Targeted Tshz3 deletion in corticostriatal circuit components segregates core autistic behaviors.

We previously linked TSHZ3 haploinsufficiency to autism spectrum disorder (ASD) and showed that embryonic or postnatal Tshz3 deletion in mice results in behavioral traits relevant to the two core domains of ASD, namely social interaction deficits and repetitive behaviors. Here, we provide evidence that cortical projection neurons (CPNs) and striatal cholinergic interneurons (SCINs) are two main and complementary players in the TSHZ3-linked ASD syndrome. In the cerebral cortex, TSHZ3 is expressed in CPNs and in a proportion of GABAergic interneurons, but not in cholinergic interneurons or glial cells. In the striatum, TSHZ3 is expressed in all SCINs, while its expression is absent or partial in the other main brain cholinergic systems. We then characterized two new conditional knockout (cKO) models generated by crossing Tshz3flox/flox with Emx1-Cre (Emx1-cKO) or Chat-Cre (Chat-cKO) mice to decipher the respective role of CPNs and SCINs. Emx1-cKO mice show altered excitatory synaptic transmission onto CPNs and impaired plasticity at corticostriatal synapses, with neither cortical neuron loss nor abnormal layer distribution. These animals present social interaction deficits but no repetitive patterns of behavior. Chat-cKO mice exhibit no loss of SCINs but changes in the electrophysiological properties of these interneurons, associated with repetitive patterns of behavior without social interaction deficits. Therefore, dysfunction in either CPNs or SCINs segregates with a distinct ASD behavioral trait. These findings provide novel insights onto the implication of the corticostriatal circuitry in ASD by revealing an unexpected neuronal dichotomy in the biological background of the two core behavioral domains of this disorder.

Postsynaptic autism spectrum disorder genes and synaptic dysfunction.

This review provides an overview of the synaptic dysfunction of neuronal circuits and the ensuing behavioral alterations caused by mutations in autism spectrum disorder (ASD)-linked genes directly or indirectly affecting the postsynaptic neuronal compartment. There are plenty of ASD risk genes, that may be broadly grouped into those involved in gene expression regulation (epigenetic regulation and transcription) and genes regulating synaptic activity (neural communication and neurotransmission). Notably, the effects mediated by ASD-associated genes can vary extensively depending on the developmental time and/or subcellular site of expression. Therefore, in order to gain a better understanding of the mechanisms of disruptions in postsynaptic function, an effort to better model ASD in experimental animals is required to improve standardization and increase reproducibility within and among studies. Such an effort holds promise to provide deeper insight into the development of these disorders and to improve the translational value of preclinical studies.

Haploinsufficiency of the mouse Tshz3 gene leads to kidney defects.

Renal tract defects and autism spectrum disorder (ASD) deficits represent the phenotypic core of the 19q12 deletion syndrome caused by the loss of one copy of the TSHZ3 gene. Although a proportion of Tshz3 heterozygous (Tshz3+/lacZ) mice display ureteral defects, no kidney defects have been reported in these mice. The purpose of this study was to characterize the expression of Tshz3 in adult kidney as well as the renal consequences of embryonic haploinsufficiency of Tshz3 by analyzing the morphology and function of Tshz3 heterozygous adult kidney. Here, we described Tshz3 expression in the smooth muscle and stromal cells lining the renal pelvis, the papilla and glomerular endothelial cells (GEnCs) of the adult kidney as well as in the proximal nephron tubules in neonatal mice. Histological analysis showed that Tshz3+/lacZ adult kidney had an average of 29% fewer glomeruli than wild-type kidney. Transmission electron microscopy of Tshz3+/lacZ glomeruli revealed a reduced thickness of the glomerular basement membrane and a larger foot process width. Compared to wild type, Tshz3+/lacZ mice showed lower blood urea, phosphates, magnesium and potassium at 2 months of age. At the molecular level, transcriptome analysis identified differentially expressed genes related to inflammatory processes in Tshz3+/lacZ compare to wild-type (control) adult kidneys. Lastly, analysis of the urinary peptidome revealed 33 peptides associated with Tshz3+/lacZ adult mice. These results provide the first evidence that in the mouse Tshz3 haploinsufficiency leads to cellular, molecular and functional abnormalities in the adult mouse kidney.

