Gene expression profiling of postnatal lung development in the marsupial gray short-tailed opossum (Monodelphis domestica) highlights conserved developmental pathways and specific characteristics during lung organogenesis

Abstract

Background: After a short gestation, marsupials give birth to immature neonates with lungs that are not fully developed and in early life the neonate partially relies on gas exchange through the skin. Therefore, significant lung development occurs after birth in marsupials in contrast to eutherian mammals such as humans and mice where lung development occurs predominantly in the embryo. To explore the mechanisms of marsupial lung development in comparison to eutherians, morphological and gene expression analysis were conducted in the gray short-tailed opossum (Monodelphis domestica).

Results: Postnatal lung development of Monodelphis involves three key stages of development: (i) transition from late canalicular to early saccular stages, (ii) saccular and (iii) alveolar stages, similar to developmental stages overlapping the embryonic and perinatal period in eutherians. Differentially expressed genes were identified and correlated with developmental stages. Functional categories included growth factors, extracellular matrix protein (ECMs), transcriptional factors and signalling pathways related to branching morphogenesis, alveologenesis and vascularisation. Comparison with published data on mice highlighted the conserved importance of extracellular matrix remodelling and signalling pathways such as Wnt, Notch, IGF, TGFβ, retinoic acid and angiopoietin. The comparison also revealed changes in the mammalian gene expression program associated with the initiation of alveologenesis and birth, pointing to subtle differences between the non-functional embryonic lung of the eutherian mouse and the partially functional developing lung of the marsupial Monodelphis neonates. The data also highlighted a subset of contractile proteins specifically expressed in Monodelphis during and after alveologenesis.

Conclusion: The results provide insights into marsupial lung development and support the potential of the marsupial model of postnatal development towards better understanding of the evolution of the mammalian bronchioalveolar lung.

Keywords: Lung; Marsupial; Monodelphis domestica; RNA-seq.

Fig. 1

H&E staining of lung tissue samples collected from Monodelphis at different time points of postnatal development. Based on morphology as described in the text, the lungs of new born at day 1 (a) were in the canalicular stage, lungs collected at day 8 (b) and day 14 (c) were at early saccular stage, and at saccular stage by day 29 (d) and day 35 (e), while at day 61 (f) lungs were mature with an increased alveolar number similar to an adult lung (g). a Is a full embryo cross-section. Whole lung mounts (b to f), scale bar 1 mm. See also Additional file 1: Figure S1 for pictures at higher resolution where individual cell nuclei are easier to identify

Fig. 2

Global gene expression analysis of Monodelphis lung. a Hierarchical clustering dendogram of postnatal log expression data. b First two principal components PCA plot. c Heatmap of lung specific gene expression

Fig. 3

Heatmap and hierarchical clustering of 1242 genes with the most significantly differential expression during postnatal lung development (P < 0.05). Centred and scaled log RNA-seq expression data. Rows in the heatmap represent genes and columns represent samples. The gene tree on the left was generated by hierarchical clustering. Four gene clusters were selected to represent genes with different expression dynamics (k-mean clustering, k = 4, right side). Expression profile of the four major gene expression clusters was during (a) the later alveolar stage of development, b saccular stage of development, c earlier postnatal period with a gradual reduction of expression during the course of development, and (d) early stages of development. The limits of each cluster are represented by grey and white bars along the right side of the heatmap. Cluster A is the largest gene cluster and the expression profile of this cluster is summarized by a dark grey band (min, max log expression) rather than showing independent gene expression as in cluster (b to d). The light grey shaded area represents the range log expression of all the data (1242 genes)

Fig. 4

Validation of RNA-seq data. Comparison of gene expression profiles obtained by q-PCR (line) and RNA-seq quantification (bar). For this validation, 10 genes were chosen to represent expression dynamics of clusters identified in Fig. 3. This includes genes from cluster A (SPINK5, CA3, ARSF, MYOTand DSG1), cluster C (HPX, FGA, SERPINC1) and cluster D (TNC, HTRA1) in Fig. 3. In general, there is high concordance between expression results from q-PCR and RNA-seq data

Fig. 5

Differentially expressed genes related to lung morphology and development. Heat map (centered and scaled RNA-seq expression data) showing the expression profile of genes related to (a) the extracellular matrix (ECM and MMPs) and (b) regulatory factors (signalling molecules and transcriptional factors). Rows represent genes and columns represent development phases. Gene symbols are listed on the right side. While a majority of ECM molecules exhibit higher expression during development (day 3 to 35), regulatory molecules are either highly expressed during early (top), intermediate (bottom) or late (middle) phases of development

Fig. 6

a Principal Component Analysis and clustering of the combined gene expression datasets from Monodelphis (red, postnatal day D3, D8, D14, D29, D35, D63 and adult) and mice GSE20954 (black, days post-conception X18, X18, X22, X30 and X50). b Principal Component Analysis of the combined gene expression datasets in A plus a second, more comprehensive, mice dataset GSE74243 (blue: embryonic day 16.5 to 19.5, postnatal day P0 to P56, average expression of replicates at each time point for the B6 strain). c, d Hierarchical co-clustering and bootstrap values of combined data in A and B (correlation distance, clustering method complete). The tree is annotated with Bootstrap Probability (green) and Approximately Unbiased p-value (red)

Fig. 7

Clustering of correlated gene expression profiles during lung development in monodelphis and mice. Heatmap of 207 genes with highly correlated (correlation > 0.8) gene expression changes during lung development in Monodelphis (red labels) and mice GSE20954 (black labels) and GSE74243 (blue); centered and scaled log expression values. GSE74243 contains data from 3 strains of mice (green AJ, blue B6 and orange C3). Here we used the average of the 3 animal replicates to summarise expression values for each strain at each type point. The grouping of mice data is represented at the bottom