx1 has been implicated as a candidate gene responsible for defective pharyngeal arch remodeling in DiGeorge/Velocardiofacial syndrome. Tbx1(+/-) mice mimic aspects of the DiGeorge phenotype with variable penetrance, and null mice display severe pharyngeal hypoplasia. Here, we identify enhancer elements in the Tbx1 gene that are conserved through evolution and mediate tissue-specific expression. We describe the generation of transgenic mice that utilize these enhancer elements to direct Cre recombinase expression in endogenous Tbx1 expression domains. We use these Tbx1-Cre mice to fate map Tbx1-expressing precursors and identify broad regions of mesoderm, including early cardiac mesoderm, which are derived from Tbx1-expressing cells. We test the hypothesis that fibroblast growth factor 8 (Fgf8) functions downstream of Tbx1 by performing tissue-specific inactivation of Fgf8 using Tbx1-Cre mice. Resulting newborn mice display DiGeorge-like congenital cardiovascular defects that involve the outflow tract of the heart. Vascular smooth muscle differentiation in the great vessels is disrupted. This data is consistent with a model in which Tbx1 induces Fgf8 expression in the pharyngeal endoderm, which is subsequently required for normal cardiovascular morphogenesis and smooth muscle differentiation in the aorta and pulmonary artery.     

Loss of Gbx2 results in neural crest cell patterning and pharyngeal arch artery defects in the mouse embryo

Several syndromes characterized by defects in cardiovascular and craniofacial development are associated with a hemizygous deletion of chromosome 22q11 in humans and involve defects in pharyngeal arch and neural crest cell development. Recent efforts have focused on identifying 22q11 deletion syndrome modifying loci. In this study, we show that mouse embryos deficient for Gbx2 display aberrant neural crest cell patterning and defects in pharyngeal arch-derived structures. Gbx2(-/-) embryos exhibit cardiovascular defects associated with aberrant development of the fourth pharyngeal arch arteries including interrupted aortic arch type B, right aortic arch, and retroesophageal right subclavian artery. Other developmental abnormalities include overriding aorta, ventricular septal defects, cranial nerve, and craniofacial skeletal patterning defects. Recently, Fgf8 has been proposed as a candidate modifier for 22q11 deletion syndromes. Here, we demonstrate that Fgf8 and Gbx2 expression overlaps in regions of the developing pharyngeal arches and that they interact genetically during pharyngeal arch and cardiovascular development.     

Gain of function of Tbx1 affects pharyngeal and heart development in the mouse

Mammalian development is highly sensitive to Tbx1 gene dosage reduction. Gene function insights can also be learned from increased or ectopic expression. The authors generated a novel mouse transgenic line, named COET, which expresses Tbx1 upon Cre‐mediated recombination. The authors crossed this transgenic line with Tbx1^Cre animals to activate expression in the Tbx1‐expression domain. Compound mutant COET;Tbx1^Cre/+ animals died after birth and showed heart enlargement. At E18.5, compound mutants showed ventricular septal defects and thymic abnormalities. The authors crossed compound mutants into a Tbx1 null background to understand whether this phenotype is caused by gene overdosage. Results showed that gene dosage reduction at the endogenous locus could not rescue heart and thymic defects, although the transgene rescued the loss of function phenotype. Thus, the transgenic phenotype appears to be due to gain of function. Resultant data demonstrate that Tbx1 expression must be tightly regulated to be compatible with normal embryonic development. genesis 47:188–195, 2009. © 2009 Wiley‐Liss, Inc.     

Eya1 acts upstream of Tbx1, Neurogenin 1, NeuroD and the neurotrophins BDNF and NT-3 during inner ear development

Cell fate specification during inner ear development is dependent upon regional gene expression within the otic vesicle. One of the earliest cell fate determination steps in this system is the specification of neural precursors, and regulators of this process include the Atonal-related basic helix-loop-helix genes, Ngn1 and NeuroD and the T-box gene, Tbx1. In this study we demonstrate that Eya1 signaling is critical to the normal expression patterns of Tbx1, Ngn1, and NeuroD in the developing mouse otocyst. We discuss a potential mechanism for the absence of neural precursors in the Eya1-/- inner ears and the primary and secondary mechanisms for the loss of cochleovestibular ganglion cells in the Eya1bor/bor hypomorphic mutant.     