Camk2a-Cre and Tshz3 Expression in Mouse Striatal Cholinergic Interneurons: Implications for Autism Spectrum Disorder.

Camk2a-Cre mice have been widely used to study the postnatal function of several genes in forebrain projection neurons, including cortical projection neurons (CPNs) and striatal medium-sized spiny neurons (MSNs). We linked heterozygous deletion of TSHZ3/Tshz3 gene to autism spectrum disorder (ASD) and used Camk2a-Cre mice to investigate the postnatal function of Tshz3, which is expressed by CPNs but not MSNs. Recently, single-cell transcriptomics of the adult mouse striatum revealed the expression of Camk2a in interneurons and showed Tshz3 expression in striatal cholinergic interneurons (SCINs), which are attracting increasing interest in the field of ASD. These data and the phenotypic similarity between the mice with Tshz3 haploinsufficiency and Camk2a-Cre-dependent conditional deletion of Tshz3 (Camk2a-cKO) prompted us to better characterize the expression of Tshz3 and the activity of Camk2a-Cre transgene in the striatum. Here, we show that the great majority of Tshz3-expressing cells are SCINs and that all SCINs express Tshz3. Using lineage tracing, we demonstrate that the Camk2a-Cre transgene is expressed in the SCIN lineage where it can efficiently elicit the deletion of the Tshz3-floxed allele. Moreover, transcriptomic and bioinformatic analysis in Camk2a-cKO mice showed dysregulated striatal expression of a number of genes, including genes whose human orthologues are associated with ASD and synaptic signaling. These findings identifying the expression of the Camk2a-Cre transgene in SCINs lineage lead to a reappraisal of the interpretation of experiments using Camk2a-Cre-dependent gene manipulations. They are also useful to decipher the cellular and molecular substrates of the ASD-related behavioral abnormalities observed in Tshz3 mouse models.

Disruption of NEUROD2 causes a neurodevelopmental syndrome with autistic features via cell-autonomous defects in forebrain glutamatergic neurons.

While the transcription factor NEUROD2 has recently been associated with epilepsy, its precise role during nervous system development remains unclear. Using a multi-scale approach, we set out to understand how Neurod2 deletion affects the development of the cerebral cortex in mice. In Neurod2 KO embryos, cortical projection neurons over-migrated, thereby altering the final size and position of layers. In juvenile and adults, spine density and turnover were dysregulated in apical but not basal compartments in layer 5 neurons. Patch-clamp recordings in layer 5 neurons of juvenile mice revealed increased intrinsic excitability. Bulk RNA sequencing showed dysregulated expression of many genes associated with neuronal excitability and synaptic function, whose human orthologs were strongly associated with autism spectrum disorders (ASD). At the behavior level, Neurod2 KO mice displayed social interaction deficits, stereotypies, hyperactivity, and occasionally spontaneous seizures. Mice heterozygous for Neurod2 had similar defects, indicating that Neurod2 is haploinsufficient. Finally, specific deletion of Neurod2 in forebrain excitatory neurons recapitulated cellular and behavioral phenotypes found in constitutive KO mice, revealing the region-specific contribution of dysfunctional Neurod2 in symptoms. Informed by these neurobehavioral features in mouse mutants, we identified eleven patients from eight families with a neurodevelopmental disorder including intellectual disability and ASD associated with NEUROD2 pathogenic mutations. Our findings demonstrate crucial roles for Neurod2 in neocortical development, whose alterations can cause neurodevelopmental disorders including intellectual disability and ASD.

Construct Validity and Cross Validity of a Test Battery Modeling Autism Spectrum Disorder (ASD) in Mice.

