Development of Vessels, Airways and Cartilage Rings: The role of T-box genes

Tbx4 and Tbx5 are two closely related genes that belong to the T-box family of transcription factor genes. Loss of Tbx4 results in absence of chorio-allantoic fusion and a failure of formation of the primary vascular plexus of the allantois leading to embryonic death at E10.5. Using a candidate gene approach we identified a number of genes downstream of Tbx4 in the allantois including, extracellular matrix molecules Vcan, Has2, Itgα5; transcription factors Snai1 and Twist, and signaling molecules Bmp2, Bmp7, Notch2, Jag1 and Wnt2In addition, we show that the canonical Wnt signaling pathway contributes to the vessel-forming potential of the allantois. Ex vivo, the Tbx4 mutant phenotype can be rescued using agonists of the Wnt signaling pathway and an inhibitor of the canonical Wnt signaling pathway phenocopies the Tbx4mutant phenotype in wildtype allantoises. In vivo, Tbx4 and Wnt2 double heterozygous placentas show decreased vasculature suggesting interactions between Tbx4 and the canonical Wnt signaling pathway in the process of allantois-derived blood vessel formation. Both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing respiratory system. Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Although many genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. We investigated the roles of Tbx4 and Tbx5 in lung and trachea development using conditional mutant alleles and two different Cre recombinase transgenic lines. Loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Mutants deficient in Tbx4 and Tbx5 show severely reduced lung branching at mid-gestation. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes in the epithelium, Bmp4 and Spry2, is downregulated. Lung branching undergoes arrest ex vivo when Tbx4 and Tbx5 are both completely lacking. Lung-specific Tbx4 heterozygous; Tbx5 conditional null mice die soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal-bronchial cartilage rings. Sox9 a master regulator of cartilage formation, is expressed in the trachea but mesenchymal cells fail to condense and consequently do not develop cartilage normally at birth. Tbx4;Tbx5 double heterozygous mutants show decreased lung branching and fewer tracheal cartilage rings, suggesting a genetic interaction. Finally, we show that Tbx4 and Tbx5 interact with Fgf10 during the process of lung growth and branching but not during tracheal bronchial cartilage development

A Retrotransposon Insertion in the 5' Regulatory Domain of Ptf1a Results in Ectopic Gene Expression and Multiple Congenital Defects in Danforth’s Short Tail Mouse

Danforth's short tail mutant (Sd) mouse, first described in 1930, is a classic spontaneous mutant exhibiting defects of the axial skeleton, hindgut, and urogenital system. We used meiotic mapping in 1,497 segregants to localize the mutation to a 42.8-kb intergenic segment on chromosome 2. Resequencing of this region identified an 8.5-kb early retrotransposon (ETn) insertion within the highly conserved regulatory sequences upstream of Pancreas Specific Transcription Factor, 1a (Ptf1a). This mutation resulted in up to tenfold increased expression of Ptf1a as compared to wild-type embryos at E9.5 but no detectable changes in the expression levels of other neighboring genes. At E9.5, Sd mutants exhibit ectopic Ptf1a expression in embryonic progenitors of every organ that will manifest a developmental defect: the notochord, the hindgut, and the mesonephric ducts. Moreover, at E 8.5, Sd mutant mice exhibit ectopic Ptf1a expression in the lateral plate mesoderm, tail bud mesenchyme, and in the notochord, preceding the onset of visible defects such as notochord degeneration. The Sd heterozygote phenotype was not ameliorated by Ptf1a haploinsufficiency, further suggesting that the developmental defects result from ectopic expression of Ptf1a. These data identify disruption of the spatio-temporal pattern of Ptf1a expression as the unifying mechanism underlying the multiple congenital defects in Danforth's short tail mouse. This striking example of an enhancer mutation resulting in profound developmental defects suggests that disruption of conserved regulatory elements may also contribute to human malformation syndromes

Novel domains of expression for orphan receptor tyrosine kinase Ror2 in the human and mouse reproductive system.

