Maxime Bouchard

Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates

Glutamatergic and GABAergic neurons mediate much of the excitatory and inhibitory neurotransmission, respectively, in the vertebrate nervous system. The process by which developing neurons select between these two cell fates is poorly understood. Here we show that the homeobox genes Tlx3 and Tlx1 determine excitatory over inhibitory cell fates in the mouse dorsal spinal cord. First, we found that Tlx3 was required for specification of, and expressed in, glutamatergic neurons. Both generic and region-specific glutamatergic markers, including VGLUT2 and the AMPA receptor Gria2, were absent in Tlx mutant dorsal horn. Second, spinal GABAergic markers were derepressed in Tlx mutants, including Pax2 that is necessary for GABAergic differentiation, Gad1/2 and Viaat that regulate GABA synthesis and transport, and the kainate receptors Grik2/3. Third, ectopic expression of Tlx3 was sufficient to suppress GABAergic differentiation and induce formation of glutamatergic neurons. Finally, excess GABA-mediated inhibition caused dysfunction of central respiratory circuits in Tlx3 mutant mice.

Pax2/8-regulated Gata3 expression is necessary for morphogenesis and guidance of the nephric duct in the developing kidney

The mammalian pro- and mesonephros are transient embryonic kidneys essential for urogenital system development. The nephric (Wolffian) duct, which is a central constituent of both structures, elongates caudally along a stereotypical path to reach the hindlimb level where it induces metanephros (adult kidney) formation, while the remaining duct gives rise to the male genital tract (epidydimis, vas deferens). The transcription factors Pax2 and Pax8 are essential for the initiation of pro- and mesonephros development. In a cDNA microarray screen for genes specifically expressed in the pro/mesonephros and regulated by Pax proteins, we identified Gata3, a transcription factor gene associated with hypoparathyroidism, deafness and renal anomaly (HDR) syndrome. Gata3 is already expressed in the pronephric anlage, together with Pax2 and Pax8, suggesting that it may be a direct Pax2/8 target gene. Inactivation of Gata3 by insertion of an Ires-GFP reporter gene resulted in a massive increase in nephric duct cellularity, which was accompanied by enhanced cell proliferation and aberrant elongation of the nephric duct. Interestingly, however, the nephrogenic cord extended, with delayed kinetics, along the entire caudal path up to the level of the hindlimb bud, indicating that extension of the nephric duct and cord is controlled by different guidance cues. At the molecular level, the nephric duct of Gata3-/- embryos is characterized by the loss of Ret expression and signaling, which may contribute to the guidance defect of the nephric duct. Together, these results define Gata3 as a key regulator of nephric duct morphogenesis and guidance in the pro/mesonephric kidney.

Distinct regulators control the expression of the mid-hindbrain organizer signal FGF8

Local expression of FGF8 at the mid/hindbrain boundary (MHB) governs the development of multiple neurons and support cells. Here we show that the paired-domain protein Pax2 is necessary and sufficient for the induction of FGF8 in part by regulating the expression of Pax5&8. A network of transcription and secreted factors, including En1, Otx2, Gbx2, Grg4 and Wnt1&4, that is established independently of Pax2, further refines the expression domain and level of FGF8 at the MHB through opposing effects on Pax2 activity. Our results indicate that the expression of local organizing factors is controlled by combinatorial interaction between inductive and modulatory factors.

Pax2 and Pax8 Regulate Branching Morphogenesis and Nephron Differentiation in the Developing Kidney

Pax genes are important regulators of kidney development. In the mouse, homozygous Pax2 inactivation results in renal agenesis, a phenotype that has largely precluded the analysis of Pax gene function during metanephric kidney development. To address this later function, kidney development was analyzed in embryos that were compound heterozygous for Pax2 and for Pax8, a closely related member of the Pax gene family. Both genes are coexpressed in differentiating nephrons and collecting ducts. At the morphological level, Pax2(+/-)Pax8(+/-) metanephric kidneys are severely hypodysplastic and characterized by a reduction in ureter tips and nephron number in comparison with wild-type or Pax2(+/-) kidneys. In developing nephrons, the molecular analysis of Pax2(+/-)Pax8(+/-) kidneys reveals a strong reduction in the expression levels of Lim1, a key regulator of nephron differentiation, accompanied by an increase in apoptosis. At a more mature stage, the reduction of Pax2/8 gene dosage severely affects distal tubule formation, revealing a role for Pax genes in the differentiation of specific nephron segments. At the ureter tips, the expression of Wnt11, a target of glial cell-derived neurotrophic factor-Ret signaling, is significantly reduced, whereas the expression levels of Ret and GDNF remain normal. Together, these results demonstrate a crucial role for Pax2 and Pax8 in nephron differentiation and branching morphogenesis of the metanephros.