Modeling in other organism species is one of the crucial stages in ascertaining the association between gene and psychiatric disorder. Testing Autism Spectrum Disorder (ASD) in mice is very popular but construct validity of the batteries is not available. We presented here the first factor analysis of a behavioral model of ASD-like in mice coupled with empirical validation. We defined fourteen measures aligning mouse-behavior measures with the criteria defined by DSM-5 for the diagnostic of ASD. Sixty-five mice belonging to a heterogeneous pool of genotypes were tested. Reliability coefficients vary from .68 to .81. The factor analysis resulted in a three- factor solution in line with DSM criteria: social behavior, stereotypy and narrowness of the field of interest. The empirical validation with mice sharing a haplo-insufficiency of the zinc-finger transcription factor TSHZ3/Tshz3 associated with ASD shows the discriminant power of the highly loaded items.

Postnatal Tshz3 Deletion Drives Altered Corticostriatal Function and Autism Spectrum Disorder-like Behavior.

BACKGROUND: Heterozygous deletion of the TSHZ3 gene, encoding for the teashirt zinc-finger homeobox family member 3 (TSHZ3) transcription factor that is highly expressed in cortical projection neurons (CPNs), has been linked to an autism spectrum disorder (ASD) syndrome. Similarly, mice with Tshz3 haploinsufficiency show ASD-like behavior, paralleled by molecular changes in CPNs and corticostriatal synaptic dysfunctions. Here, we aimed at gaining more insight into "when" and "where" TSHZ3 is required for the proper development of the brain, and its deficiency crucial for developing this ASD syndrome. METHODS: We generated and characterized a novel mouse model of conditional Tshz3 deletion, obtained by crossing Tshz3flox/flox with CaMKIIalpha-Cre mice, in which Tshz3 is deleted in CPNs from postnatal day 2 to 3 onward. We characterized these mice by a multilevel approach combining genetics, cell biology, electrophysiology, behavioral testing, and bioinformatics. RESULTS: These conditional Tshz3 knockout mice exhibit altered cortical expression of more than 1000 genes, ∼50% of which have their human orthologue involved in ASD, in particular genes encoding for glutamatergic synapse components. Consistently, we detected electrophysiological and synaptic changes in CPNs and impaired corticostriatal transmission and plasticity. Furthermore, these mice showed strong ASD-like behavioral deficits. CONCLUSIONS: Our study reveals a crucial postnatal role of TSHZ3 in the development and functioning of the corticostriatal circuitry and provides evidence that dysfunction in these circuits might be determinant for ASD pathogenesis. Our conditional Tshz3 knockout mouse constitutes a novel ASD model, opening the possibility for an early postnatal therapeutic window for the syndrome linked to TSHZ3 haploinsufficiency.

Analysis of the Teashirt Target Genes in Ureteric Bud Development.

Microarrays and RNA-seq (RNA sequencing) are powerful techniques to assess transcript abundance in biological samples and to improve our understanding of the relationship between genotype and phenotype. Tshz3+/- heterozygous mouse is a model for a human 19q12 syndrome characterized by autistic traits and renal tract defects (Caubit et al., Nat Genet 48:1359-1369, 2016). To unravel renal tract pathological mechanisms, we took advantage of Tshz3 mouse and performed comparative genome-wide expression profiling on embryonic ureter and/or kidney.

TSHZ3 deletion causes an autism syndrome and defects in cortical projection neurons.

TSHZ3, which encodes a zinc-finger transcription factor, was recently positioned as a hub gene in a module of the genes with the highest expression in the developing human neocortex, but its functions remained unknown. Here we identify TSHZ3 as the critical region for a syndrome associated with heterozygous deletions at 19q12-q13.11, which includes autism spectrum disorder (ASD). In Tshz3-null mice, differentially expressed genes include layer-specific markers of cerebral cortical projection neurons (CPNs), and the human orthologs of these genes are strongly associated with ASD. Furthermore, mice heterozygous for Tshz3 show functional changes at synapses established by CPNs and exhibit core ASD-like behavioral abnormalities. These findings highlight essential roles for Tshz3 in CPN development and function, whose alterations can account for ASD in the newly defined TSHZ3 deletion syndrome.

Tshz1 Regulates Pancreatic ß-Cell Maturation.

