Travelling down the tube: developmental biology of the ureter and urinary bladder

Transcription

1 Travelling down the tube: developmental biology of the ureter and urinary bladder Adrian S. Woolf Institute of Human Development University of Manchester

2 Children s Hospital and University of Manchester, UK

3 Who are they and what is it?

4 The Nobel Prize in Physics 2010 Andre Geim and Konstantin Novoselov University of Manchester, UK Discovered graphene a new class of material.2d atomic crystals

5 Topics Covered in this Lecture Formation of smooth muscle in lower renal tract Urinary tract epithelial differentiation Neuro-muscular differentiation of the urinary bladder

6 End-Stage Renal Failure (ESRF) in Children Of the 800 children in the United Kingdom with ESRF, about half were born with renal tract malformations. As a cause of childhood ESRF, renal tract malformations are x6 more common than any of the following: The congenital nephrotic syndromes; Metabolic diseases (cystinosis, primary hyperoxaluria) The nephronophthisis spectrum of disorders. North American Pediatric Renal Trials and Collaborative Studies database confirms that renal tract malformations are the commonest causes of chronic renal failure in children.

7 The Spectrum of Human Kidney Malformations 1. Agenesis (= the kidney never formed) 2. Dysplasia (= undifferentiated kidneys which may contain cysts) 3. Hypoplasia (= too few nephrons per kidney)

8 Spectrum of Kidney Malformations Kerecuk L et al Nat Clin Pract Nephrol 4: , 2008

9 Anomalies of the Ureter often Accompany Kidney Malformations Multicystic Dysplastic Pelvi-Ureteric Junction Vesico-Ureteric Reflux Kidney with Obstruction Atretic Ureter

10 Metanephric Cell Lineages Five weeks human gestation = embryonic day 10.5 mouse

11 Harmonised Kidney and Lower Renal Tract Development Metanephric kidney Fetal urinary bladder

12 Fetal Ureteric Peristalsis 1 Relaxed 2 Contraction begins * 4 Wave nears bladder 3 Wave half way down * * Caubit C, Lye CM et al Development 135: , 2008

13 Topics Covered in this Lecture Formation of smooth muscle in lower renal tract Urinary tract epithelial differentiation Neuro-muscular differentiation of the urinary bladder

14 Teashirt Transcription Factors teashirt is implicated in the development of the Drosophila kidney where it is expressed in mesenchymal stellate cells precursors intercalating between principal cells Stellate cells express teashirt (yellow) Data from Helen Skaer and Barry Denholm, Cambridge, UK

15 Developing Urinary Tract Muscle Expresses Teashirt-3 (Tshz3) Caubit C, Lye CM et al Development 135: , 2008

16 Teashirt-3 Expression at the Beginning of Renal Tract Development Caubit X, Lye CM et al Development 135: , 2008

17 Congenital Hydronephrosis in Teashirt-3 Homozygous Null Mutant Mice (Phenotype Apparent from Embryonic Day 16) Wild-type Null Mutant Caubit X, Lye CM et al Development 135: , 2008

18 There is no Anatomical Block to Urine Flow in Tshz3 Mutant Mice Wild-type Null Mutant Cubit X, Lye CM et al Development 135: , 2008

19 In Late Gestation, Proximal Ureters of Tshz3 Mutants Lack Smooth Muscle Proximal ureter Distal ureter Cubit X, Lye CM et al Development 135: , 2008

20 Organ Culture of E15 Ureters for Six Days - The Top of the Tshz3 Mutant Ureter Fails to Undergo Peristalsis Caubit C, Lye CM et al Development 135: , 2008

21 Lye CM et al J Am Soc Nephrol 21:24-30, 2010

22 Failure to Initiate Expression of Smooth Muscle Contractile Proteins In thetshz3 Mutant Mouse Proximal Ureter Caubit X, Lye CM et al Development 135: , 2008

23 Intact SHH and BMP4 Signals in Tshz3 Null Mutant Ureters Yu J et al Development 129: Caubit C, Lye CM et al Development 135: , 2008

24 Teashirt-3 and Myocardin are downstream to BMP4 in smooth muscle differentiation Wildtype Ureter Myocardin Tshz3 and Myocardin mrna levels pre and post BMP4 Tshz3 mutant ureter Caubit C, Lye CM et al Development 135: , 2008

25 Ureter Myogenesis: Putting Teashirt-3 into Context Sonic Hedgehog growth factor Lye CM et al J Am Soc Nephrol 21:24-30, 2010 Jenkins D et al J Anat 211: , 2007

26 TSHZ3 and Human Renal Malformations? TSHZ3 is expressed in normal human embryos in the proximal ureter. Jenkins D et al Nephrol Dial Transplant 2010;25: Translocations and a chromosomal deletion disrupting the 19q12 region where TSHZ3 resides reported in patients with hydonephrosis and multicystic dysplastic kidney Groenen PM et al Genomics 38: , 1996 Kulharya AS et al Am J Med Genet 77: , 1998

27 TSHZ3 and Human Disease? In 48 children with pelvi-ureteric junction obstruction, we found no mutations of TSHZ3. Non-significant increased frequency of missense (E469G) polymorphism versus 633 controls. Glutamic acid, a negatively charged and hydrophilic molecule, is changed to glycine, a nonpolar and hydrophobic residue Jenkins D et al Nephrol Dial Transplant 2010;25:54-60

28 Topics Covered in this Lecture Formation of smooth muscle in lower renal tract Urinary tract epithelial differentiation Neuro-muscular differentiation of the urinary bladder