Gata3 Acts Downstream of β-Catenin Signaling to Prevent Ectopic Metanephric Kidney Induction

Metanephric kidney induction critically depends on mesenchymal–epithelial interactions in the caudal region of the nephric (or Wolffian) duct. Central to this process, GDNF secreted from the metanephric mesenchyme induces ureter budding by activating the Ret receptor expressed in the nephric duct epithelium. A failure to regulate this pathway is believed to be responsible for a large proportion of the developmental anomalies affecting the urogenital system. Here, we show that the nephric duct-specific inactivation of the transcription factor gene Gata3 leads to massive ectopic ureter budding. This results in a spectrum of urogenital malformations including kidney adysplasia, duplex systems, and hydroureter, as well as vas deferens hyperplasia and uterine agenesis. The variability of developmental defects is reminiscent of the congenital anomalies of the kidney and urinary tract (CAKUT) observed in human. We show that Gata3 inactivation causes premature nephric duct cell differentiation and loss of Ret receptor gene expression. These changes ultimately affect nephric duct epithelium homeostasis, leading to ectopic budding of interspersed cells still expressing the Ret receptor. Importantly, the formation of these ectopic buds requires both GDNF/Ret and Fgf signaling activities. We further identify Gata3 as a central mediator of β-catenin function in the nephric duct and demonstrate that the β-catenin/Gata3 pathway prevents premature cell differentiation independently of its role in regulating Ret expression. Together, these results establish a genetic cascade in which Gata3 acts downstream of β-catenin, but upstream of Ret, to prevent ectopic ureter budding and premature cell differentiation in the nephric duct.

Pax2 and Pax8 cooperate in mouse inner ear morphogenesis and innervation.

BackgroundPax2;5;8 transcription factors play diverse roles in vertebrate and invertebrate organogenesis, including the development of the inner ear. Past research has suggested various cochlear defects and some vestibular defects in Pax2 null mice but the details of the cochlear defects and the interaction with other Pax family members in ear development remain unclear.ResultsWe show that Pax2;8 double null mice do not develop an ear past the otocyst stage and show little to no sensory as well as limited and transient neuronal development, thus indicating that these two family members are essential for overall ear morphogenesis and sustained neurosensory development. In support of functional redundancy between Pax proteins, Pax2 can be substituted by a Pax5 minigene, a gene normally not expressed in the embryonic mouse ear. There is no detectable morphological defect in Pax8 null mice suggesting that Pax2 expression can compensate for Pax8. Conversely, Pax8 cannot compensate for Pax2 leading to a cochlear phenotype not fully appreciated previously: Cochlear development is delayed until E15.5 when the cochlea extrudes as a large sack into the brain case. Immunocytochemistry and tracing from the brain show that a cochlear spiral ganglia form as a small addition to the inferior vestibular ganglion. However, the empty cochlear sack, devoid of any sensory epithelium development as indicated by the absence of Sox2 or MyoVII expression, nevertheless develop a dense innervation network of small neurons situated in the wall of the cochlear sack.ConclusionsCombined these data suggest that Pax2 is needed for organ of Corti formation and is directly or indirectly involved in the coordination of spiral ganglion formation which is partially disrupted in the Pax2 null ears. All three Pax genes can signal redundantly in the ear with their function being determined primarily by the spatio-temporal expression driven by the three distinct promoters of these genes.

The characterization of novel Pax genes of the sea urchin and Drosophila reveal an ancient evolutionary origin of the Pax2/5/8 subfamily

The developmental control genes of the Pax family can be grouped into different subclasses according to structure and sequence homology. Here we describe the isolation and characterization of three novel Pax genes of the sea urchin for which no homologues are yet known in other animal phyla. One of these genes, suPaxB, codes for the previously characterized transcription factor TSAP which is involved in the developmental regulation of two pairs of late histone genes. Furthermore, conserved members of the Pax2/5/8 subfamily, which have so far been described only in vertebrates, were isolated not only from the sea urchin, but also from Drosophila and C. elegans. Hence, the Pax2/5/8 transcription factors constitute an ancient subfamily of highly conserved Pax proteins. During Drosophila embryogenesis, the Pax258 gene is shown to be expressed in the precursor cells of the external sensory organs, thus suggesting a role for Pax258 in the early development of the peripheral nervous system of insects.