The homeodomain transcription factor Pdx1 controls pancreas organogenesis, specification of endocrine pancreas progenitors, and the postnatal growth and function of pancreatic ß-cells. Pdx1 expression in human-derived stem cells is used as a marker for induced pancreatic precursor cells. Unfortunately, the differentiation efficiency of human pancreatic progenitors into functional ß-cells is poor. In order to gain insight into the genes that Pdx1 regulates during differentiation, we performed Pdx1 chromatin immunoprecipitation followed by high-throughput sequencing of embryonic day (e) 13.5 and 15.5 mouse pancreata. From this, we identified the transcription factor Teashirt zinc finger 1 (Tshz1) as a direct Pdx1 target. Tshz1 is expressed in developing and adult insulin- and glucagon-positive cells. Endocrine cells are properly specified in Tshz1-null embryos, but critical regulators of ß-cell (Pdx1 and Nkx6.1) and α-cell (MafB and Arx) formation and function are downregulated. Adult Tshz1(+/-) mice display glucose intolerance due to defects in glucose-stimulated insulin secretion associated with reduced Pdx1 and Clec16a expression in Tshz1(+/-) islets. Lastly, we demonstrate that TSHZ1 levels are reduced in human islets of donors with type 2 diabetes. Thus, we position Tshz1 in the transcriptional network of maturing ß-cells and suggest that its dysregulation could contribute to the islet phenotype of human type 2 diabetes.

TSHZ3 and SOX9 regulate the timing of smooth muscle cell differentiation in the ureter by reducing myocardin activity.

Smooth muscle cells are of key importance for the proper functioning of different visceral organs including those of the urogenital system. In the mouse ureter, the two transcriptional regulators TSHZ3 and SOX9 are independently required for initiation of smooth muscle differentiation from uncommitted mesenchymal precursor cells. However, it has remained unclear whether TSHZ3 and SOX9 act independently or as part of a larger regulatory network. Here, we set out to characterize the molecular function of TSHZ3 in the differentiation of the ureteric mesenchyme. Using a yeast-two-hybrid screen, we identified SOX9 as an interacting protein. We show that TSHZ3 also binds to the master regulator of the smooth muscle program, MYOCD, and displaces it from the coregulator SRF, thereby disrupting the activation of smooth muscle specific genes. We found that the initiation of the expression of smooth muscle specific genes in MYOCD-positive ureteric mesenchyme coincides with the down regulation of Sox9 expression, identifying SOX9 as a possible negative regulator of smooth muscle cell differentiation. To test this hypothesis, we prolonged the expression of Sox9 in the ureteric mesenchyme in vivo. We found that Sox9 does not affect Myocd expression but significantly reduces the expression of MYOCD/SRF-dependent smooth muscle genes, suggesting that down-regulation of Sox9 is a prerequisite for MYOCD activity. We propose that the dynamic expression of Sox9 and the interaction between TSHZ3, SOX9 and MYOCD provide a mechanism that regulates the pace of progression of the myogenic program in the ureter.

The tiptop/teashirt genes regulate cell differentiation and renal physiology in Drosophila.

The physiological activities of organs are underpinned by an interplay between the distinct cell types they contain. However, little is known about the genetic control of patterned cell differentiation during organ development. We show that the conserved Teashirt transcription factors are decisive for the differentiation of a subset of secretory cells, stellate cells, in Drosophila melanogaster renal tubules. Teashirt controls the expression of the water channel Drip, the chloride conductance channel CLC-a and the Leukokinin receptor (LKR), all of which characterise differentiated stellate cells and are required for primary urine production and responsiveness to diuretic stimuli. Teashirt also controls a dramatic transformation in cell morphology, from cuboidal to the eponymous stellate shape, during metamorphosis. teashirt interacts with cut, which encodes a transcription factor that underlies the differentiation of the primary, principal secretory cells, establishing a reciprocal negative-feedback loop that ensures the full differentiation of both cell types. Loss of teashirt leads to ineffective urine production, failure of homeostasis and premature lethality. Stellate cell-specific expression of the teashirt paralogue tiptop, which is not normally expressed in larval or adult stellate cells, almost completely rescues teashirt loss of expression from stellate cells. We demonstrate conservation in the expression of the family of tiptop/teashirt genes in lower insects and establish conservation in the targets of Teashirt transcription factors in mouse embryonic kidney.

