Mechanisms of Organogenesis

Project Leader
Prof. Seppo Vainio, Ph.D., Vice Scientific Director of the Institute of Biomedicine
Laboratory of Developmental Biology, Oulu Center for Cell-Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu

Background and Significance

It has been speculated that ontogenesis recapitulates to certain degree the process of evolution. Relatively limited mRNA encoding gene families coordinate ontogenesis in all multicellular animal species. We also know that the same regulatory circuits are used several times and in different contexts and organs to drive the step-by-step process of morphogenesis and the associated cell differentiations. The ontogenesis control genes when reactivated ectopically later in the life cycle in the stem cell niches and other regions have been speculated to be the reason behind many diseases such as cancer. It is by now well established that the developmental control genes remain active in the adult in the organs that have the capacity to regenerate as a response to injury for example. The stem cell researchers speculate that perhaps many of the described degenerative diseases may involve changes in the mode of the tissue and organ renewal processed in the resident stem cells.

Other major issue of significance when considering the relevance of the ontogenesis studies is that in utero insults, such as stress may also predispose later in life to the acquired diseases such as diabetes, metabolic syndrome, cardiovascular diseases and cancer. There is evidence that the time in utero and breast feeding period appears to be some how, capable of epigenetically programming the behavioural fundaments of certain measurable characters later, earlier considered to be in part set up by the classic Mendelian inheritance. In connection to these findings the discoveries that the genome expresses around 98% of the so called non coding RNA species that are in part secreted in lipid encapsulated vesicles sets great new challenges to our views on how we are predisposed to diseases and how we inherit the characters, issues that are targeted in our research lines.

Recent Progress

The vasculature is critical for renal function, but little is known about how the complex network is being generated. Due to this reason we targeted the cell types that are behind kidney vasculature development. Tie1Cre;R26R(YFP)-based fate mapping in combination with a novel time-lapse embryonic kidney organ culture set up revealed the mode of sequence by which the kidney vasculature development occurs and correlation with the CD31(+) cells. When the Tie1(+) or the CD31(+) endothelial cells (EC) where depleted either with the Tie1Cre-induced diphtheria toxin susceptibility or cell sorting by taking use of also a novel dissociation and reaggregation approach this revealed striking regeneration. Depletion of the CD146(+) ECs  abolished the EC network  regeneration. Elegant fate mapping with a developed universal mapping strategy that was based on green fluorescent protein (GFP)-marked CD146(+)/CD31(-) cells purified by FACS indicated in the reaggregates that these cells are CD31(+) cells in origin. These assembled with the EC structures with the CD31(+) wild-type ECs. We also showed that the EC network development requires VEGF signalling, and that VEGF and erythropoietin expression does not require external hypoxia. Based on the data we conclude that the ex vivo kidney organ culture set up provides a great and novel way to target renal EC development. The data demonstrates that CD146(+) cells are the progenitors that assemble the kidney vasculature.

Our work where we took use of the Wnt11 knock out mouse models indicated that deficiencies in Wnt11 function leads to anomalies in the kidney tubular system and lead to secondary glomerular cysts. Wnt11 is expressed in the developing kidney in the emerging tubular system and this suggested a role in the epithelial ductal system ontogenesis. We found that in the C57Bl6 strain the Wnt-11 knock out mice also survived to the postnatal stages and some could even reach adulthood. The urine and plasma analyses of such survivors revealed compromised kidney function. The tubules of Wnt11-/- mice appeared enlarged and the tubular convolution was also not normal. The Wnt11 -/- mice had notable secondary glomerular cysts unlike in controls. The Wnt11 failure reduced Wnt9b, Six2, Foxd1 and Hox10 marker gene expression. These factors have been implicated in kidney development suggesting roles in the Wnt11 deficiency caused phenotypes. The Dvl2, that is an important PCP pathway component, was down regulated by more than 90 % in case of Wnt11 deficiency. We conclude that the Wnt11 signal coordinates also the tubular system development and that the knock out serves as a model for studying the mechanisms behind tubular anomalies and glomerular cyst formation.

Besides Wnt11 we found that Wnt5a is expressed in ureteric bud (UB) and that its absence leads to duplex collecting system, uni- or bilateral kidney agenesis, hypoplasia with altered pattern of ureteric tree organization and lobularization defects with partly fused ureter trunks not noted in controls. The Wnt5a deficiency reduced the UB tip number. The absence of Wnt5a associated with anomalies in the ultrastructure of the collecting duct cells and the basement membrane (BM) that was thicker when compared to controls. Consistent with these findings laminin and of type IV collagen expression was reduced in case of Wnt5a impairment. The P4ha1 gene and the C-P4H enzyme activity were also higher when compared to controls. The kidney associated phenotypes where severe also in the compound Wnt5a+/-;P4ha1+/- embryos. We identified a R260H WNT5A variant from a renal human disease cohort. Functional studies with a corresponding mouse variant reduced Wnt5a-signalling in  the experimental setting. We suggested that Wnt5a has a novel function during kidney ontogenesis, especially in patterning of UB derived collecting duct development. Changes in WNT5A signalling may contribute to congenital diseases in the kidney.

