BACKGROUND: Gonadal sex determination (GSD) in humans is a complex biological process that takes place in early stages of embryonic development when the bipotential gonadal primordium (BGP) differentiates towards testes or ovaries. This decision is directed by one of two distinct pathways embedded in a GSD network activated in a population of coelomic epithelial cells, the Sertoli progenitor cells (SPC) and the granulosa progenitor cells (GPC). In males, the pathway is activated when the Sex-Determining Region Y (SRY) gene starts to be expressed, whereas in females the WNT4/ β-catenin pathway promotes the differentiation of the GPCs towards ovaries. The interactions and dynamics of the elements that constitute the GSD network are poorly understood, thus our group is interested in inferring the general architecture of this network as well as modeling the dynamic behavior of a set of genes associated to this process under wild-type and mutant conditions. METHODS: We reconstructed the regulatory network of GSD with a set of genes directly associated with the process of differentiation from SPC and GPC towards Sertoli and granulosa cells, respectively. These genes are experimentally well-characterized and the effects of their deficiency have been clinically reported. We modeled this GSD network as a synchronous Boolean network model (BNM) and characterized its attractors under wild-type and mutant conditions. RESULTS: Three attractors with a clear biological meaning were found; one of them corresponding to the currently known gene expression pattern of Sertoli cells, the second correlating to the granulosa cells and, the third resembling a disgenetic gonad. CONCLUSIONS: The BNM of GSD that we present summarizes the experimental data on the pathways for Sertoli and granulosa establishment and sheds light on the overall behavior of a population of cells that differentiate within the developing gonad. With this model we propose a set of regulatory interactions needed to activate either the SRY or the WNT4/ β-catenin pathway as well as their downstream targets, which are critical for further sex differentiation. In addition, we observed a pattern of altered regulatory interactions and their dynamics that lead to some disorders of sex development (DSD). Crowther Audrey audrey.crowther@huskers.unl.edu University of Nebraska-Lincoln 2016-08-22T09:27:00Z 2017-02-13T09:58:53Z

Sex-determining Region Y gene

Gene: SRY

UniProt ID: Q05066

Gene ID: 6736

 2016-09-15T21:31:46Z

R-spondin-1

Gene: RSPO1

UniProt ID: Q2MKA7

Gene ID: 284654

 2016-08-22T10:37:27Z

Fibroblast growth factor 9

Gene: FGF9

UniProt ID: P31371

Gene ID: 2254

 2016-08-26T22:31:27Z

Prostaglandin D₂

 2016-08-26T22:38:00Z

Wilms tumor protein -KTS isoform

Gene: WT1

UniProt ID: P19544

Gene ID: 7490

 2017-02-12T21:29:55Z

Transcription factor GATA-4

Gene: GATA4

UniProt ID: P43694

Gene ID: 2626

 2017-02-12T21:29:55Z

Protein Wnt-4

Gene: WNT4

UniProt ID: P56705

Gene ID: 54361

 2016-11-09T10:42:44Z

urogenital ridge

This node encompasses the following genes: LHX1, LHX9, EMX2, PAX2 and, PAX8

 2016-08-22T10:07:03Z

Doublesex- and mab-3-related transcription factor 1

Gene: DMRT1

UniProt ID: Q9Y5R6

Gene ID: 1761

 2016-08-22T10:36:23Z

Forkhead box protein L2

Gene: FOXL2

UniProt ID: P58012

Gene ID: 668

 2016-08-27T13:13:48Z

Wilms tumor protein +KTS isoform

Gene: WT1

UniProt ID: P19544

Gene ID: 7490

 2016-08-22T10:15:28Z

Dickkopf-related protein 1

Gene: DKK1

UniProt ID: O94907

Gene ID: 22943

 2016-08-22T10:35:34Z

Steroidogenic factor 1

Gene: NR5A1

UniProt ID: Q13285

Gene ID: 2516

 2016-08-26T22:15:53Z

Catenin beta-1

Gene: CTNNB1

UniProt ID: P35222

Gene ID: 1499

 2016-08-22T10:37:27Z

Chromobox protein homolog 2

Gene: CBX2

UniProt ID: Q14781

Gene ID: 84733

 2016-09-15T21:31:46Z

Nuclear receptor subfamily 0 group B member 1

Gene: NR0B1

UniProt ID: P51843

Gene ID: 190

 2016-08-22T10:34:01Z

Muellerian-inhibiting factor

Gene: AMH

UniProt ID: P03971

Gene ID: 268

 2016-08-22T10:37:27Z

Transcription factor SOX-9

Gene: SOX9

UniProt ID: P48436

Gene ID: 6662

 2016-09-15T21:31:46Z

Desert hedgehog protein

Gene: DHH

UniProt ID: O43323

Gene ID: 50846

 2016-08-22T10:27:21Z

SRY activates SRY.