BackgroundThe noncanonical Wnt receptor and tyrosine kinase Ror2 has been associated with recessive Robinow syndrome (RRS) and dominant brachydactyly type B1. The phenotypes of mouse mutants implicate Ror2 in the development of the heart, lungs, bone, and craniofacial structures, which are affected in RRS. Following a recently identified role of Ror2 in the migration of mouse primordial germ cells, we extensively characterized its expression throughout the fetal internal reproductive system and the postnatal ductal system.ResultsWe show that Ror2 gene products are present in the germ cells and somatic cells of the testis and the ovary of both the mouse and human fetus. In reproductive tract structures, we find that Ror2 is expressed in the mesonephros, developing Wolffian and Müllerian ducts, and later in their derivatives, the epididymal epithelium and uterine epithelium.ConclusionsThis study sets the stage to explore function for this tyrosine kinase receptor in novel regions of expression in the developing reproductive system in both mouse and human

Novel domains of expression for orphan receptor tyrosine kinase Ror2 in the human and mouse reproductive system.

BackgroundThe noncanonical Wnt receptor and tyrosine kinase Ror2 has been associated with recessive Robinow syndrome (RRS) and dominant brachydactyly type B1. The phenotypes of mouse mutants implicate Ror2 in the development of the heart, lungs, bone, and craniofacial structures, which are affected in RRS. Following a recently identified role of Ror2 in the migration of mouse primordial germ cells, we extensively characterized its expression throughout the fetal internal reproductive system and the postnatal ductal system.ResultsWe show that Ror2 gene products are present in the germ cells and somatic cells of the testis and the ovary of both the mouse and human fetus. In reproductive tract structures, we find that Ror2 is expressed in the mesonephros, developing Wolffian and Müllerian ducts, and later in their derivatives, the epididymal epithelium and uterine epithelium.ConclusionsThis study sets the stage to explore function for this tyrosine kinase receptor in novel regions of expression in the developing reproductive system in both mouse and human

Expression of <i>Tbx4</i> and <i>Tbx5</i> in the developing lung and trachea.

<p>(A–L) <i>Tbx4</i> and <i>Tbx5</i> expression analyzed using ISH on lungs. <i>Tbx5</i> is first expressed at E9.0 (B,D) when the specification of lung primordia occurs, as seen by <i>Nkx2.1</i> expression (A,C). Red arrows point to the posterior extent of the third pharyngeal pouch which marks the anterior of the expression domain of both <i>Nkx2.1</i> and <i>Tbx5</i>. Right views (A,B), ventral views (C,D). <i>Tbx4</i> is first expressed at E9.5 along with <i>Tbx5</i> in the newly formed lung buds (E,F). Expression is seen in lung whole mounts at E11.5 (G,H), E13.5 (I,J) and in lung mesenchyme in cryosections at E15.5 (K,L). (M–S) <i>Tbx4</i> and <i>Tbx5</i> expression analyzed using ISH on tracheas. <i>Tbx4</i> and <i>Tbx5</i> are expressed throughout tracheal mesenchyme (m) at E13.5 (M,M′,N,N′) but not in the epithelium (e) or the mesothelium (arrowheads). Caudal view of cut tracheas after whole mount ISH (M–P). ISH on cryosections (M′–P′). At E13.5, <i>Sox9</i> is expressed in the mesenchyme on the ventral side (O,O′) and <i>SM22α</i> is expressed on the dorsal side (P,P′) of the trachea. D-dorsal; V-ventral; R-right; L-left. At E15.5, <i>Tbx4</i> and <i>Tbx5</i> are expressed around the condensing cartilage mesenchyme and in the intercartilage mesenchyme (Q,R). <i>Sox9</i> is expressed in the condensing cartilage rings (S). Asterisks indicate areas of cartilage condensation. Insets in (Q) and (S) show ISH on E15.5 sagittal cryosections with <i>Tbx4</i> and <i>Sox9</i> probe, respectively. Scale bars represent 50 µm.</p

Early loss of <i>Tbx5</i> leads to aberrant lung bud and trachea specification.