29 Mature Urothelium In Vivo Superficial Intermediate Basal urothelial cells Lumen Stroma Superficial urothelial umbrella cells are coated by plaques of Asymmetric Unit Membrane (AUM) which limit transcellular permeability Jenny Southgate, University of York, UK

30 Uroplakins are Major Urothelial Proteins Fetal ureters Uroplakin a-smooth muscle actin Lye CM. PhD Thesis 2008

31 Uroplakins Asymmetric unit membrane Family of uroplakin proteins

32 Uroplakin III Null Mutant Mice Have Malformed Ureters and Bladders Wildtype Mutant Vesico-ureteric junctions Urothelial surfaces Kidneys Hu P et al J Cell Biol 151:961-72, 2000 Kong XT et al J Cell Biol 167: , 2004

33 Uroplakin III Protein Expression in the Human Fetus Uroplakin in brown Nuclei in blue Seven week bladder (urogenital sinus) Thirteen week ureter Thirteen week renal pelvis Jenkins D et al J Am Soc Nephrol 16: , 2005

34 De Novo UPIIIA Missense Mutations Cause Human Renal Tract Malformations A B 273 P/P 273 P/P 273 P/L UPIIIa wild-type UPIIIa Pro273Leu Jenkins D et al J Am Soc Nephrol 16: , 2005

35 Xenopus Uroplakin III Triggers Frog Development In unfertilized frog eggs, xupiii is protein is localised to the lipid rafts on the egg cell surface. After fertilization, xupiii is phosphorylated on tyrosine residue-249 in the carboxyl-terminal cytoplasmic tail. An antibody against xupiii blocks sperm-egg interaction and blocks egg activation. Sakakibara K et al J Biol Chem 280: , 2005

36 Embryonic Urothelial Cells Express both Uroplakins and SHH Jenkins D et al J Anat 211: , 2007

37 HARMONISED URINARY TRACT DEVELOPMENT Onset of urine production by fetal kidney Uroplakins sense a (yet-to-be defined) component of fetal urine Urothelia differentiate and establish a paracrine signalling centre Urothelial growth factors pattern adjacent smooth muscle Ureteric peristalsis propels urine to the bladder

38 Topics Covered in this Lecture Urinary tract epithelial differentiation Formation of smooth muscle in lower renal tract Neuro-muscular differentiation of the urinary bladder

39 Bernardo Ochoa Defined the Urofacial (Ochoa) Syndrome During the last 40 years over 100 patients have been reported with a dysfunctional lower urinary tract associated with a peculiar distortion of the facial expression. Genetic studies have demonstrated that this condition is inherited as an autosomal recessive trait, mapped to chromosome 10q23-q24. Patients with this syndrome as well as those with subclinical neurological bladder, occult neuropathic bladder, nonneurogenic neurogenic bladder (or Hinman syndrome) may be affected by the same congenital disorder of neurological origin. Ochoa B Pediatr Nephrol 19:6-12, 2004.

40 Individuals with Urofacial (Ochoa) Syndrome Who are Smiling Aydogdu O et al Eur J Pediatr 169: , 2010

41 Cystograms Showing a Spectrum of Dysmorphology in Uro-facial Syndrome Hyper-reflexic, incompletely emptying, trabeculated bladders with vesicoureteric reflux Aydogdu O et al Eur J Pediatr 169: , 2010

42 Urofacial Syndrome Family at Manchester Children s Hospital Whole genome SNP mapping in index case defined an autozygous region of 16Mb on chromosome 10q Homozygous mutations of HPSE2 found in this and then in several other families. HPSE2 encodes a haparanaselike protein. In health, HPSE2 is expressed in both brain and the urinary tract. Daly SB et al Am J Hum Genet 86: , 2010

43 What Does Heparanase Do? Heparanase activity cleaves heparan sulphate, thus modifying side chains of proteoglycans. Heparanase activity is implicated in organogenesis, angiogenesis, inflammation and tumour metastasis. Heparanase expression is induced in many hematological and solid tumors, associated with poor prognosis.

44 Heparanase 2 ( ) Interacts with Heparan Sulfate with High Affinity and Inhibits Heparanase 1 ( ) Activity Levy-Adam F et al J Biol Chem 285: , 2010

45 Immunolocalisation of heparanase-2 in fetal human urinary bladder Stuart HM, Roberts N et al submitted

46 Innervation of the Urinary Bladder Fowler CJ et al Nature Rev Neurosci 9: , 1998

47 Hpse2 null mutant mice have megabladders Wild type Homozygous mutant Woolf AS & Roberts N, unpublished data

48 Experimental downregulation of hpse2 In Xenopus tropicalis Wild-type * Normal proctodeum Splice morphant 1 Splice morphant 2 * Malformed proctodeum * Roberts N, Hilton E et al unpublished data

49 Experimental downregulation of hpse2 In Xenopus tropicalis Impaired neurogenesis control morpholino hpse2 morpholino

50 Summary Urinary tract epithelial differentiation Uroplakins Formation of smooth muscle in lower renal tract.teashirt-3 Neuro-muscular differentiation of the urinary bladder.heparanase-2

51 Acknowledgements for Ureter and Bladder Studies Manchester Renal Genetic clinics: Bronwyn Kerr, Helen Stuart, Kate Hillman Uroplakins: Dagan Jenkins Teashirt-3: Clare Lye, David Long and Laurent Fasano s team Urofacial syndrome/hpse2: Bill Newman, Helen Stuart, Emma Hilton, Neil Roberts, Eddie McKenzie Wellcome Trust/MRC Developmental Biology Resource Funding: BBSRC, Manchester BRC, KRUK, MRC