The Müllerian duct (MD) is the anlage of the oviduct, uterus and upper part of the vagina, the main parts of the female reproductive tract. We targeted the roles of the Wnt4+ cells in construction of the female sex duct by using time lapse organ culture of the novel gonadal rudiment and the (EGFPCre)-based fate mapping technologies. The studies revealed that the MD is indeed derived initially from the Wnt4+ progenitor cells. It appeared that the Wnt4 signal is required early in the MD-forming cells but also later since the anti-Wnt4 function-blocking antibodies inhibited the ductal elongation also later as well. We found that some of the Wnt4mCh/mCh hypomorphic inherited allele positive mice survived to adulthood. They were able to assemble the MD in around 45% of the cases. The MD was however severely malformed so that the associated epithelial cell polarization and basement membrane were deregulated relative to controls. The oviduct had coiled poorly, the endometrial glands had been lost, the myometrium was dedifferentiated and a hydro-uterus was typically present as well. We concluded that the Wnt4 signal is important not only for the female sex determination, oocyte maturation but also later in ontogenesis of the female reproductive tract.

In our other collaborative work we targeted the role of the Wnt signalling pathway in the zonation of the adrenal gland. In specific the capsular R-Spondin3 (RSPO3) signals to the underlying steroidogenic compartment to determine the glomerulosa fate via the induced β-catenin signalling.  Absence of the RSPO3 factor reduced Sonic Hedgehog signalling suggesting a cause behind the growth impairment. The Rspo3 has also a function later in the adult to maintain the zona glomerulosa. This data indicates that the adrenal capsule serves as a central signalling centre to coordinate cell replacement when cells are damaged and also to maintain zonation during the life cycle.

We revealed further details with our collaborators of how the Wnt4 signal is involved in the zonation of the adrenal gland. The PKA activation prevented zona glomerulosa (ZF) differentiation through Wnt4 mediated repression and inhibition of the Wnt signalling pathway. The data point that PKA activation serves to inhibit the Wnt signalling and via promotion of the concurrent cell lineage commitment. Besides the normal ontogenesis roles the Wnt signalling pathway appears to be involved in tumourigenesis. Thus judgement is based on the data that partial inactivation of PKA catalytic activity stimulated β-catenin-induced tumorigenesis for example. Together low PKA activity but high Wnt signalling provides a poor prognosis for the adrenocortical carcinoma (ACC) patients. It appears that the PKA functions as a tumour suppressor in the adrenal cortex, through repression of WNT signalling activity.

Future Goals

The capacity to (re)program cells to specific cell lineages opened tremendous opportunities in medicine. We can to date identify the key factors even from the single cells. This has set the stage for detailed analysis of the organogenesis mechanisms as well. The methods to be able to store the nephron progenitor cells offered concrete ways to archive the kidney cells. Such cells serve as the “building blocks” to reconstruct the kidney and to model diseases at the level of the organoid models. With the gene editing tools large-scale ontogenesis programming research is a realistic vision. The renal diseased cells, their gene corrected versions offer together with the micro fluidistic organoid cultures new ways to model human physiology and to screen novel drugs. The single cell resolution approaches and the 4D imaging tools enlarge our horizons at present. The pioneers of the Finnish kidney school, Lauri Saxén and Sulo Toivonen who established the ex vivo organ cultures would be striked to see that their models are again in fashion after 50 years of their work. Finally the expressed non-coding RNA species and their presence in the secreted vesicles can be expected to influence our views of the fundamental control mechanisms in nature.

Publications 2016-

Drelon C, Berthon A, Sahut-Barnola I, Mathieu M, Dumontet T, Rodriguez S, Batisse-Lignier M, Tabbal H, Tauveron I, Lefrançois-Martinez AM, Pointud JC, Gomez-Sanchez CE, Vainio S, Shan J, Sacco S, Schedl A, Stratakis CA, Martinez A, Val P. PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development. Nat Commun 7:12751, 2016.

Halt KJ, Pärssinen HE, Junttila SM, Saarela U, Sims-Lucas S, Koivunen P, Myllyharju J, Quaggin S, Skovorodkin IN, Vainio SJ. CD146(+) cells are essential  for kidney vasculature development. Kidney Int 90:311-24, 2016.

Nagy II, Xu Q, Naillat F, Ali N, Miinalainen I, Samoylenko A, Vainio SJ. Impairment of Wnt11 function leads to kidney tubular abnormalities and secondary glomerular cystogenesis. BMC Dev Biol. 16:30, 2016.

Pietilä I, Prunskaite-Hyyryläinen R, Kaisto S, Tika E, van Eerde AM, Salo AM,  Garma L, Miinalainen I, Feitz WF, Bongers EM, Juffer A, Knoers NV, Renkema KY, Myllyharju J, Vainio SJ. Wnt5a Deficiency Leads to Anomalies in Ureteric Tree Development, Tubular Epithelial Cell Organization and Basement Membrane Integrity Pointing to a Role in Kidney Collecting Duct Patterning. PLoS One. 11:e0147171, 2016.