<p>(A–I) Foreguts were isolated between 8–16 somite stages and analyzed by ISH following culture. <i>Nkx2.1</i> (A), <i>Tbx4</i> (B) and <i>Tbx5</i> (C) expression in lung buds (arrows) and tracheal primordia (yellow arrowhead) was confirmed in control cultures. Excision of <i>Tbx5</i> using 4-OH tamoxifen in conditional null foreguts with <i>CreER</i> leads to the loss of <i>Nkx2.1</i> expression in one of the lung buds at 3 (E) or 4 days (H). Conditional double nulls (F,I) carrying <i>CreER</i> show a phenotype similar to the conditional <i>Tbx5</i> nulls, suggesting additional removal of <i>Tbx4</i> does not exacerbate the phenotype. <i>Nkx2.1</i> expression was absent in the foregut tube of conditional <i>Tbx5</i> null and conditional double null foreguts after 3 or 4 days of culture (yellow arrowheads in E,F,H,I) compared to controls (D,G). Conditional <i>Tbx5</i> nulls show reduced <i>Wnt2</i> expression (K) and absence of <i>Wnt2b</i> expression (M) in the developing lung buds as compared to controls (J,L). Black arrowheads (J) point to <i>Wnt2</i> expression in the heart in the controls. ht, heart; th, thyroid primordia.</p

Multiple Roles and Interactions of <em>Tbx4</em> and <em>Tbx5</em> in Development of the Respiratory System

<div><p>Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Both <em>Tbx4</em> and <em>Tbx5</em> are expressed throughout the mesenchyme of the developing lung and trachea; and, although multiple genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. In this study, we investigated the roles of <em>Tbx4</em> and <em>Tbx5</em> in lung and trachea development using conditional mutant alleles and two different Cre recombinase transgenic lines. Loss of <em>Tbx5</em> leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Mutants deficient in <em>Tbx4</em> and <em>Tbx5</em> show severely reduced lung branching at mid-gestation. Concordant with this defect, the expression of mesenchymal markers <em>Wnt2</em> and <em>Fgf10</em>, as well as Fgf10 target genes <em>Bmp4</em> and <em>Spry2</em>, in the epithelium is downregulated. Lung branching undergoes arrest <em>ex vivo</em> when <em>Tbx4</em> and <em>Tbx5</em> are both completely lacking. Lung-specific <em>Tbx4</em> heterozygous;<em>Tbx5</em> conditional null mice die soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings. <em>Sox9</em>, a master regulator of cartilage formation, is expressed in the trachea; but mesenchymal cells fail to condense and consequently do not develop cartilage normally at birth. <em>Tbx4</em>;<em>Tbx5</em> double heterozygous mutants show decreased lung branching and fewer tracheal cartilage rings, suggesting a genetic interaction. Finally, we show that <em>Tbx4</em> and <em>Tbx5</em> interact with <em>Fgf10</em> during the process of lung growth and branching but not during tracheal/bronchial cartilage development.</p> </div

Loss of <i>Tbx4</i> and <i>Tbx5</i> affects the <i>Fgf10</i> signaling pathway and <i>Wnt2</i> expression.

<p>(A–X) Marker analysis of control and <i>Tbx4</i>- and <i>Tbx5</i>-deficient lungs using whole mount ISH (A–O, S–X) and IHC (P–R): Fewer foci of <i>Fgf10</i> expression were seen in the <i>Tbx4</i> and <i>Tbx5</i>-deficient lungs (B,C) compared to control (A). cr, cranial; m, medial; cd, caudal; a, accesory lobes. Fgf10 target genes <i>Bmp4</i> (F,G,H) and <i>Spry2</i> (I,J) and canonical <i>Wnt2</i> (K,L,M) were downregulated in <i>Tbx4</i> and <i>Tbx5</i>-deficient lungs compared to controls. <i>Fgfr2</i> (D,E), <i>Etv5</i> (N,O), PECAM (P,Q,R), <i>Shh</i> (S,T), <i>Ptc</i> (U,V) and <i>Nkx2.1</i> (W,X) were expressed similarly in controls and <i>Tbx4</i> and <i>Tbx5</i>-deficient lungs.</p