Teashirt-3, a novel regulator of muscle differentiation, associates with BRG1-associated factor 57 (BAF57) to inhibit myogenin gene expression.

In adult muscles and under normal physiological conditions, satellite cells are found in a quiescent state but can be induced to enter the cell cycle by signals resulting from exercise, injury-induced muscle regeneration, or specific disease states. Once activated, satellite cells proliferate, self-renew, and differentiate to form myofibers. In the present study, we found that the zinc finger-containing factor Teashirt-3 (TSHZ3) was expressed in quiescent satellite cells of adult mouse skeletal muscles. We showed that following treatment with cardiotoxin TSHZ3 was strongly expressed in satellite cells of regenerating muscles. Moreover, immunohistochemical analysis indicated that TSHZ3 was expressed in both quiescent and activated satellite cells on intact myofibers in culture. TSHZ3 expression was maintained in myoblasts but disappeared with myotube formation. In C2C12 myoblasts, we showed that overexpression of Tshz3 impaired myogenic differentiation and promoted the down-regulation of myogenin (Myog) and up-regulation of paired-box factor 7 (Pax7). Moreover, knockdown experiments revealed a selective effect of Tshz3 on Myog regulation, and transcriptional reporter experiments indicated that TSHZ3 repressed Myog promoter. We identified the BRG1-associated factor 57 (BAF57), a subunit of the SWI/SNF complex, as a partner of TSHZ3. We showed that TSHZ3 cooperated with BAF57 to repress MYOD-dependent Myog expression. These results suggest a novel mechanism for transcriptional repression by TSHZ3 in which TSHZ3 and BAF57 cooperate to modulate MyoD activity on the Myog promoter to regulate skeletal muscle differentiation.

Teashirt 3 regulates development of neurons involved in both respiratory rhythm and airflow control.

Neonatal breathing in mammals involves multiple neuronal circuits, but its genetic basis remains unclear. Mice deficient for the zinc finger protein Teashirt 3 (TSHZ3) fail to breathe and die at birth. Tshz3 is expressed in multiple areas of the brainstem involved in respiration, including the pre-Bötzinger complex (preBötC), the embryonic parafacial respiratory group (e-pF), and cranial motoneurons that control the upper airways. Tshz3 inactivation led to pronounced cell death of motoneurons in the nucleus ambiguus and induced strong alterations of rhythmogenesis in the e-pF oscillator. In contrast, the preBötC oscillator appeared to be unaffected. These deficits result in impaired upper airway function, abnormal central respiratory rhythm generation, and altered responses to pH changes. Thus, a single gene, Tshz3, controls the development of diverse components of the circuitry required for breathing.

Ureter myogenesis: putting Teashirt into context.

After the basic shape of the mammalian ureter is established, its epithelia mature and a coat of smooth muscle cells differentiate around nascent urothelia. The ureter actively propels tubular fluid from the renal pelvis to the bladder, and this peristalsis, which starts in the fetal period, requires coordinated smooth muscle contraction. Teashirt-3 (Tshz3) is expressed in smooth muscle cell precursors that form the wall of the forming mammalian ureter. The Teashirt gene family was first identified in Drosophila where Teashirt (Tsh) protein acts as a transcription factor directing embryonic anterior-posterior patterning and leg and eye development. In fly embryonic renal tubules, Tsh is expressed in mesodermally derived stellate cells intercalating between principal cells, and a paralogue, tiptop, is expressed in forming tubules. Teashirt is a component of several gene networks in flies and it is notable that similar networks control mammalian renal tract development. Null mutation of Tshz3 in mice leads to failure of functional muscularization in the top of the ureter and this is followed by congenital hydronephrosis. A signaling pathway can be envisaged, starting with sonic hedgehog secreted by the nascent ureteric urothelium and ending with ureteric smooth muscle cell differentiation, with Tshz3 downstream of bone morphogenetic protein 4 and upstream of myocardin and smooth muscle cell contractile protein synthesis. The phenotype of Tshz3 mutant mice resembles that of human congenital pelviureteric junction obstruction, and we suggest these individuals may have mutations of genes encoding molecules in the differentiation pathway mediated by Tshz3.