Inhibition of Mammalian Target of Rapamycin Augments Lipopolysaccharide-Induced Lung Injury and Apoptosis

Acute lung injury during bacterial infection is associated with neutrophilic inflammation, epithelial cell apoptosis, and disruption of the alveolar-capillary barrier. TLR4 is required for lung injury in animals exposed to bacterial LPS and initiates proinflammatory responses in part via the transcription factor NF-κB. Ligation of TLR4 also initiates a proapoptotic response by activating IFN-β and STAT1-dependent genes. We recently demonstrated that mammalian target of rapamycin (mTOR), a key controller of cell growth and survival, can physically interact with STAT1 and suppress the induction of STAT1-dependent apoptosis genes. We therefore hypothesized that the mTOR inhibitor rapamycin would increase LPS-induced apoptosis and lung injury in vivo. Rapamycin increased lung injury and cellular apoptosis in C57BL/6J mice exposed to intratracheal LPS for 24 h. Rapamycin also augmented STAT1 activation, and the induction of STAT1-dependent genes that mediate cellular apoptosis (i.e., Fas, caspase-3). LPS-induced lung injury was attenuated in STAT1 knockout mice. In addition, LPS and IFN-β–induced apoptosis was absent in cultured cells lacking STAT1, and, unlike in wild-type cells, a permissive effect of rapamycin was not observed. In contrast to its effect on STAT1, rapamycin inhibited NF-κB activation in vivo and reduced selected markers of inflammation (i.e., neutrophils in the bronchoalveolar lavage fluid, TNF-α). Therefore, although it inhibits NF-κB and neutrophilic inflammation, rapamycin augments LPS-induced lung injury and apoptosis in a mechanism that involves STAT1 and the induction of STAT1-dependent apoptosis genes.

Identification of Pax2-regulated genes by expression profiling of the mid-hindbrain organizer region.

The paired domain transcription factor Pax2 is required for the formation of the isthmic organizer (IsO) at the midbrain-hindbrain boundary, where it initiates expression of the IsO signal Fgf8. To gain further insight into the role of Pax2 in mid-hindbrain patterning, we searched for novel Pax2-regulated genes by cDNA microarray analysis of FACS-sorted GFP+ mid-hindbrain cells from wild-type and Pax2–/– embryos carrying a Pax2GFP BAC transgene. Here, we report the identification of five genes that depend on Pax2 function for their expression in the mid-hindbrain boundary region. These genes code for the transcription factors En2 and Brn1 (Pou3f3), the intracellular signaling modifiers Sef and Tapp1, and the non-coding RNA Ncrms. The Brn1 gene was further identified as a direct target of Pax2, as two functional Pax2-binding sites in the promoter and in an upstream regulatory element of Brn1 were essential for lacZ transgene expression at the mid-hindbrain boundary. Moreover, ectopic expression of a dominant-negative Brn1 protein in chick embryos implicated Brn1 in Fgf8 gene regulation. Together, these data defined novel functions of Pax2 in the establishment of distinct transcriptional programs and in the control of intracellular signaling during mid-hindbrain development.

Nephric duct insertion is a crucial step in urinary tract maturation that is regulated by a Gata3-Raldh2-Ret molecular network in mice

Urinary tract development depends on a complex series of events in which the ureter moves from its initial branch point on the nephric duct (ND) to its final insertion site in the cloaca (the primitive bladder and urethra). Defects in this maturation process can result in malpositioned ureters and hydronephrosis, a common cause of renal disease in children. Here, we report that insertion of the ND into the cloaca is an unrecognized but crucial step that is required for proper positioning of the ureter and that depends on Ret signaling. Analysis of Ret mutant mice at birth reveals hydronephrosis and defective ureter maturation, abnormalities that our results suggest are caused, at least in part, by delayed insertion of the ND. We find a similar set of malformations in mutants lacking either Gata3 or Raldh2. We show that these factors act in parallel to regulate ND insertion via Ret. Morphological analysis of ND extension in wild-type embryos reveals elaborate cellular protrusions at ND tips that are not detected in Ret, Gata3 or Raldh2 mutant embryos, suggesting that these protrusions may normally be important for fusion with the cloaca. Together, our studies reveal a novel Ret-dependent event, ND insertion, that, when abnormal, can cause obstruction and hydronephrosis at birth; whether ND defects underlie similar types of urinary tract abnormalities in humans is an interesting possibility.