Microarray analysis of the Df1 mouse model of the 22q11 deletion syndrome

The 22q11 deletion syndrome (22q11DS; DiGeorge/velo-cardio-facial syndrome) primarily affects the structures comprising the pharyngeal arches and pouches resulting in arch artery, cardiac, parathyroid, thymus, palatal and craniofacial defects. Tbx1 haploinsufficiency is thought to account for the main structural anomalies observed in the 22q11DS. The Df1 deleted mouse provides a model for 22q11DS, the deletion reflecting Tbx1 haploinsufficiency in the context of the deletion of 21 adjacent genes. We examined the expression of genes in Df1 embryos at embryonic day (E) 10.5, a stage when the arch-artery phenotype is fully penetrant. Our aims were threefold, with our primary aim to identify differentially regulated genes. Second, we asked whether any of the genes hemizygous in Df1 were dosage compensated to wild type levels, and third we investigated whether genes immediately adjacent to the deletion were dysregulated secondary to a position effect. Utilisation of oligonulceotide arrays allowed us to achieve our aims with 9 out of 12 Df1 deleted genes passing the stringent statistical filtering applied. Several genes involved in vasculogenesis and cardiogenesis were validated by real time quantitative PCR (RTQPCR), including Connexin 45, a gene required for normal vascular development, and Dnajb9 a gene implicated in microvascular differentiation. There was no evidence of any dosage compensation of deleted genes, suggesting this phenomenon is rare, and no dysregulation of genes mapping immediately adjacent to the deletion was detected. However Crkl, another gene implicated in the 22q11DS phenotype, was found to be downregulated by microarray and RTQPCR.     

Fgf8b-containing spliceforms, but not Fgf8a, are essential for Fgf8 function during development of the midbrain and cerebellum

The single Fgf8 gene in mice produces eight protein isoforms (Fgf8a-h) with different N-termini by alternative splicing. Gain-of-function studies have demonstrated that Fgf8a and Fgf8b have distinct activities in the developing midbrain and hindbrain (MHB) due to their different binding affinities with FGF receptors. Here we have performed loss-of-function analyses to determine the in vivo requirement for these two Fgf8 spliceforms during MHB development. We showed that deletion of Fgf8b-containing spliceforms (b, d, f and h) leads to loss of multiple key regulatory genes, including Fgf8 itself, in the MHB region. Therefore, specific inactivation of Fgf8b-containing spliceforms, similar to the loss of Fgf8, in MHB progenitors results in deletion of the midbrain, isthmus, and cerebellum. We also created a splice-site mutation abolishing Fgf8a-containing spliceforms (a, c, e, and g). Mice lacking Fgf8a-containing spliceforms exhibit growth retardation and postnatal lethality, and the phenotype is variable in different genetic backgrounds, suggesting that the Fgf8a-containing spliceforms may play a role in modulating the activity of Fgf8. Surprisingly, no discernable defect was detected in the midbrain and cerebellum of Fgf8a-deficient mice. To determine if Fgf17, which is expressed in the MHB region and possesses similar activities to Fgf8a based on gain-of-function studies, may compensate for the loss of Fgf8a, we generated Fgf17 and Fgf8a double mutant mice. Mice lacking both Fgf8a-containing spliceforms and Fgf17 display the same defect in the posterior midbrain and anterior cerebellum as Fgf17 mutant mice. Therefore, Fgf8b-containing spliceforms, but not Fgf8a, are essential for the function of Fgf8 during the development of the midbrain and cerebellum.     

