Hammerschmidt, Matthias - C 07

Like in mammals, full-thickness wounds in the skin of adult zebrafish form large collagenous granulation tissues, involving an essential cross talk between innate immune cells (in particular macrophages) and fibroblasts. However, in contrast to mammals, the zebrafish granulation tissue perfectly regresses afterwards, leaving no scar behind (Fig. 1; Ref. 1). The mechanisms of this perfect regression, and the molecular and cellular basis of the different regeneration potentials in fish versus mammals, remain largely elusive. 

In principle, fish should either possess anti-fibrotic factors absent in mammals or should lack pro-fibrotic factors present in mammals. To identify such differentiating factors, we have performed single cell RNA-seq for different stages of cutaneous wound healing in adult zebrafish and have started to compare them with corresponding scRNA-seq data acquired by the Eming laboratory for mouse wounds (Fig. 2; Ref. 2). Yet, to our surprise, we found multiple mammalian pro-fibrotic factors also up-regulated in zebrafish wounds. One of them is Lysylhydroxylase 2 (encoded by the plod2 gene) made by wound fibroblasts, which promotes the formation of stable DHLNL collagen cross links (Fig. 2; Ref. 2), indicating that the extracellular matrix of the fish granulation tissue can be degraded even though collagens are similarly stably cross-linked as in scarring mammalian wounds. Therefore, more systematic cross-species comparisons will be necessary. Such comparative functional analyses will be facilitated by a better understanding of some of the cellular mechanisms underlying granulation tissue regression in fish wounds. This proposal aims to provide such basic insights while testing first selected genes. The specific aims of the project are: 

1. Analysis of the mechanisms by which fibroblasts and extracellular matrix are lost during zebrafish granulation tissue
2. Dissection of the impacts of neutrophils and macrophages on fish granulation tissue regression.
3. Gain-of-function analysis of selected genes with annotated pro-fibrotic functions in mammals that are absent from zebrafish wounds (Ref. 2), for instance amphiregulin (areg), an EGF-like secreted growth factor that in mammals is made by wound macrophages and by cancer-associated fibroblasts, promoting scarring and carcinogenesis (Fig. 2; Refs. 3,4)

Fibrosis represents a major burden affecting millions of people worldwide, with significant morbidity and mortality. It is estimated that 45% of deaths in the Western world can be attributed to diseases where fibrosis plays a major etiological role. Skin fibrosis and scarring, a consequence of an exaggerated healing response, although usually not life-threatening, also represents a serious clinical problem causing physical and psychological challenges for millions of patients. Elucidating the mechanisms underlying perfect and scar-free cutaneous wound healing in zebrafish should pave the path towards anti-fibrotic therapeutic treatments alleviating both dermal fibrosis and even more deleterious fibrosis for instance in heart, liver, kidneys or lungs of human patients.

Aim 1: 

• Fibroblast death: Tunel stainings; transgene-driven blockage of fibroblast cell death with Tg(col1a2:Cre) x Tg(hsp70:loxP-Stop-loxP-bcl2) + heatshock for temporally confined transgene activation at 4 dpw.
• Fibroblast emigration: in vivo imaging of fibroblasts with Tg(col1a2:mCherry); transgene-driven blockage of fibroblast motility with Tg(col1a2:Cre) x Tg(hsp70:loxO-Stop-loxP-dnRac1) + heatshock at 4 dpw.
• Collagen degradation and phagocytosis: in vivo imaging of GFP-fused collagen-type I fiber
• (TgKi(col1a2:col1a2-GFP)) vanishment / shrinkage and uptake by macrophages (Tg(mpeg:mCherry)).

Aim 2: 

• Transgene-driven conditional ablation of neutrophiles and/or macrophages shortly before wounding: Tg(mpx:NTR-mCherry) (neutrophils) and/or Tg(mpeg:NTR-Cherry) (macrophages) + bath incubation in prodrug Metronidazole / Nifurpirinol / Ronidazole.

Aim 3: 

• Transgene-driven global (Tg(hsp70:areg-p2A-GFP) + daily heatshocks from 0-4 dpw; Ref. 2) or macrophage or fibroblast-specific (Tg(mpeg:Cre) or Tg(col1a2:Cre) x Tg(hsp70:loxP-stop-loxP-areg-p2A-GFP) + daily heatshocks from 0-4 dpw) – forced expression of areg -> more and/or more persistent granulation tissue / fibrosis?

1. Richardson R, Slanchev K, Kraus C, Knyphausen P, Eming S, Hammerschmidt M (2013). Adult Zebrafish as a Model System for Cutaneous Wound-Healing Research. J Invest Dermatol. 133(6):1655-65. doi: 10.1038/jid.2013.16.
2. Küçükaylak I, Morcillo FJM, Halwas K, Reiche N, Metzger M, Comelli P, Brinckmann J, Eming SA, Hammerschmidt M (2025) Exploring mechanisms of scar-free skin wound healing in adult zebrafish in comparison to mouse. bioRxiv [Preprint]. doi: 10.1101/2025.09.09.675222
3. Zhang MY, Fang S, Gao H, Zhang X, Gu D, Liu Y, Wan J, Xie J (2021). A critical role of AREG for bleomycin-induced skin fibrosis. Cell Biosci. 23;11(1):40. doi: 10.1186/s13578-021-00553-0.
4. Zhou J, Xu Y, Li Y, Zhang Q, Zhong L, Pan W, Ji K, Zhang S, Chen Z, Liu Y, Fan L, Liu C, Chen Q, Wang Z (2025). Cancer-associated fibroblasts derived amphiregulin promotes HNSCC progression and drug resistance of EGFR inhibitor. Cancer Lett. 622:217710. doi: 10.1016/j.canlet.2025.217710

For information about Prof. Hammerschmidt's research and work, please visit the following page: AG Hammerschmidt

• Center for Molecular Medicine Cologne (CMMC)
• Institute of Zoology, Developmental Biology Unit, University of Cologne, Cologne, Germany