CBX2 wtih NR5A1 and WT1mKTS activates SRY.

SOX9 activates SRY.

GATA4 with CBX2, NRA1, and WT1pKTS activates SRY.

CTNNB1 inhibits SRY.

GATA4 cooperates with NR5A1 to upregulate SRY gene expression.

WTmKTS activates the SRY promoter.

CTNNB1 indirectly inhibits SRY.

GATA4 cooperates with NR5A1 to upregulate SRY gene expression.

CBX2 upregulates SRY gene expression.

SOX9 is a predicted activator for SRY.

WT1pKTS has been suggested to maintain a critical level of SRY expression.

SRY is a predicted activator for SRY.

 S_6 1 S_13 1 S_15 1 S_11 1 S_14 1 S_18 1 S_14 1 S_1 1 S_14 1 S_15 1 S_13 1 S_5 1 S_14 1

DKK1 inhibits RSPO1.

CTNNB1 and WNT4 activate RSPO1.

RSPO1 has been identified as a Wnt/β-catenin gene target.

RSPO1 has been identified as a Wnt/β-catenin gene target.

High expression of DKK1 leads to downregulation of RSPO1 signaling.

 S_7 1 S_12 1 S_14 1 S_12 1

SOX9 activates FGF9.

WNT4 inhibits FGF9.

WNT4 and FGF9 play antagonistic roles in sex differentiation.

SOX9 indirectly activates FGF9.

 S_18 1 S_7 1

SOX9 activates PGD2.

PGD2 contains a SOX9 binding site within the 5′-flanking region.

 S_18 1

GATA4 activates WT1mKTS.

UGR activates WT1mKTS.

GATA4 is a predicted activator for WT1mKTS.

Pax2 (one of the proteins represented by UGR) activates the WT1 promoter.

 S_8 1 S_6 1

WNT4 activates GATA.

UGR activates GATA4.

SRY activates GATA4.

NR5A1 activates GATA4.

NR5A1 is a predicted activator for GATA4.

WNT4 is a predicted activator for GATA.

UGR is a predicted activator for GATA4.

SRY is a predicted activator for GATA4.

 S_7 1 S_1 1 S_13 1 S_8 1

GATA4 activates WNT4.

NR0B1 activates WNT4.

RSPO1 activates WNT4.

FGF9 inhibits WNT4.

DKK1 inhibits WNT4.

CTNNB1 activates WNT4.

GATA4 mutant lines do not express WNT4.

DKK1 is a secreted inhibitor that inhibits canonical β-catenin signaling by targeting WNT4.

WNT4 is a downstream gene target of CTNNB1 .

NR0B1 is a predicted activator for WNT4.

Reduced RSPO1 expression leads to reduced WNT4 mRNA.

FGF9 and WNT4 play antagonistic roles in sex differentiation..

 S_16 1 S_12 1 S_3 1 S_14 1 S_12 1 S_3 1 S_6 1 S_12 1 S_3 1 S_2 1 S_12 1 S_3 1

UGR activates UGR.

NR5A1 and WNT4 inhibit UGR.

NR5A1 is a predicted inhibitor for UGR.

WNT4 is a predicted inhibitor for UGR.

UGR is a predicted activator for UGR.

 S_8 1 S_13 1 S_7 1

SOX9 and SRY are activators for DMRT1.

FOXL2 inhibits DMRT1.

SOX9 is a predicted activator for DMRT1.

SRY is a predicted activator for DMRT1.

FOXL2 and DMRT1 play antagonistic roles in sex differentiation.

 S_1 1 S_10 1 S_18 1 S_10 1

SOX9 inhibits FOXL2.

DMRT1 inhibits FOXL2.

WNT4 with CTNNB1 activates FOXL2.

WNT4 and FOXL2 have complementary roles in ovarian development.

FOXL2 activity is partly dependent on CTNNB1 expression.

FOXL2 and DMRT1 play antagonistic roles in sex differentiation.

SOX9 activity is predicted to downregulate FOXL2 expression.

 S_7 1 S_14 1 S_9 1 S_18 1

WNT4 with CTNNB1 inhibits WT1pKTS.

UGR activates WT1pKTS.

GATA4 activates WT1pKTS.

WNT4 with CTNNB1 is a predicted inhibitor for WT1pKTS.

WNT4 with CTNNB1 is a predicted inhibitor for WT1pKTS.

GATA4 is a predicted activator for WT1pKTS.

Pax2 (one of the proteins represented by UGR) activates the WT1 promoter.

 S_6 1 S_7 1 S_14 1 S_8 1 S_7 1 S_14 1

SOX9 and SRY are predicted activators for DKK1.