Six1 and Six4 are essential for Gdnf expression in the metanephric mesenchyme and ureteric bud formation, while Six1 deficiency alone causes mesonephric-tubule defects

Interaction between the ureteric-bud epithelium and the metanephric mesenchyme is important for kidney development. Six1 and Six4 are the mammalian homologs of Drosophila sine oculis, and they are coexpressed in the nephrogenic mesenchyme. Six1-deficient mice show varying kidney defects, while Six4-deficient mice have no apparent abnormalities. Here, we report Six1/Six4-deficient mice that we generated in order to elucidate the functions of Six4 in Six1-deficient kidney development. The Six1/Six4-deficient mice exhibited more severe kidney phenotypes than the Six1-deficient mice; kidney and ureter agenesis was observed in all the neonates examined. The Six1/Six4-deficient metanephric mesenchyme cells were directed toward kidney lineage but failed to express Pax2, Pax8, or Gdnf, whereas the expression of these genes was partially reduced or unchanged in the case of Six1 deficiency. Thus, Six4 cooperates with Six1 in the metanephric mesenchyme to regulate the level of Gdnf expression; this could explain the absence of the ureteric bud in the Six1/Six4-deficient mice. In contrast, Six1 deficiency alone caused defects in mesonephric-tubule formation, and these defects were not exacerbated in the Six1/Six4-deficient mesonephros. These results highlight the fact that Six1 and Six4 have collaborative functions in the metanephros but not in the mesonephros.     

Tbx20-related genes, mid and H15, are required for tinman expression, proper patterning, and normal differentiation of cardioblasts in Drosophila

Tbx20-related T-box genes have been implicated in the regulation of heart development in several vertebrate species. In the present report, we demonstrate that a pair of genes representing Drosophila orthologs of Tbx20, midline (mid) and H15, have important functions during the development of the Drosophila equivalent of the heart, i.e. the dorsal vessel. We show that mid is among the earliest known genes that are specifically expressed in all cardioblasts during early embryogenesis, and H15 expression is subsequently activated in the same cells. Mutant embryos lacking the activity of mid, or both mid and H15, are able to form dorsal vessels with largely normal numbers of cardioblasts and pericardial cells. Furthermore, the mutant cardioblasts express several general cardioblast markers such as Mef2 and Toll at normal levels. However, the expression of tinman (tin), which normally occurs in four out of six cardioblasts in each hemisegment of the dorsal vessel, is almost abolished. Conversely, the expression of the Dorsocross (Doc) T-box genes, which is normally restricted to the two Tin-negative cardioblasts in each hemisegment, is strongly expanded into the majority of cardioblasts in mid mutant and mid+H15-deficient embryos. Altogether, the data from the loss-of-function phenotypes demonstrate that mid, and to a lesser degree H15, have important roles in establishing the metameric patterning of cardioblast identities, but not in specifying cardioblasts as such. Ectopic expression of mid causes ectopic tin expression and, less efficiently, produces extra cardioblasts. We propose that one of the major functions of mid and H15 during cardioblast development is the re-activation of tin expression at a stage when the induction of tin by Dpp in the dorsal mesoderm has ceased. Through this activity, mid and H15 are required for the normal functional diversification of cardioblasts and the expression of tin-dependent terminal differentiation genes within the dorsal vessel.     

Foxh1 and Foxa2 are not required for formation of the midgut and hindgut definitive endoderm

The definitive endoderm forms during gastrulation and is rapidly transformed into the gut tube which is divided along the anterior-posterior axis into the foregut, midgut, and hindgut. Lineage tracing and genetic analysis have examined the origin of the definitive endoderm during gastrulation and demonstrated that the majority of definitive endoderm arises at the anterior end of the primitive streak (APS). Foxh1 and Foxa2 have been shown to play a role in specification of the APS and definitive endoderm. However, prior studies have focused on the role of these factors in specification of foregut definitive endoderm, while their role in the specification of midgut and hindgut definitive endoderm is less understood. Furthermore, previous analyses of these mutants have utilized definitive endoderm markers that are restricted to the anterior endoderm, expressed in extraembryonic endoderm, or present in other germ layers. Here, we characterized the expression of several novel definitive and visceral endoderm markers in Foxh1 and Foxa2 null embryos. In accordance with previous studies, we observed a deficiency of foregut definitive endoderm resulting in incorporation of visceral endoderm into the foregut. Interestingly, this analysis revealed that formation of midgut and hindgut definitive endoderm is unaffected by loss of Foxh1 or Foxa2. This finding represents a significant insight into specification and regionalization of mouse definitive endoderm.     