Analysis of TSHZ2 and TSHZ3 genes in congenital pelvi-ureteric junction obstruction.

BACKGROUND: Congenital pelvi-ureteric junction obstruction (PUJO) affects 0.3% of human births. It may result from aberrant smooth muscle development in the renal pelvis, resulting in hydronephrosis. Mice that are null mutant for the Teashirt3 (Tshz3) gene exhibit congenital PUJO with defective smooth muscle differentiation and absent peristalsis in the proximal ureter. METHODS: Given the phenotype of Tshz3 mutant mice, we considered that Teashirt genes, which code for a family of transcription factors, might represent candidate genes for human PUJO. To evaluate this possibility, we used in situ hydridization to analyse the three mammalian Tshz genes in mouse embryonic ureters and determined whether TSHZ3 was expressed in the human embryonic ureter. TSHZ2 and TSHZ3 were sequenced in index cases with non-syndromic PUJO. RESULTS: Tshz2 and Tshz3 genes were detected in mouse ureters and TSHZ3 was expressed in the human embryonic renal pelvis. Direct sequencing of TSHZ2 and TSHZ3 did not identify any mutations in an initial cohort of 48 PUJO index cases, excluding these genes as a major cause of this condition. A polymorphic missense change (E469G) in TSHZ3 was identified at a residue highly conserved throughout evolution in all Teashirt proteins, although subsequently no significant difference between the E469G allele frequency in Albanian and Macedonian PUJO index cases (3.2%) versus 633 control individuals (1.7%) was found (P = 0.18). CONCLUSIONS: Mutations in TSHZ2 and TSHZ3 are not a major cause of PUJO, at least in Albanian and Macedonian populations. Expression of these genes in the human fetal ureter emphasizes the importance of analysing these genes in other groups of patients with renal tract malformations.

Teashirt 3 is necessary for ureteral smooth muscle differentiation downstream of SHH and BMP4.

Ureteric contractions propel foetal urine from the kidney to the urinary bladder. Here, we show that mouse ureteric smooth muscle cell (SMC) precursors express the transcription factor teashirt 3 (TSHZ3), and that Tshz3-null mutant mice have congenital hydronephrosis without anatomical obstruction. Ex vivo, the spontaneous contractions that occurred in proximal segments of wild-type embryonic ureter explants were absent in Tshz3 mutant ureters. In vivo, prior to the onset of hydronephrosis, mutant proximal ureters failed to express contractile SMC markers, whereas these molecules were detected in controls. Mutant embryonic ureters expressed Shh and Bmp4 transcripts as normal, with appropriate expression of Ptch1 and pSMAD1/5/8 in target SM precursors, whereas myocardin, a key regulator for SMC differentiation, was not expressed in Tshz3-null ureters. In wild-type embryonic renal tract explants, exogenous BMP4 upregulated Tshz3 and myocardin expression. More interestingly, in Tshz3 mutant renal tract explants, exogenous BMP4 did not improve the Tshz3 phenotype. Thus, Tshz3 is required for proximal ureteric SMC differentiation downstream of SHH and BMP4. Furthermore, the Tshz3 mutant mouse model of ;functional' urinary obstruction resembles congenital pelvi-ureteric junction obstruction, a common human malformation, suggesting that TSHZ, or related, gene variants may contribute to this disorder.

Tshz1 is required for axial skeleton, soft palate and middle ear development in mice.

Members of the Tshz gene family encode putative zinc fingers transcription factors that are broadly expressed during mouse embryogenesis. Tshz1 is detected from E9.5 in the somites, the spinal cord, the limb buds and the branchial arches. In order to assess the function of Tshz1 during mouse development, we generated Tshz1-deficient mice. Tshz1 inactivation leads to neonatal lethality and causes multiple developmental defects. In the craniofacial region, loss of Tshz1 function leads to specific malformations of middle ear components, including the malleus and the tympanic ring. Tshz1(-/-) mice exhibited Hox-like vertebral malformations and homeotic transformations in the cervical and thoracic regions, suggesting that Tshz1 and Hox genes are involved in common pathways to control skeletal morphogenesis. Finally, we demonstrate that Tshz1 is required for the development of the soft palate.