SOX9 is a predicted activator for DKK1.

SRY is a predicted activator for DKK1.

 S_18 1 S_1 1

CBX2 activates NR5A1.

NR0B1 with WNT4 inhibits NR5A1.

WT1mKTS activates NR5A1.

UGR activates NR5A1.

GATA4 activates NR5A1.

WT1mKTS binds to the NR5A1 promoter.

GATA4 binds to NR5A1.

WNT4 indirectly inhibits NR5A1.

CBX2 binds to the NR5A1 promoter.

NR5A1 is expressed in the UGR.

NR0B1 inhibits NR5A1-mediated transactivation.

 S_5 1 S_16 1 S_7 1 S_8 1 S_16 1 S_7 1 S_6 1 S_16 1 S_7 1 S_15 1 S_16 1 S_7 1

SOX9 with AMH inhibits CTNNB1.

RSPO1 and WNT4 activate CTNNB1.

AMH is a predicted activator for CTNNB1.

SRY inhibits CTNNB1.

WNT4 may be involved in CTNNB1 relocalization to the cell membrane during ovarian development.

SOX9 expression downregulates RSPO1 and CTNNB1 signaling in an unknown mechanism.

AMH is a predicted activator for CTNNB1.

In the absence of RSPO1, CTNNB1 transcriptional activity decreases.

SRY expression downregulates RSPO1 and CTNNB1 signaling in an unknown mechanism.

 S_2 1 S_1 1 S_18 1 S_17 1 S_7 1 S_1 1 S_18 1 S_17 1

NR0B1 with CTNNB1 and WNT4 inhibits CBX2.

UGR activatesCBX2.

NR0B1 with CTNNB1 and WNT4 is a predicted inhibitor for CBX2.

NR0B1 with CTNNB1 and WNT4 is a predicted inhibitor for CBX2.

UGR is a predicted activator for CBX2.

NR0B1 with CTNNB1 and WNT4 is a predicted inhibitor for CBX2.

 S_8 1 S_16 1 S_7 1 S_14 1

NR5A1 with SOX9 inhibits NR0B1.

WT1mKTS activates NR0B1.

WNT4 with CTNNB1 activates NR0B1.

WT1mKTS activates the NR0B1 promoter.

NR5A1 represses NR0B1 gene transcription.

CTNNB1 forms a complex with TCL and activates NR0B1 transcription.

WNT4 upregulates NR0B1 gene expression.

SOX9 indirectly inhibits NR0B1.

 S_5 1 S_13 1 S_18 1 S_7 1 S_14 1 S_13 1 S_18 1

SOX9 with GATA4 and NR5A1 or GATA4 and NR5A1 and WT1mKTs activates AMH.

NROB1 with CTNNB1 inhibits AMH.

NR5A1 positively regulates gene expression of AMH.

WT1mKTS associates with SF-1 to synergistically activate AMH.

GATA4 works synergistically with SF-1 to upregulate AMH expression.

CTNNB1 has been implicated in regulating AMH signaling.

SOX9 binds within the proximal promoter of AMH.

NROB1 inhibits SF-1 and WT1 activation of the AMH gene promoter.

 S_18 1 S_13 1 S_5 1 S_6 1 S_13 1 S_6 1 S_16 1 S_14 1

CBX2 with SRY activates SOX9.

FGF9 with SOX9 activates SOX9.

CTNNB1, FOXL2, and WNT4 inhibit SOX9.

GATA4 with NR5A1 and SRY activates SOX9.

PGD2 activates SOX9.

NR5A1 with SRY activates the enhancer of Sox9 (TES).

GATA4 upregulates SOX9 transcription.

Repression of WNT4 either directly or indirectly stabilizes SOX9 expression.

CTNNB1 indirectly inhibits SOX9.

CBX2 upregulates SOX9 transcription.

FGF9 is upregulated by SOX9. Once FGF9 secretion reaches a critical threshold, SOX9 expression stabilizes.

SRY activates the enhancer of Sox9 (TES).

PGD2 signaling activates SOX9 nuclear translocation.

FGF9 is upregulated by SOX9. Once FGF9 secretion reaches a critical threshold, SOX9 expression stabilizes.

FOXL2 inhibits the action of testis determinants, such as SOX9.

 S_15 1 S_1 1 S_10 1 S_7 1 S_14 1 S_1 1 S_10 1 S_7 1 S_14 1 S_4 1 S_10 1 S_7 1 S_14 1 S_6 1 S_13 1 S_1 1 S_10 1 S_7 1 S_14 1 S_3 1 S_18 1 S_10 1 S_7 1 S_14 1

SOX9 activates DHH.

SOX9 activates the secretion of DHH by Sertoli cells.

 S_18 1