Patterning of the third pharyngeal pouch into thymus/parathyroid by Six and Eya1

Previous studies have suggested a role of the homeodomain Six family proteins in patterning the developing vertebrate head that involves appropriate segmentation of three tissue layers, the endoderm, the paraxial mesoderm and the neural crest cells; however, the developmental programs and mechanisms by which the Six genes act in the pharyngeal endoderm remain largely unknown. Here, we examined their roles in pharyngeal pouch development. Six1-/- mice lack thymus and parathyroid and analysis of Six1-/- third pouch endoderm demonstrated that the patterning of the third pouch into thymus/parathyroid primordia is initiated. However, the endodermal cells of the thymus/parathyroid rudiments fail to maintain the expression of the parathyroid-specific gene Gcm2 and the thymus-specific gene Foxn1 and subsequently undergo abnormal apoptosis, leading to a complete disappearance of organ primordia by E12.5. This thus defines the thymus/parathyroid defects present in the Six1 mutant. Analyses of the thymus/parathyroid development in Six1-/-;Six4-/- double mutant show that both Six1 and Six4 act synergistically to control morphogenetic movements of early thymus/parathyroid tissues, and the threshold of Six1/Six4 appears to be crucial for the regulation of the organ primordia-specific gene expression. Previous studies in flies and mice suggested that Eya and Six genes may function downstream of Pax genes. Our data clearly show that Eya1 and Six1 expression in the pouches does not require Pax1/Pax9 function, suggesting that they may function independently from Pax1/Pax9. In contrast, Pax1 expression in all pharyngeal pouches requires both Eya1 and Six1 function. Moreover, we show that the expression of Tbx1, Fgf8 and Wnt5b in the pouch endoderm was normal in Six1-/- embryos and slightly reduced in Six1-/-;Six4-/- double mutant, but was largely reduced in Eya1-/- embryos. These results indicate that Eya1 appears to be upstream of very early events in the initiation of thymus/parathyroid organogenesis, while Six genes appear to act in an early differentiation step during thymus/parathyroid morphogenesis. Together, these analyses establish an essential role for Eya1 and Six genes in patterning the third pouch into organ-specific primordia.     

Developmental expression of the amphioxus <Emphasis Type="Italic">Tbx1</Emphasis>/<Emphasis Type="Italic">10</Emphasis> gene illuminates the evolution of vertebrate branchial arches and sclerotome

We have isolated an amphioxus T-box gene that is orthologous to the two vertebrate genes, Tbx1 and Tbx10, and examined its expression pattern during embryonic and early larval development. AmphiTbx1/10 is first expressed in branchial arch endoderm and mesoderm of developing neurulae, and in a bilateral, segmented pattern in the ventral half of newly formed somites. Branchial expression is restricted to the first three branchial arches, and disappears completely by 4 days post fertilization. Ventral somitic expression is restricted to the first 10–12 somites, and is not observed in early larvae except in the most ventral mesoderm of the first three branchial arches. No expression can be detected by 4 days post fertilization. Integrating functional, phylogenetic and expression data from amphioxus and a variety of vertebrate model organisms, we have reconstructed the early evolutionary history of the Tbx1/10 subfamily of genes within the chordate lineage. We conclude that Tbx1/10-mediated branchial arch endoderm and mesoderm patterning functions predated the origin of neural crest, and that ventral somite specification functions predated the origin of vertebrate sclerotome, but that Tbx1 was later co-opted during the evolution of developmental programs regulating branchial neural crest and sclerotome migration.Edited by M. Akam