Prunskaite-Hyyryläinen R, Skovorodkin I, Xu Q, Miinalainen I, Shan J, Vainio SJ. Wnt4 coordinates directional cell migration and extension of the Müllerian duct essential for ontogenesis of the female reproductive tract. Hum Mol Genet. 25(6):1059-73, 2016.

Rak-Raszewska A, Vainio S. Nephrogenesis in organoids to develop novel drugs and progenitor cell based therapies. Eur J Pharmacol. 790:3-11, 2016.

Vidal V, Sacco S, Rocha AS, da Silva F, Panzolini C, Dumontet T, Doan TM, Shan J, Rak-Raszewska A, Bird T, Vainio S, Martinez A, Schedl A. The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3. Genes  Dev 30(12):1389-94, 2016.

Xu Q, Krause M, Samoylenko A, Vainio S. Wnt Signaling in Renal Cell Carcinoma. Cancers (Basel). 8(6), 2016.

Saarela U, Akram SU, Desgrange A, Rak-Raszewska A, Shan J, Cereghini S, Ronkainen VP, Heikkilä J, Skovorodkin I, Vainio S. Novel Fixed Z-Dimension (FiZD) Kidney Primordia and an Organoid Culture System for Time-lapse Confocal Imaging. Development. epub ahead of print, 2017.

Monsivaisa D, Clementia C, Penga J, Fullertona P, Prunskaite-Hyyryläinen R, Vainio S, Matzuk M. BMP7 induces uterine receptivity and blastocyst attachment. Endochrinology, in press.

Reint G, Rak-Raszewska A, Vainio S.  Kidney Development and Perspectives for Organ Engineering. Cell and Tissue Research, in press.

Patents 2016

Strategy to engineering the mammalian skin stem cells to serve as novel biosensors, PCT-application, Ref: 013226_TISA and Ref: 2150199PC.

Research Group Members

Project Leader:
Seppo Vainio, professor (Oulu University)

Senior and Post-doctoral Investigators:
Aleksandra Rak-Raszewska, Ph.D. (FiDiPro, Academy of Finland)
Ilya Skovorodkin, Ph.D. (Academy of Finland, Center of Excellence)
Timo Pikkarainen, Ph.D. (Academy of Finland, Center of Excellence)
Anatoliy Samoylenko, Ph.D. (FiDiPro, Academy of Finland)
Nsrein Ali, Ph.D. (Tekes)
Genevieve Bart, Ph.D. (Tekes)
Mirja Krause, Ph.D. (Tekes)
Ilkka Pietilä, Ph.D. (Academy of Finland)
Jingdong Shan, Ph.D. (Biocenter Oulu)
Renata Prunskaite-Hyyryläinen, Ph.D. (EURenomics)
Florence Naillat, Ph.D. (Adademy of Finland)

Ph.D. Students:
Zenglai Tan, M.Sc. (EU, Marie Curie, RenalTract)
Prateek Singh, M.Sc., (Biocenter Oulu)
Susanna Kaisto, M.Sc. (Biocenter Oulu, Oulu-Ulm)
Abhishek Sharma, M.Sc. (Sigrid Jusélius Foundation)
Ulla Saarela, M.Sc. (FBMM)
Qi Xu, M.Sc. (Biocenter Oulu)
Tuomas Nurmi, M.Sc. Student (Sigrid Jusélius Foundation)
Ganna Reint, M.Sc. (CIMO)
Kimmo Halt, M.D. (Oulu University Hospital)

Susan E Quaggin, M.D., Northwestern University Feinberg School of Medicine, Director, Feinberg Cardiovascular Research Institute, Chief, Division of Medicine-Nephrology, Charles H. Mayo, M.D. Professor in Medicine-Nephrology.

Laboratory Technicians 3 (Academy of Finland, Center of Excellence)

Foreign Scientists 12

National and International Activities

Group Members Who Spent More Than Two Weeks in Foreign Laboratories During 2016

Dr. Mirja Krause, The Ritchie Centre, Hudson Institute of Medical Research Core, Clayton, Victoria, Australia
Dr. Florence Naillat, University Cambridge, United Kingdom, UK
Dr. Ilkka Pietilä, Department of Medical Cell, Uppsala Biomedicinska centrum, BMC, Uppsala, Sweden.
Dr. Renata Prunskaite-Hyyryläinen, Baylor College of Medicine, USA

EU Projects (present and progress)

EURenOmics EU project, partner
RenalTract Marie Curie ITN, partner
Centre of Excellence in Cell-Extracellular Matrix Research, Academy of Finland Program for 2012-2017, partner

Co-operation With Finnish and Foreign Companies

Involvement of companies on the Tekes projects

Last updated: 15.10.2018