Developmental Biology

a worm

Dr. Inge The

Research summary

Previous research

Before I joined the division of Developmental biology at Utrecht University, I was an Assistant Professor at University of Massachusetts Medical School in Worcester, U.S.A. I investigated the role of tout velu, a homolog of the EXT tumor suppressor genes, in Hedgehog signaling and gradient formation in Drosophila melanogaster. Hedgehog is a secreted protein that is important during development of many organisms. In Drosophila as well as mammals, Hedgehog family members are required in patterning and contribute to formation of many organs during development. Loss of Hedgehog leads to several developmental defects, while overactivation of the Hedgehog signaling pathway is found responsible for the formation of many cancer types. My main contribution is the finding that the EXT proteins regulate Hedgehog levels in the extracellular matrix by modification of Heparan Sulfate Proteoglycans. Since the levels of secreted Hedgehog protein is important for cell fate specification, loss of the EXT gene will lead to aberrant Hedgehog levels and therefore signaling.

Current research

When I joined Utrecht University I decided to follow a new research interest, the regulation of cell proliferation vs. differentiation. For these studies I am using C. elegans as a model system, which is a new and exciting development for me. On this project I am collaborating with the group of Sander van den Heuvel.

Developmental control of G1 to S transition

My work focuses on cell-cycle entry and exit in C. elegans development. Initiation of the cell division cycle requires activation of Cyclin dependent kinases (CDK’s). CDK/Cyclin complexes phosphorylate specific substrates to promote entry into the cell cycle. Together with the van den Heuvel lab, we are involved in several projects that address the following questions:

  1. What upstream signals control CDK activation and cell cycle entry?
  2. Which controls act in parallel to the CDK’s in cell cycle initiation?
  3. What are critical downstream targets of CDK phosphorylation?
  4. How is cell-cycle exit initiated and maintained coincident with terminal differentiation?

Genetic identification of critical downstream targets of CDK4/Cyclin D

Cells generally decide during the G1 phase of the cell cycle whether or not to proceed through another division cycle. Our genetic analysis previously showed that CDK-4 and Cyclin D are essential for G1/S progression in C. elegans. LIN-35, the sole Retinoblastoma tumor suppressor (Rb) protein family member in C. elegans, is a critical target for inactivation by CDK-4/Cyclin D (Boxem et al Dev 2001). These results are analogous to those obtained in other metazoans, including mammals.

As an important additional observation, we found that LIN-35 Rb is not the only critical substrate of Cyclin D/CDK-4. To identify additional critical targets, we screened for mutants that, in combination with lin-35 Rb loss of function,bypass the requirement for cyd-1/cdk-4. Thus far, a single mutant, he121, has been found to rescue the phenotype of lin-35; cyd-1 mutants to wild type. Triple mutant animals lin-35; he121 cyd-1 (or cdk-4) are large, fertile and of normal morphology. Thus the combination of lin-35 Rbinactivation and he121 mutation completely overcomes the requirement for the Cdk4/Cyclin D kinase in regulating cell cycle entry. This makes he121 an interesting gene and potential downstream target of CDK-4/Cyclin D.

Identifying which gene is affected by the he121 mutation is not trivial. The mutation by itself does not cause an apparent phenotype. Hence, mapping of the he121 mutation requires two other mutations, lin-35 and cyd-1 or cdk-4 loss of function, in the background. We have narrowed the location within a few map units on chromosome 2, and the laboratory of Edwin Cuppen has sequenced the entire genome of the he121 strain. We are currently analyzing candidate genes that contain homozygous mutations or polymorphisms. In addition, we are screening for additional alleles and additional mutations that suppress cyd-1 or cdk-4 mutants.

Teaching

I co-developed Developmental Biology courses at the Bachelor's and Master's levels, and I currently teach in these and other courses (see education).

People

Former lab members:

  • Martine Prinsen (Research technician)
  • Vivian Su (PhD student)
  • Ujjaini Dasgupta (Postdoctoral fellow)
  • Kelly Donovan (Research technician)
  • Hau Hung (Research technician)
  • Elena Jelezkova (Research technician)

Selected Publications

The I, Ruijtenberg S, Bouchet BP, Cristobal A, Prinsen MB, van Mourik T, Koreth J, Xu H, Heck AJ, Akhmanova A, Cuppen E, Boxem M, Muñoz J, van den Heuvel S. Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells. Nat. Commun. 2015 Jan 6;6:5906.

Korzelius J, The I, Ruijtenberg S, Prinsen MB, Portegijs V, Middelkoop TC, Groot Koerkamp MJ, Holstege FC, Boxem M, van den Heuvel S. Caenorhabditis elegans cyclin D/CDK4 and cyclin E/CDK2 induce distinct cell cycle re-entry programs in differentiated muscle cells. PLoS Genet. 2011 Nov;7(11)

Ruijtenberg S, van den Heuvel S, The I. Regulation of DNA synthesis and replication checkpoint activation during C. elegans development. InTech Open Access Books. Edited by Jelena Kusic-Tisma. 2011 Sep. ISBN 978-953-307-775-8

Korzelius J, The I, Ruijtenberg S, Portegijs V, Xu H, Horvitz HR, van den Heuvel S. C. elegans MCM-4 is a general DNA replication and checkpoint component with an epidermis-specific requirement for growth and viability. Dev. Biol. 2011 Feb 15;350(2):358-69.

Dasgupta U, Dixit BL, Rusch M, Selleck S, The I. Functional conservation of the human EXT1 tumor suppressor gene and its Drosophila homolog tout velu. Dev. Genes Evol. 2007 Aug;217(8):555-61.

Wildwater M, The I, van den Heuvel S. Coordination of cell proliferation and differentiation: finding a GEM in the root? Dev. Cell. 2007 Jun;12(6):841-2.

Su VF, Jones KA, Brodsky M, The I. Quantitative analysis of Hedgehog gradient formation using an inducible expression system. BMC Dev. Biol. 2007 May 7;7:43.

Micchelli CA, The I, Selva E, Mogila V, Perrimon N. Rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling. Development. 2002 Feb;129(4):843-51.

The I, Perrimon N. Morphogen diffusion: the case of the wingless protein. Nat. Cell Biol. 2000 May;2(5):E79-82.

The I, Bellaiche Y, Perrimon N. Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan. Mol. Cell. 1999 Oct;4(4):633-9.

Bellaiche Y, The I, Perrimon N. Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion. Nature. 1998 Jul 2;394(6688):85-8.

Guo HF, The I, Hannan F, Bernards A, Zhong Y. Requirement of Drosophila NF1 for activation of adenylyl cyclase by PACAP38-like neuropeptides. Science. 1997 May 2;276(5313):795-8.

The I, Hannigan GE, Cowley GS, Reginald S, Zhong Y, Gusella JF, Hariharan IK, Bernards A. Rescue of a Drosophila NF1 mutant phenotype by protein kinase A. Science. 1997 May 2;276(5313):791-4.

Ishioka C, Ballester R, Engelstein M, Vidal M, Kassel J, The I, Bernards A, Gusella JF, Friend SH. A functional assay for heterozygous mutations in the GTPase activating protein related domain of the neurofibromatosis type 1 gene. Oncogene. 1995 Mar 2;10(5):841-7.

The I, Murthy AE, Hannigan GE, Jacoby LB, Menon AG, Gusella JF, Bernards A. Neurofibromatosis type 1 gene mutations in neuroblastoma. Nat Genet. 1993 Jan;3(1):62-6.